BEHAVIORAL AND BRAIN SCIENCES (1995) 18, 403-414
Printed in the United States of America
Future directions for rhodopsin
structure and function studies
Paul A. Hargrave
Department of Ophthalmology, and Department of Biochemistry and
Molecular Biology, School of Medicine, University of Florida, Gainesville,
FL 32610
Electronic mail: hargrave@eyel.eye.ufl.edu
Abstract: To understand how the photoreceptor protein rhodopsin performs in its role as a receptor, its structure needs to be
determined at the atomic level. Upon receiving a photon of light, rhodopsin undergoes a change in conformation that allows it to bind
and activate the C-protein, transducin. An important future goal should be to determine the structure of both the inactive and the
photoactivated state of rhodopsin, R*. This should provide the groundwork necessary for experiments on how rhodopsin achieves its
signaling state R*, and how R* functions to activate transducin. To do this, the crystal structure of both rhodopsin and R* must be
determined. Few membrane proteins have been successfully crystallized, so this is not a trivial undertaking. Two- or threedimensional crystals of rhodopsin must be prepared that are well ordered, to produce a high-resolution structure. Rhodopsin must be
purified to homogeneity and the appropriate detergent(s) selected for crystallization experiments. Long-term thermal stability of the
rhodopsin-detergent complex must be achieved in the presence of a precipitant. Two-dimensional crystals may offer advantages in
investigating the structure of R*, but the structure obtained may be limited in resolution. It is necessary to work with rhodopsin in the
dark, unless suitable light-stable retinal derivatives are developed. Protein engineering of rhodopsin offers attractive opportunities to
improve its ability to crystallize, but is presently hindered by the absence of a high-yielding expression system. Knowledge of the
structure of rhodopsin will have general importance. Because rhodopsin is a member of the family of G-protein-coupled receptors,
knowledge of the structure and the mechanism of action of rhodopsin suggests by analogy how other members of the receptor family
may function.
Keywords: detergent; C-protein-coupled receptor; membrane protein; membrane protein crystallization; photoreceptor; protein
crystallization; protein structure; retinal; rhodopsin; X-ray structure
1. Introduction1
What does rhodopsin do as a photoreceptor protein, and
how does it do it? The answer to these questions will
provide the details of rhodopsin's structure, and how it
acts to carry out the functions of a photoreceptor protein.
We know the details in rough outline - the folding and
insertion of the opsin polypeptide chain into the membrane, its binding of 11-cis retinal, the vectorial transport
to the outer segment, absorption of a photon by the
retinal, the change in conformation of the protein, and the
subsequent binding and activation of transducin. But how
is this all actually achieved at the molecular level? How
does the protein primary structure adopt the appropriate
conformation to accomplish these results? To answer this
it will be necessary first to know the details of rhodopsin's
structure in its inactive state in the dark, and its structure
when activated by light, in its signaling state. It would be
important to determine the structure of Metarhodopsin I
(which contains all-trans retinal but cannot activate transducin) and the difference between Metarhodopsin I and
Metarhodopsin II (R*). These are necessary and achievable goals that will provide the baseline for understanding
rhodopsin function. Rhodopsin is more easily obtained in
the amounts needed for physical studies than other members of this large class of G-protein-coupled receptors. By
analogy, insights into the function of rhodopsin should be
© J995 Cambridge University Press
0140-525X195 $9.00+.10
valuable for understanding the signaling mechanism of
the entire class of receptors.
Mutants of rhodopsin, naturally occurring and otherwise, have proven useful in demonstrating what particular
regions or amino acids in rhodopsin are most functionally
significant (see Berson 1993; Dryja 1992; Khorana 1992;
Nathans 1992; Oprian 1992). This important area must be
left for consideration elsewhere. In the present target
article I will discuss the prospects for obtaining a highresolution atomic structure for rhodopsin, which I believe
will be critical for understanding the function of Gprotein-linked receptors generally.
2. What do we think we know about the structure
of rhodopsin?
There has been a virtual explosion of information about
rhodopsin during the past twenty years. Original references to the literature are to be found in several recent
reviews (Applebury 1991; Applebury & Hargrave 1986;
Chabre 1985; Chabre & Deterre 1989; Findlay 1986;
Hargrave & McDowell 1992; 1993; Khorana 1992; Liebman et al. 1987; Nathans 1992). Although many gaps in
our knowledge have been filled, we are still a long way
from the goals outlined above. We view rhodopsin as an
intrinsic membrane protein, with one-half of its mass
403
Hargrave: Rhodopsin structure and function
Ac-N-
Figure 1. A topographic model for bovine rhodopsin in the rod cell disk membrane. Rhodopsin's polypeptide chain is shown
traversing the lipid bilayer 7 times yielding 7 hydrophobic helical segments (I to VII) that are separated from each other by
hydrophilic segments. Loops il-t4 and the carboxyl-terminal sequence face the cytoplasniic surface; loops el-e3 and the aminoterminal sequence are sequestered in the disk membrane lumen. (Based on Dratz & Hargrave 1983; Ovchinnikov et al. 1988a).
embedded in the lipid bilayer and the remaining half
approximately equally distributed between the two hydrophilic surfaces facing the cytoplasm and the intradiscal
environment. We believe it to be topographically organized so that the polypeptide chain traverses the lipid
bilayer seven times, alternately exposing hydrophilic sequences at membrane surfaces and burying hydrophobic
sequences in the lipid bilayer (Fig. 1). The amino terminus, to which carbohydrate is attached at two sites, is
located in the intradiscal environment; the carboxyl terminus, containing serine and threonine residues that may
be phosphorylated by rhodopsin kinase, is exposed to the
rod cell cytoplasm. The transmembrane segments are
thought to be largely helical in structure, although several
of them may be irregular due to presence of a proline in
their sequence. Rhodopsin's chromophore, ll-cis retinal,
is attached by Schiff base linkage to the side chain of lysine
296 (Lys296), located midway in the membrane-spanning
seventh helix. The retinal is enveloped in a pocket formed
by the amino acids lining the interior surfaces of rhodopsin's helices.
Further characteristic features emerge when the sequences of many vertebrate rhodopsins are compared.
Present are two highly conserved cysteines that are involved in a stabilizing disulfide bond linking helix III with
loop el. One or more cysteines are generally present in
the C-terminal sequence following helix VII. These cysteines become palmitoylated and presumably insert into
the lipid bilayer forming a fourth cytoplasmic loop region.
Additional amino acids are found to be conserved in all
rhodopsins studied to date. These conserved residues are
good candidates for structurally-functionally essential residues that are required components of what it means to be
404
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
a rhodopsin. Some of these residues are also conserved in
other members of the class of G-protein-coupled receptors (Probst et al. 1992) and may be assumed to be
required for features that are common to members of the
receptor class, such as membrane translocation or signal
transmission.
3. What do we think we know about the function
of rhodopsin?
Within a millisecond of absorbing light and isomerizing
its ll-cis retinal to all-trans, rhodopsin forms Metarhodopsin I (MI). This intermediate is not able to bind and
activate transducin, but Metarhodopsin II (Mil) is (Fig.
2). Deprotonation of the retinylidene-Schiffbase accompanies the conversion of MI to Mil (reviewed by Hofmann 1986). This deprotonation event is essential for the
activation of transducin.
The protonated retinylidene-Schiffbase is stabilized by
an ion pair with the carboxyl group of glutamic acid 113
(Glull3) (Nathans 1990; Sakmar et al. 1989; Zhukovsky &
Oprian 1989). Studies with mutant rhodopsins show that
the maintenance of the interaction between Lys296 and
Glull3 is essential in keeping rhodopsin inactive. Opsins
lacking either of these charged groups constitutively activate transducin (Robinson et al. 1992). Thus it appears, in
part, that when MI loses the proton from its Schiff base,
this breaks a key ionic interaction that allows it to assume
the more open protein structure, MIL
The cytoplasmic surface of rhodopsin and MI is unable
to bind and activate transducin. But the conformational
change that accompanies the formation of Mil causes a
Hargrave: Rhodopsin structure and function
7 ATP
Rhodopsin
Kinase
all-trans
retinal
7ADP
Figure 2. The rhodopsin cycle. Rhodopsin (R) is activated by
light (hv) and thereby forms Metarhodopsin I (MI), which exists
in equilibrium with Metarhodopsin II (Mil, which is equivalent
to R*). R* causes transducin (T) to become activated (to T*) by
CDP-» GTP exchange. R* becomes phosphorylated (R*(P,)7),
allowing it to bind arrestin, thereby blocking the ability of R* to
continue to activate transducin. Upon loss of all-trans retinal,
phosphorylated opsin {OiP^) is dephosphorylated and rebinds
11-cw retinal, regenerating rhodopsin.
rearrangement of surface residues that provides the appropriate surface array for binding transducin. Amino
acids in loops t2, t'3, and t4 are involved in this proteinprotein recognition event (Konig et al. 1989; reviewed in
Hargrave & McDowell 1993). Further molecular details
concerning how rhodopsin achieves all of these transformations, remain to be elucidated.
4. What will most advance our knowledge of how
rhodopsin acts as a photoreceptor?
To better understand how rhodopsin acts to transduce the
reception of a photon into activation of a G-protein, it will
be necessary to have an understanding of rhodopsin
structure at the level of atomic resolution. This will
require both a knowledge of where rhodopsin's amino
acids are located before absorption of a photon, and where
rhodopsin's amino acids become relocated to following
absorption of a photon, when rhodopsin becomes transformed to MI and then to its signaling state, MIL
All proteins are stabilized by a variety of individual
interactions of their amino acids, involving disulfide
bonds, hydrogen bonds, ionic bonds, and Van der Waals's
forces. In rhodopsin's inactive state these forces contribute to maintain a stable three-dimensional structure in
which the protein is constrained from binding and activating transducin. All proteins have a certain range of dynamic movement at any temperature, but this must be
quite small for vertebrate rhodopsin, since "noise" (signaling in the absence of photon absorption) is extremely
low (Baylor 1987).
The juxtaposition of amino acids in rhodopsin's signaling state serves to define the binding site for transducin.
The particular three-dimensional array that permits this
binding is not present in MI but is formed when rhodopsin adopts the Mil conformation. Thus, a knowledge of
the structure of Mil will show what amino acid interactions have been broken in rhodopsin and have been
formed to stabilize the receptor signaling state, MIL Such
information would provide the boundary conditions and
would be essential in beginning to understand how the
isomerization of 11-cts retinal leads to the structural
reorganization that allows binding and activation of
transducin.
5. What are the prospects for obtaining a highresolution structure for rhodopsin?
There are basically three methods for obtaining atomic
level structural data for proteins: (1) X-ray diffraction of
three-dimensional crystals (Kiihlbrandt 1988); (2) electron diffraction, electron microscopy, and image processing of two-dimensional crystals (Amos et al. 1982;
Kiihlbrandt 1992); and (3) nuclear magnetic resonance
(NMR). Rhodopsin, at 40 kDa, is too large for analysis by
currently available NMR techniques and cannot be examined in the membrane by liquid-state NMR. That leaves
crystallography.
What are the prospects for getting a high-resolution
structure for any membrane protein or protein complex?
At present there are atomic resolution structures for more
than 1,700 proteins, but only four classes of membrane
proteins are represented: bacteriorhodopsin from Halobacterium halobium (Henderson et al. 1990); the bacterial
photosynthetic reaction centers (Arnoux et al. 1989; Deisenhofer & Michel 1989; Feher et al. 1989) bacterial
porins (Cowan et al. 1992; Weiss et al. 1991); and light
harvesting complex from green plants (Kiihlbrandt et al.
1994). _
6. What are the chances of getting crystals of
rhodopsin?
The chances are excellent! Several investigators have
already devoted many years to this task and crystals have
been obtained. Both two-dimensional (Corless et al.
1982; Demin et al. 1987; Dratz et al. 1985; Schertler et al.
1993; Schertler & Hargrave 1995) and three-dimensional
crystals have been obtained (de Grip et al. 1992; Demin et
al. 1987; Yurkova et al. 1990; E. Dratz, personal communication).
Two-dimensional crystals, presumably of rhodopsin,
have been induced to form in the frog disk membrane
following extraction of some of the lipid with the detergent Tween 80 (Corless et al. 1982). The molecules in
these crystals appear to be 20-25A in width and 70-80A in
length and to have a cross-sectional area similar to that of
bacteriorhodopsin. When observed, crystals made up
~5%-10% of the membrane. Although they were not
definitively demonstrated to be composed of rhodopsin,
it seems unlikely that they would be composed of minor
membrane proteins. Two-dimensional (2-D) crystals of
bovine rhodopsin that diffract to a resolution of about 25A
have also been formed, using the Tween 80 extraction
method (Demin et al. 1987). In a different study, twodimensional crystals of bovine rhodopsin were induced to
form within the disk membrane in the presence of ammonium sulfate and an amphiphilic compound, chlorhexidine (Dratz et al. 1985). Analysis of these crystals by
electron microscopy also showed a surface area for rhodopsin similar to that of bacteriorhodopsin, further supBEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
405
Hargrave: Rhodopsin structure and function
porting the presence of seven transmembrane segments
in rhodopsin. However, none of the crystal preparations
obtained by these investigators were of sufficient quality
to give higher-resolution data; for example, data that
would allow individual helices of the protein to be resolved or the position and identity of individual amino
acids to be defined.
Visualization of the helices of bovine rhodopsin has
been obtained by cryoelectron microscopy of twodimensional crystals formed from purified rhodopsin in
phosphatidyl choline (Schertler et al. 1993). Rhodopsin
was solubilized in n-octyl tetraoxy-ethylene (C8E4) and
crystals were formed as the detergent was removed by
dialysis. Analysis of the crystals as frozen hydrated specimens allowed collection of data adequate for calculation of
a 9A resolution map. The map shows an elongated arcshaped feature flanked by four resolved peaks of density.
Orientation of the helices is clearly different from that of
bacteriorhodopsin. More recently, tubular structures
containing rhodopsin crystals have been formed in good
quantity by extracting frog disk membranes with Tween
80 (Schertler & Hargrave 1995). Electron micrographs of
the frozen hydrated crystals allowed a projection structure to be calculated to 6A resolution. This showed an
arrangement of helices similar to that in the map obtained
previously for bovine rhodopsin (Schertler et al. 1993).
Helices 4, 6, and 7 are nearly perpendicular to the
membrane plane, but helix 5 is more tilted or bent than
anticipated from the 9A map. The rhodopsin molecule can
be described as a bundle of four tilted helices alongside
three perpendicular helices that are arranged in a straight
line. The next step will be to collect data from tilted
specimens to allow calculation of a three-dimensional
map so that helix tilt angles can be determined.
Three-dimensional crystals of rhodopsin have been
obtained over a pH range from 5.5 to 7.0, using five
different detergents and two different precipitants (de
Grip et al. 1992). Crystals have also been obtained from
octyl polyoxyethylene (o-POE), using ammonium sulfate,
sodium phosphate, or sodium citrate as precipitants
(Yurkova et al. 1990). Unfortunately, the crystals formed
to date have been too small, fragile, and disordered to
allow high-resolution diffraction analysis. This also has
been the experience of other investigators who have
worked with rhodopsin (E. Dratz, personal communication). The crystals obtained from o-POE were needle
shaped with maximum dimension 70 u,m X 70 |x?n X 1000
|im; too small for X-ray analysis (Yurkova etal. 1990). Such
observations are certainly not unique to rhodopsin and
appear to be experienced with the majority of membrane
proteins. It is often difficult enough to obtain ordered
crystals from well-behaved soluble, globular proteins, but
these difficulties are compounded when dealing with
non-water-soluble proteins that have to be handled in
detergent solutions.
The conditions that will eventually succeed in producing large, stable, and highly diffracting crystals of rhodopsin will probably be unique for rhodopsin and will have to
be determined empirically. The particular properties and
behavior of rhodopsin as a protein will dictate what
conditions will eventually succeed. For that reason, detailed knowledge of rhodopsin's properties as a protein
are a prerequisite to such an undertaking. The body of
experience obtained from the crystallization of all pre406
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
vious proteins provides only guidelines for procedures to
be used as the studies with rhodopsin proceed. We will
consider the steps in the process of obtaining protein
crystals and look for the most productive approaches that
might be utilized with respect to rhodopsin.
7. Preparation of rhodopsin-containing
membranes
Cattle retinas are conveniently obtained in quantity from
animals slaughtered under conditions minimizing brightlight exposure. From each retina it is often possible to
obtain more than 700 fxg of rhodopsin. Purified rod cell
outer segments that contain membrane-bound rhodopsin
are conveniently obtained by a number of protocols that
include homogenization, differential centrifugation, and
density gradient centrifugation (McDowell 1993; Papermaster & Dreyer 1974). Following hypotonic lysis of the
rod cell outer segments and washing at low ionic strength,
membranes are prepared that contain nearly 95% of their
protein content as rhodopsin plus opsin. The components
other than rhodopsin are opsin, the "rim protein," peripherin/rds, ROM-1, remaining membrane-associated proteins, plasma membrane proteins, and phospholipids
(reviewed in Hargrave & McDowell 1993; Molday 1989).
These membrane preparations can be solubilized in any
of a variety of detergents and submitted to chromatographic steps that lead to purified rhodopsin.
8. Purification of detergent-solubilized rhodopsin
For purposes of crystallization it seems most desirable
that a reproducible well-defined homogeneous preparation of rhodopsin is obtained, free of opsin and other
components. There are two main methods in use for the
preparative chromatography of rhodopsin; hydroxyapatite chromatography and lectin affinity chromatography.
When rhodopsin is purified by calcium phosphate
chromatography using the commercial detergent preparation Emulphogene, it shows a single protein band by
electrophoresis and a spectral ratio of 280nm/498nm of
1.75 (Shichi et al. 1969). However, rhodopsin purified by
this method contains variable amounts of lipid (Papermaster & Dreyer 1974). When chromatography on hydroxyapatite is carried out using the cationic detergent
tridecyltrimethylammonium bromide (TrTAB), the protein binds to the column and can be conveniently washed
free of lipid (Hong & Hubbell 1973). Rhodopsin of the
same high-spectral purity is then eluted using a salt
gradient and contains from 0.2 to 0.8 moles of phosphate
(phospholipid) per mole of protein. One disadvantage of
this method is that TrTAB is not commercially available
and must be synthesized by the investigator. However, it
is dialyzable, and rhodopsin purified in TrTAB can be
conveniently prepared in another detergent by addition
of a nondialyzable detergent to the rhodopsin TrTAB
solution followed by dialysis (Hong & Hubbell 1973).
Probably the most widely used method to prepare
rhodopsin employs chromatography on concanavalin A
Sepharose. Several methods have been described (de
Grip 1982a; Litman 1982). Rhodopsin, in a variety of
detergents, is applied to the affinity matrix in a buffer
containing salts of Mg+2, Ca +2 , and Mn +2 (to stabilize the
Hargrave: Rhodopsin structure and function
bound concanavalin A tetramer). Washing with detergentbuffer removes lipids and contaminating proteins. Rhodopsin is eluted with a-methyl mannoside in detergentbuffer. Dialysis removes the sugar and presumably the
heavy metal ions. One must be alert to possible contamination by variable amounts of divalent metal ions, especially in light of a report that zinc becomes tightly associated with rhodopsin (Shuster et al. 1992). Another
contaminant that can be introduced by this purification
method is concanavalin A itself. It can be removed by
passing the purified rhodopsin over a column containing
an antibody to concanavalin A (de Grip 1982a) or by
mannose agarose affinity chromatography. However, a
more attractive approach is to prepare rhodopsin in a mild
detergent, such as octyl or nonyl glucoside, that does not
cause concanavalin A to be removed from the column
(Litman 1982).
9. Homogeneity of rhodopsin
Purified rhodopsin should be assessed for homogeneity
by sodium dodecylsulphate (SDS) polyacrylamide gel
electrophoresis (SDS-PAGE). The stained protein band
on SDS-PAGE is always broader and less sharp than that
of most other proteins. This appears to be due to heterogeneity of glycosylation. Vertebrate rhodopsins contain
two sites of glycosylation, but not all molecules are identical in their carbohydrate content. About 70% of the oligosaccharides on bovine rhodopsin contain Man3GlcNAc3,
10% Man4GlcNAc3, and 20% Man5GlcNAc3 (Fukuda et
al. 1979; Margrave et al. 1984; Liang et al. 1979). The
distribution of these three components between the two
sites in rhodopsin is unknown. This heterogeneity could
make a difference in the ability of differently glycosylated
molecules to pack and interact in crystals and represents a
potential source of crystal disorder. One approach to
dealing with this source of heterogeneity would be removal of the oligosaccharides by endoglycosidase digestion (Plantner et al. 1991). Peptide-N-glycosidase F is
capable of removing both oligosaccharide chains completely, leaving deglycosylated rhodopsin as the sole
product. The rhodopsin species with 0, 1, and 2 oligosaccharide chains are easily distinguished by SDS-PAGE
(Plantner et al. 1991). If required, reaction mixtures
might be further purified by passing through a column of
concanavalin A Sepharose, allowing passage of successfully deglycosylated rhodopsin molecules.
When rhodopsin is separated by SDS-PAGE, a series of
molecular weight bands is often generated corresponding
to dimer, trimer, and higher molecular weight oligomers.
This generation of a multiplicity of bands can be considered a unique characteristic of the protein, but is also a
nuisance when analyzing the protein for homogeneity,
since the presence of contaminating proteins can be
obscured. Rhodopsin is thought to be a monomer in disk
membranes (Cone 1972; Downer 1985). The production
of oligomers by SDS-PAGE is an artifact. Oligomers are
probably produced after solubilization of rhodopsin by
conditions that promote rhodopsin-rhodopsin collisions
that alter detergent-lipid interaction and promote interaction of rhodopsin monomers that leads to aggregation.
Oligomer formation may be eliminated by incubating
rhodopsin solutions or rhodopsin-containing membranes
at low protein concentration (< 1 mg/mL) in the presence
of high concentrations of SDS (5%) and high concentrations of reducing agent, avoiding elevated temperatures
(using room temperature or 37°C), and avoiding the use of
stacking gels that concentrate rhodopsin during PAGE
(Papermaster & Dreyer 1974).
Since cattle are rarely completely dark adapted, we
must consider the possibility that rhodopsin prepared
from such light-exposed retinas could contain phosphorylated opsin as a contaminant. The various species of
phosphorylated rhodopsin can be conveniently detected
by isoelectric focusing. Rhodopsin itself has a pi of 6.0,
and the different phosphorylated species have progressively more acidic pis (Aton et al. 1984; Kiihn &
McDowell 1977). It would be important to assess whether
a rhodopsin sample, prepared for purposes of crystallization, contained species other than that of the unphosphorylated protein with a pi of 6.0. If such phosphorylated rhodopsins are found, they can be conveniently
removed by passing the detergent-solubilized rhodopsin
sample over a Fe +3 -Chelex column (Andersson & Porath
1986; J. H. McDowell, personal communication) or by
anion-exchange chromatography of rhodopsin at neutral
pH (W. de Grip, personal communication).
Rhodopsin is also posttranslationally modified by palmitoylation of two adjacent carboxyl-terminal cysteine
residues (Ovchinnikov et al. 1988a; Papac et al. 1992).
Partial or complete loss of palmitate would result in a
significant change in the hydrophobicity and probably
also the organization of the carboxyl-terminal region of
rhodopsin. Disorganization of the carboxyl-terminal 40
amino acids of rhodopsin could result in a major problem
for crystallization. It is known that the cysteine thioester
linkage is labile to hydroxylamine, alkali, and to reducing
agents (O'Brien & Zatz 1984). It has been shown by
O'Brien and colleagues that simply storing solubilized
rhodopsin at 4°C for four days led to a decrease in fatty
acid content from 2.26 moles to 0.83 moles/mole rhodopsin (O'Brien et al. 1987). The rate of loss is temperaturedependent and is accelerated with increase in temperature. This suggests that in crystallization experiments that
take several weeks to complete, rhodopsin will be heterogeneous with respect to palmitate content and may lose
one or both of its palmitates during the course of the
experiment. In experiments reported by de Grip, it was
found necessary to include 5 mM dithioerythritol in the
rhodopsin buffer in order to prevent damage to rhodopsin
due to detergent impurities (de Grip et al. 1992). Presence of this reducing agent and the long times for crystallization would be expected to lead to loss of palmitate
from its thioester linkage to rhodopsin.
10. What are the characteristics of an ideal
detergent for the crystallization of
rhodopsin?
An ideal detergent would provide an environment for
rhodopsin that simulated its membrane environment so
well that its properties in detergent solution would be the
same as those measured in the disk membrane. Rhodopsin would be as thermally stable in this detergent as in the
membrane. It would bleach with the same kinetics, form
a stable opsin, and the opsin would be fully regenerable to
BEHAVIORAL AND BRAIN SCIENCES (1995) 18.3
407
Hargrave: Rhodopsin structure and function
formation. Too large a micelle may interfere with proteinprotein interactions. An unstable micelle leads to nonspecific hydrophobic interactions, aggregation, and
precipitation.
11. Measurement of properties of rhodopsindetergent complexes
CHO
Figure 3. Model for a rhodopsin-detergent complex. Upper
left: Cross-sectional diagram of a rhodopsin molecule encircled
by a detergent micelle. Polar (P) surfaces of rhodopsin, including its carbohydrate chains (CHO) interact with the aqueous
environment and with detergent head groups (DH). Buried
hydrophobic regions interact with hydrophobic detergent tails
(DT). Lower right: Two rhodopsin-detergent complexes are
shown forming interactions between their polar surfaces that
presumably are required for crystal formation. Larger detergent
micelles would make these protein-protein interactions less
effective. Figures based on (Michel 1991).
rhodopsin upon binding 11-cts retinal. No such detergent
has yet been found. In addition, such an ideal detergent
must have other important properties.
An appropriate detergent for protein crystallization
must be a defined chemical substance that is available
commercially in pure form or that can be synthesized in
quantity economically and relatively easily. It must be
pure and chemically stable. The detergent should have
good solubility at room temperature and at 4°C, and over
a reasonable range of pH and ionic strength. Although the
ability to solubilize efficiently membrane-bound rhodopsin would be helpful, this is not required, since a detergent can usually be exchanged.
It is generally assumed that detergent molecules form a
ring around solubilized membrane proteins, binding to
hydrophobic surfaces previously occupied by lipid fatty
acyl chains (Fig. 3). This leaves the hydrophilic ends of the
protein exposed to the aqueous environment. The detergent should produce small monodisperse micelles and
create the smallest stable protein-detergent complex possible (Garavito 1991). Such a small complex might be
expected to enhance ionic interactions between hydrophilic protein surfaces that may promote good crystal
408
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Numerous detergents have been used in the study of the
properties of rhodopsins (de Grip 1982b; de Grip et al.
1992; Fong et al. 1982) and a variety of parameters have
been measured to assess the characteristics of the
rhodopsin-detergent complexes. Since crystallization trials may require two to four weeks, it is the stability, in
particular the long-term stability of the rhodopsindetergent complex, that is most pertinent to the consideration of its crystallization.
Thermal stability is the primary parameter that has
been measured to assess the stability of the rhodopsindetergent complex. In one study the rhodopsin-detergent
complexes were heated at various temperatures and the
denaturation of rhodopsin followed by measurement of
decrease in absorption at 500 nm (de Grip 1982b; de Grip
et al. 1992). Temperatures at which the half-time of
denaturation is 10 min for each detergent were determined (Table 1). Among the detergents tested, the one
imparting the greatest stability to rhodopsin is Pdodecylmaltose.
Table 1. Thermal stability of bovine rhodopsin and opsin
in detergents
i
CMC"
5O
Detergent"
(mM)
rhodopsin
(°C)
P-l-octylglucose
a-1-octylglucose
a-1-octylmannose
C 8 P0E
C8HESO
P-1-nonylglucose
C9POE
C9-N-methylglucamide
P-1-decylmaltose
C1OPOE
C10-N-methylglucamide
P- 1-dodecylmaltose
p-1-dodecylphosphomaltose
C 12 E 8
C 12 E 10
CHAPS
CHAPSO
23.0
13.0
1.5
8.5
18.0
6.5
2.2
16.5
1.7
0.7
3.5
0.2
3.5
0.06
0.02
6.5
7.0
50
53
52
42
42
53
49
51
56
52
51
60
48
48
50
56
56
^1/2
opsin
(min)
=10
ND«
40
ND
ND
20
ND
ND
90
15
85
>40h
ND
ND
140
35 h
20 h
"Detergent concentration was 30 mM in PIPES buffer pH 6.5.
^Critical micelle concentration.
°^5o presents the temperature where the half time of rhodopsin
denaturation, measured as 500 nm decrease, amounts to 10 min.
''Half life of opsin (measured as regenerability with 11-cts
retinal) at 20°C.
C
ND = not determined.
Note: Reproduced from de Grip et al., 1992.
Hargrave: Rhodopsin structure and function
Table 2. Calorimetric parameters of thermal denaturation of rhodopsin and opsin in detergent solutions
Unbleached ROS disk
membranes
Bleached ROS disk
membranes
AHca.
Detergent
Concentration
(mM)
AHcaI
(kcal/mole)
CO
disk membranes
digitonin
dodecyl maltoside
octylglucoside
TrTAB
10
6
90
200
172
155
158
122
109
71.7
62.5
61.8
56.0
39.0
±
±
±
±
±
3
12
4
1
13
±
±
±
±
±
.2
.2
.1
.3
.1
(kcal/mole)
T,n
(°C)
129 ± 3
50 ± 3
54 ± 8
ND°
ND
55.8 ± .4
44.5 ± .1
39.1 ± .1
ND
ND
"ND = not detectable.
Thermal stability of rhodopsin has also been measured
by differential scanning calorimetry (Khan et al. 1991;
McDowell et al. 1992; Miljanich et al. 1985; Shnyrov &
Berman 1988). By programmed heating of rhodopsin in
the membrane and in detergent solution, temperatures of
denaturation are obtained (Table 2). Such data show that
even the mildest detergent tested, digitonin, falls far
short of offering the protective environment that is available to rhodopsin in its native membrane.
Long-term stability of the rhodopsin-detergent complex under conditions used in crystallization has been
examined by de Grip et al. (1992). They examined rhodopsin for maintenance of spectral integrity, structural
stability (avoidance of formation of lower molecular
weight fragments measured by SDS-PAGE and immunoblotting), and formation of crystals when rhodopsin/
detergent/precipitant solutions were kept at 20°C for two
to three months. Some fragmentation of rhodopsin occurred when detergents or precipitants contained oxyethylene units. This suggested that the degradation was
caused by peroxidation, which seems quite likely because
it was subsequently eliminated by inclusion of a reducing
agent. One wonders whether scrupulous purification of
the detergents/precipitants to remove peroxidants, the
choice of more stable detergents, or inclusion of vitamin
E or butyl-hydroxytoluene as an antioxidant, and an argon
atmosphere (Farnsworth & Dratz 1976) might prove helpful and eliminate the need for inclusion of a reducing
agent (which may lead to depalmitoylation, as discussed
above).
12. Small amphiphiles are often helpful in
crystallization of membrane proteins
Considerable success has been achieved in crystallization
of membrane proteins by the addition of l%-5% of small
amphiphilic compounds to the protein-detergent complex. Mixed micelles are formed in which the amphiphiles intercolate into positions that would be occupied
by larger detergent molecules. They reduce the size of
the protein-detergent complex that is thought to maximize the hydrophilic protein-protein interactions needed
for good crystal lattice formation (Michel 1991). Of the
more than 100 different compounds investigated, threo1,2,3-heptane triol and benzamidine have been the most
successful in trials with bacteriorhodopsin and the lightharvesting complex. However, inclusion of the am-
phiphiles is not essential, since the proteins have been
successfully crystallized under other conditions in their
absence.
Other investigators have found that amphiphiles improved the solubility of their membrane protein and
influenced the type of crystal formed (Allen & Feher
1991; Garavito 1991). Crystals of a reaction center complex that showed a resolution of 10A were improved to 7A
when 1,6-hexanediamine was added. In this instance it
has been suggested that the diamine acted as a bridge
stabilizing the protein micelles in the crystal lattice
(Welte & Wacker 1991). Although the addition of small
amphiphiles has been applied to the formation of 3-D
crystals of rhodopsin, conditions have not yet been found
in which they yield an improvement (de Grip et al. 1992;
Demin et al. 1987). However, the quality of 2-D rhodopsin crystals in the membrane was improved by the inclusion of the surface-active agent, chlorhexidine (Dratz et
al. 1985).
13. What are the prospects for obtaining twodimensional crystals of rhodopsin that will
yield a high-resolution structure?
Currently available methods are capable of yielding highresolution structures from well-ordered two-dimensional
crystals of membrane proteins. Given the perfect microscope and the perfect membrane specimen, atomic resolution can be achieved. Bacteriorhodopsin is the classic
example where a structure has been obtained with a
resolution of 3.5 A in the plane parallel to the membrane
(Henderson et al. 1990). Interpretation of the structure
has allowed assignment of 21 amino acids from all 7 helices
that contribute directly to the environment of the retinal,
allowing a proposal to be made for the location of amino
acids that constitute the proton channel. This is the type
of information that would be very useful for vertebrate
rhodopsin.
A wide variety of examples of membrane proteins have
been examined by electron microscopy and image analysis. The two-dimensional crystals studied thus far fall
into three categories: (1) proteins that occur naturally in a
semicrystalline array; bacteriorhodopsin in purple membrane, gap junctions from hepatocytes (Unwin & Zampighi 1980); (2) crystalline arrays that can be produced
from membranes in vitro; cytochrome oxidase (Vanderkooi 1974), rhodopsin (Corless et al. 1982; Dratz et al.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
409
Hargrave: Rhodopsin structure and function
1985), acetylcholine receptor (Toyoshima & Unwin 1990),
H,K-ATPase (Hebert et al. 1992); and (3) proteins crystallized from protein-detergent complexes or protein-lipiddetergent complexes, as two-dimensional sheets; bacterial porin (Dorset et al. 1983), cytochrome reductase,
cytochrome b-cl complex (Hovmoller et al. 1983), lightharvesting complex (Kiihlbrandt 1984), photosynthetic
reaction center (Miller & Jacobs 1983), and NADH dehydrogenase (Boekema et al. 1986).
Vertebrate rhodopsin exists in a highly fluid membrane
and does not naturally form a crystalline array. Because
the lamellar part of the rod cell disk membrane is nearly
pure rhodopsin in lipid, it is attractive to attempt to
induce rhodopsin to form a crystalline array within its
native membrane (Corless etal. 1982; Demin etal. 1987;
Dratz et al. 1985). However, the level of resolution obtained by that approach has not come close to what is
required. Improvements have been made by examining
unstained membranes and by use of improved methods
for data analysis (Schertler et al. 1993; Schertler & Hargrave 1994). However, substantial improvements in resolution require that the crystals be larger, which may
require that they be formed by a different approach.
The most versatile approach to the formation of 2-D
crystals is to reconstitute detergent-solubilized proteins
into a lipid environment formed during the slow dialysis
of detergent. With this approach there is no control over
the type of detergent, the amount and type of lipid, and
the method and rate of removal of detergent. Rhodopsin's
physical and photochemical properties have been studied
in a variety of different lipids (Deese et al. 1981; de Grip
et al. 1983; Mitchell et al. 1992; Ryba & Marsh 1992).
Based on these and other studies it may be possible to
choose a lipid environment that will be more conducive to
the formation and stabilization of rhodopsin in a crystalline array.
14. It may be easier to study all forms of
rhodopsin in two-dimensional crystals
It is not only possible to examine 2-D crystals of membrane proteins at low temperature, it may be desirable.
The best electron microscopic image yet obtained from
bacteriorhodopsin was from a sample at the temperature
of liquid helium (Henderson etal. 1990). Electron microscopic data is frequently taken from frozen or refrigerated
samples. Studies of the neutron diffraction of an intermediate in the photocycle of bacteriorhodopsin involved
measuring it at -180°C (Dencher et al. 1989).
Low temperatures have been used to isolate selectively
the spectral intermediates in bleaching of rhodopsin.
Various intermediates can be produced by irradiation at
low temperature followed by warming to the temperature at which the intermediate is stable. MI can be
produced by warming to a temperature between — 40°C
and - 15°C, and Mil can be formed over the range - 15°C
to 0°C. Thus, by photolyzing the rhodopsin 2-D crystal
and subjecting it to the appropriate temperature it should
be possible to form these structural intermediates. By
immediately freezing in liquid ethane and maintaining the
crystals at liquid nitrogen temperature, the intermediates
should be preserved for examination by electron crystallography. Difference maps between two rhodopsin forms
410
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
should indicate the areas where the rhodopsin polypeptide chain has undergone a change in conformation in
proceeding from rhodopsin to MI and from MI to Mil.
Such an approach has been successfully applied to detection of structural changes between the ground state and
the M intermediate of bacteriorhodopsin (Subramaniam
et al. 1993).
15. The objective: Three-dimensional crystals of
rhodopsin
X-ray crystallography of three-dimensional crystals has
produced the highest resolution structural data for proteins. Only four classes of membrane proteins have thus
far yielded crystals of sufficient order to produce reasonably high-resolution structures. The successful methods
for production of crystals from membrane proteins have
been adaptations of methods used for soluble globular
proteins. Concentrated solutions of purified membrane
proteins in detergent may form crystals in the presence of
increasing concentrations of precipitants such as ammonium sulfate or polyethylene glycol. Critical parameters
such as the choice of detergent have been discussed
earlier. Here I wish to discuss the experimental problems
introduced by the intrinsic nature of rhodopsin itself: its
extreme sensitivity to light.
16. Working with rhodopsin in the dark
To produce and analyze crystals of rhodopsin demands
that all aspects of the process be conducted under dim red
light (light of > 620 nm) or by infrared image converter.
This means that all of the crystallization trials, selection
and examination of crystals, mounting, and X-ray analysis
be conducted under darkroom conditions. Such work is
not only tedious but has been reported to lead to failure in
identifying crystals (de Grip et al. 1992). Eventually
another problem will arise when the crystals are examined by X-ray analysis. Interaction of the measuring beam
with water in the membrane sample causes fluorescence
- light that would be expected to bleach the rhodopsin
being examined. Such considerations have led investigators to attempt to produce light-insensitive rhodopsins.
17. The search for light-stable rhodopsin: The
nonisomerizable chromophore
Locking retinal into place so that it would not be photosensitive is one approach to handling rhodopsin more
conveniently for crystallization and analysis. What is
desired is a rhodopsin that will not be subject to a change
in structure upon light exposure. Such a rhodopsin might
also have greater long-term stability, enhancing its ability
to withstand multi-week crystallization times.
Opsin will combine with a number of isomers and
derivatives of retinal to yield visual pigments of varying
stability (Derguini & Nakanishi 1986). To be optimally
useful for crystallization trials, any visual pigment formed
by combination of a retinal derivative with opsin must
have the following characteristics: (1) minimal perturbation of its native structure; (2) long-term stability in
detergent; (3) insensitivity to light, as measured by main-
Hargrave: Rhodopsin structure and function
tenance of spectral integrity; and (4) insensitivity to attack
by hydroxylamine. (This is a measure of the native conformation of the protein-retinal complex. Hydroxylamine
attacks the retinylidene-Schiff base linkage when it becomes accessible to the aqueous environment. The
retinylidene-Schiff base is unavailable for attack in native
rhodopsin.)
Ring-locked derivatives of retinal designed to eliminate
photoisomerization about the 11-12 double bond have
been investigated as possible chromophores that would
yield a rhodopsin insensitive to photoisomerization. Such
compounds have been produced with 5-membered (Ito et
al. 1982), 6-membered (Bhattacharya et al. 1992; van der
Steen et al. 1989), 7-membered (Akita et al. 1980), 8- and
9-membered rings (Hu et al. 1994) built around the
retinal 11-12 double bond (Fig. 4). Each of these compounds (except the 9-membered one) has been successfully recombined with opsin, and the pigments
formed (Rh5, Rh6, Rh7, and Rh8 respectively) have been
the subject of many interesting studies (de Grip et al.
1990; Fukada et al. 1984; Hu et al. 1994; Zankel et al.
1990). However, pigments Rh5 and Rh7 have been subject to attack by hydroxylamine and are unstable in
digitonin solution. By contrast, Rh6 is stable to hydroxylamine in the dark in detergent solution and is
nearly as thermally stable as rhodopsin itself. Although it
has unusual photostability, it does bleach in detergent
solution at a rate 0.6% that of rhodopsin (Bhattacharya et
al. 1992; de Grip et al. 1990). The rhodopsin analog has
greatly reduced yet measurable activity in stimulating
enzymes in the phototransduction pathway and in serving
as a substrate for rhodopsin kinase. It is probable that all
of the 11-cis ring-locked chromophores photoisomerize to
a limited extent about the 7- and 9-double bonds, and that
during this photoisomerization the Schiff base becomes
transiently accessible for hydrolysis. To restrict further
the potential for photoisomerization, a second ring be-
Figure 4. Structures of different retinals of interest for rhodopsin function and crystallography. (1) 11-cis retinal; (2)
all-trans retinal; (3) Ret-5, 5-membered ring-locked retinal; (4)
Ret-6, 6-membered ring-locked retinal; (5) Ret-7, 7-membered
ring-locked retinal; (6) 6-membered ring-locked retinoyl fluoride; (7) Ret-6 with an additional ring-linking carbons 9 and 11.
tween the retinal carbons 9 and 11 has been introduced
(Fig. 4, compound 7). Unfortunately the photosensitivity
of the rhodopsin pigment formed with this retinal is not
further reduced when compared to Rh6 (W. de Grip,
personal communication).
18. Truly light-stable rhodopsin?
There have been two different approaches taken to capitalizing on the greatly enhanced light stability of rhodopsin containing the 6-membered ring-locked retinal. One
approach has been chemical - to strengthen further the
association of the retinal to rhodopsin by covalent attachment (van der Steen et al. 1989). The other approach has
been biological - to engineer the protein so that it is less
susceptible to bleaching (Ridge et al. 1992).
A promising compound for locking rhodopsin in a
photo-insensitive state is the acid fluoride of the 6-membered ring-locked retinal (van der Steen et al. 1989). It
reacts with opsin to yield a blue-shifted rhodopsin analog
with 390nm absorbance maximum that is thermally stable, nonbleachable, and is completely inactive in signal
transduction (de Grip et al. 1990). It can be heated in
detergent at 60°C (under conditions that denature rhodopsin with t1/2 < 1 min.) with complete stability. The
rhodopsin analog can be illuminated for 1 hr. under
conditions that bleach rhodopsin with t1/2 = 15 sec,
without loss of absorbance (van der Steen et al. 1989).
Such photo- and thermal stability appears to come not
only from a good fit to the retinal binding site and
stabilization toward photoisomerization, but from the
irreversible amide bond that replaces the normally hydrolyzable Schiff base. However, this rhodopsin analog
has been difficult to purify (W. de Grip, personal communication): the acylation reaction is slow and incomplete,
and the acylation must be performed on the completely
reductively methylated protein in which all lysines except
Lys296 are methylated. This leads to heterogeneity and
difficulty in purification. Another disadvantage is that in
formation of the retinyl-amide linkage, the protonated
Schiff base is destroyed; thus the ion-pairing that is an
important part of rhodopsin structure and function cannot
be observed. Although crystals of this derivative might be
useful in examining many overall features of the rhodopsin molecule, it would differ in important details in the
region of the retinal attachment site.
A novel alternative approach to stabilizing further the
Rh6 rhodopsin has been to engineer the protein to make it
less photosensitive (Ridge et al. 1992). Previous studies
had identified a number of amino acids that were located
in the retinal binding pocket. Twelve mutant rhodopsins
that exhibited spectral differences in their combination
with 11-cis retinal in the ground or excited state were
recombined with the cyclohexene-locked retinal. One
such mutant rhodopsin, Trp265Phe, was completely stable to illumination. The mutant Rh6 rhodopsin was stable
to hydroxylamine and was inactive in phototransduction
or as a substrate for rhodopsin kinase (Ridge et al. 1992).
The lack of photosensitivity of this mutant resulting from a
single amino acid mutation points to an important role of
Trp265 in the signal transduction mechanism. Mutant
Trp265Phe Rh6 would appear to be an ideal candidate for
crystallization trials. However, one must not overlook the
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
411
Hargrave: Rhodopsin structure and function
need to produce sufficient quantities of the mutant rhodopsin in a large-scale expression system and the requirement for the synthetic retinal for regeneration of rhodopsin. Although expensive and laborious, the approach
appears quite promising.
Although many of the aforementioned approaches to
the crystallization of rhodopsin appear promising, additional approaches still need to be considered.
19. Rhodopsins of different species may have
advantages for crystallization
There are many instances in which a protein from one
species will fail to yield crystals of good quality and order,
but the protein that differs in only a few amino acids from a
closely related species will crystallize beautifully. For
many of the glycolytic enzymes, the protein from yeast or
from rabbit muscle has been quite satisfactory, but for
others it has been necessary to seek alternative sources
such as crayfish or chicken (Campbell et al. 1971). Although the difficulty in obtaining good crystals of bovine
rhodopsin is probably related to its characteristics as a
membrane protein, it would be good to consider that
rhodopsins from pig, horse, or sheep (Findlay 1986) could
have small differences that are important in successful
crystal formation.
The thermal stability of rhodopsin may be an important
characteristic in successful crystal formation, as we have
mentioned previously. Rhodopsins from organisms that
experience high body temperatures could offer this advantage. Examples include the desert iguana lizard
Dipsosaurus dorsalis that has the highest known body
temperature for a vertebrate, at 47°C (McFall-Nagi &
Horwitz 1990), and the Saharan silver ant Cataglyphis
bombycina, whose body temperature can reach 53.6°C
(Wehner et al. 1992). Gaining information about the
properties of rhodopsins from these exotic species offers
special challenges. However, if study of these rhodopsins
enhances our understanding of rhodopsin's stability, this
would be a valuable addition to our knowledge of rhodopsin structure-function relationships.
Invertebrate rhodopsins may have advantages for crystallization. The primary structures of octopus (Ovchinnikov et al. 1988b) and squid (Hall et al. 1991) rhodopsins
have been determined, and many of their biochemical
properties have been investigated. The proteins can be
obtained in sufficient quantity. Invertebrate rhodopsins
are more photostable than vertebrate rhodopsins inasmuch as their chromophore, 11-cw retinal, does not
dissociate from its binding site following photoisomerization. A stable metarhodopsin is formed that is then
converted back to rhodopsin upon reception of another
photon of the correct wavelength. This ability of invertebrate rhodopsins to form a stable metarhodopsin makes
them attractive candidates for more conveniently obtaining a structure for the signaling state of the receptor,
metarhodopsin. One disadvantage, however, is their
much lower thermal stability in detergents.
The octopus or squid rhodopsins may have an additional characteristic that will be of importance in crystal
formation. They are larger proteins, 50-51 kDa, due to a
large C-terminal extension of about 150 amino acids
(Ovchinnikov et al. 1988b; Hall et al. 1991). Although the
412
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
significance of this region for rhodopsin function is not
clear, the presence of such a large globular addition to a
predominantly membrane-embedded protein, may promote protein-protein association in the crystal lattice and
may reduce some of the problems associated with crystallization from detergents.
20. Can rhodopsin be made to look more like a
soluble protein, for purposes of
crystallization?
Based upon the membrane proteins that have formed the
most well-ordered crystals to date, it would appear that a
membrane protein that has the maximum amount of
hydrophilic surface area would have the best chances of
ordered crystal formation. One way to increase the
amount of hydrophilic protein on rhodopsin's surface is to
form a complex with a protein with which it interacts.
Complexes of antibody-Fab fragments with their protein antigens have been successfully crystallized and have
yielded high-resolution crystal structures (Mariuzza et al.
1987). There are a number of high-affinity monoclonal
antibodies of well-defined specificity for rhodopsin that
have been described (Adamus et al. 1991; Molday 1989).
IgG class antibodies specific for rhodopsin's carboxylterminal sequence are easily accessible to their binding
site on the opsin molecule either in its rhodopsin form (in
the dark) or following light exposure. Such antibodies
would be good choices to investigate the potential of this
approach. This approach is not trivial since it is necessary
to determine individually the proteolysis conditions suitable for production of a Fab fragment from each monoclonal antibody. Homogeneous Fab fragments must be produced that are suitable for crystallization purposes. Yields
can be low, requiring many tens of milligrams of purified
antibody as starting material. The Fab-rhodopsin complex should be formed and then purified to homogeneity.
The complex must be sufficiently stable to remain intact
during the purification and crystallization process. Although the validity of this approach has not yet been
demonstrated with membrane proteins, the idea is appealing.
The lectins concanavalin A and wheat germ agglutinin
bind to rhodopsin's oligosaccharide chains. These lectins
have been used for affinity purification of rhodopsin and
for labeling rhodopsin for microscopic analysis. They are
potential candidates for protein ligands that would impart
a larger hydrophilic surface to the rhodopsin molecule.
The lectins would bind to the intradiscal surface of rhodopsin and might serve to mask the heterogeneity of rhodopsin's oligosaccharide chains. However, concanavalin A
is a tetramer whose tendency for subunit dissociation is
likely to complicate purification and crystallization
procedures.
Protein engineering is one route that is free from the
problem of dissociation of a bound protein ligand. By
fusing the gene for rhodopsin with that for a desired
protein, the resulting larger fusion protein is a single
covalent entity. De Grip is exploring application of this
method to rhodopsin (de Grip et al. 1992). The protein for
fusion could be chosen from a list of many, such as
lysozyme, that are models often used for testing new
methods for protein crystallization. In theory, the poten-
Hargrave: Rhodopsin structure and function
tial of this approach is great, especially if one has good
intuition or guidance concerning what fusion protein
construct(s) will be most valuable to investigate. At present this approach suffers from the lack of an expression
system capable of producing hundreds of milligrams of
the required protein. Expression systems for rhodopsin
have been described for COS-1 monkey kidney cells
(Oprian et al. 1987), human embryonic kidney cells
(Nathans et al. 1989), and an insect cell line (Janssen et al.
1991), but the levels of expression still need to be improved. Overexpression of membrane proteins generally
has proven to be difficult (Schertler 1992).
21. An approach to the crystallization of
Metarhodopsin II (R*)
Although we suggested earlier that 2-D crystals might be
made by freezing out Mil using conditions under which it
is stable, this technique is unlikely to be practical for 3-D
crystals. Crystallization as the complex with transducin
may offer a more viable approach to the problem. This
route is another variation on the attempt to crystallize
rhodopsin in the presence of a bound protein ligand.
fi* may be stabilized in its complex with transducin, if
guanosine triphosphate (GTP) is absent, and if all traces of
GDP are removed (Kiihn 1980). This leads to a complex
fl* • Tc in which the T a nucleotide site is empty. This
complex is stable "almost indefinitely" under conditions
of physiological ionic strength (Bornancin et al. 1989). R*
continues to bind retinal and remains spectroscopically in
the Mil state, stabilized by the binding of transducin.
However, for purposes of obtaining a crystal structure of
this complex it would be necessary for the complex to
remain stable in detergent under conditions suitable for
purification and long-term crystallization. It is doubtful
whether these stringent conditions could be met. The
R* • Tc complex may be treated with hydroxylamine to
form the Rc • Tc complex in which rhodopsin has lost its
retinal and in which transducin is free of nucleotide
(Bornancin et al. 1989). Any dissociation of this complex
would be irreversible, leading to decay of the products
and denaturation of the proteins. Thus the complex would
have to maintain its strong association for periods of weeks
under conditions suitable for crystallization.
22. Another approach to obtaining the crystal
structure of R*
The activated state of rhodopsin, R*, is that state of
rhodopsin that binds to and causes the activation of
transducin. It is possible that this state may be adequately
simulated by more than one mutant form of rhodopsin.
Disruption of the ion pair between the protonated
Schiff base of Lys296 and the carboxyl group of Glull3
leads to formation of R*. When Glull3 is replaced by
glutamine, the resulting rhodopsin is constitutively active
once ll-cis retinal is removed from its binding pocket.
Similarly, when Lys296 is replaced by site-specific mutagenesis, the.resultingopsin becomes constitutively active
(Robinson et al. 1992). These observations suggest that
if one of these mutant opsins were to be crystallized,
the resulting structure might simulate the signaling state
of rhodopsin. However, opsins are much less stable in
detergents than rhodopsins, and the stability of the detergent complexes of these mutants may prove to be
inadequate.
In the above examples, key mutations in single amino
acids in rhodopsin were enough to shift the conformation
of R to that of R*. It is possible that mutations in other
regions of rhodopsin might also result in formation of a
constitutive mutant. It is known, for example, that rhodopsin loops t2, t3, and t4 participate in binding of
transducin (Konig et al. 1989; reviewed in Hargrave et al.
1993) and that loops 12 and t3 participate in activating it
(Franke et al. 1990; 1992). Recently, a single amino acid
mutation in the carboxyl end of loop i3 of the a 1B adrenergic receptor was found to produce the constitutively active receptor (Kjelsberg et al. 1992). This shows
that the adrenergic receptor is delicately poised and
rather easily pushed over the energy barrier toward
activation. It is likely that the activation of rhodopsin will
not be so easily achieved. Nonetheless, our knowledge of
the mechanism of activation of rhodopsin is in its early
stages, and we should be alert to the possibilities of other
participating residues whose alteration may readily lead
to the formation of R*. Our present interest is in routes
that may aid our understanding of rhodopsin structurefunction relationships.
23. Conclusion
An essential basis for understanding how a protein functions is the determination of its primary, secondary and
tertiary structures. A structure at the level of atomic
resolution provides the framework on which the underStanding of function may develop. Rhodopsin is a particularly important and attractive target for study. From
rhodopsin we will not only learn about the mechanism
of phototransduction but also, by analogy, about the
mechanism of action of receptors that couple to G-proteins. There is a large body of information about rhodopsin that will be of value in helping construct and
evaluate experimental strategies. At present, the strategies leading to formation of 2-D crystals appear most
versatile and most likely to succeed in production of
structures at low to moderate levels of resolution. This
will be extremely valuable in elucidating general features
of rhodopsin's molecular architecture but may not yield
the required level of atomic resolution to help in elucidating function.
The techniques of mutagenesis are powerful and may
be required to tailor a rhodopsin molecule more amenable to stable 3-D crystal formation. Full utilization of
these techniques awaits development of a high-yielding
expression system capable of producing the hundred
milligram amounts of material that will be needed for
innumerable trials. Whatever methods eventually succeed will triumph from application of a high degree of
creativity coupled with detailed knowledge of the system,
and an intense concentration of effort and resources over
an extended period of time.
ACKNOWLEDGMENTS
I would like to thank numerous colleagues who have read this
manuscript in draft form and have made many valuable suggestions, most of which I have adopted. During preparation of
this manuscript Dr. Hargrave's research on rhodopsin was
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
413
Hargrave: Rhodopsin structure and function
funded by grants from the National Eye Institute of the National
Institutes of Health (EY06225 and EY06226), a grant from the
International Human Frontier Science Program, and an unrestricted departmental award from Research to Prevent Blindness, Inc. Dr. Hargrave is Francis N. Bullard Professor of
Ophthalmology.
414
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
NOTE
1. This manuscript was delivered at the conference Controversies in Neuroscience III: Signal Transduction in the Retina
and Brain, at the Robert S. Dow Neurological Sciences Institute
and Good Samaritan Hospital & Medical Center, Portland,
Oregon, October 31-November 1, 1992.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18, 415-424
Printed in the United States of America
What are the mechanisms of
photoreceptor adaptation?
M. Deric Bownds
Laboratory of Molecular Biology, University of Wisconsin, Madison, Wl
53706.
Electronic mail: bownds@macc.wisc.edu
Vadim Y. Arshavsky1
Harvard Medical School and The Massachusetts Eye and Ear Infirmary,
Boston, MA 02114.
Electronic mall: vadim@macc.wisc.edu
Abstract: This article evaluates each of the reactions known to be involved in visual transduction as a potential site for the regulation of
light adaptation. Extensive evidence suggests that calcium acts as a feedback messenger at several different points and recent work
suggests a role for cGMP in regulating the primary excitatory pathway. A conclusion is that adaptation is likely to be regulated by
multiple and redundant mechanisms. The goal of future experimentation will be to determine the relative importance of each of
these.
Keywords: calcium; cyclic GMP; cyclic GMP phosphodiesterase; guanylate cyclase; light adaptation; phosphorylation; photoreceptor; rhodopsin; rod outer segments; transducin
1. Photoreceptor outer segments are a favorable
preparation for studying adaptation processes
Vertebrate photoreceptors show striking powers of adaptation, adjusting their gain to remain responsive to light
transients as ambient light intensity increases over 3-5 log
units. A primary locus of these adaptation processes is the
outer segment portion of the photoreceptor, on which this
target article focuses. It is clear that these structures
determine the basic parameters that define subsequent
stages of visual information processing in the retina (cf.
Shapley & Enroth-Cugell 1984). The enzymes that regulate excitation and adaptation have been characterized
mainly by studies on cattle and frog rod outer segments
(ROS), and are described in several recent reviews
(Chabre & Deterre 1989; Detwiler & Gray-Keller 1992;
Hargrave & McDowell 1992; Hurley 1992; Koutalos & Yau
1993; Lagnado & Baylor 1992; Pfister et al. 1993; Pugh &
Lamb 1990; Stryer 1991; Yarfitz & Hurley 1994; Yau 1994).
The milligram to gram quantities of ROS necessary for
purification and analysis of transduction enzymes are
more easily obtained from cattle retinas. Larger amphibian ROS, although more difficult to prepare in quantity,
have the advantage that pure and physiologically active
suspensions can be obtained. This permits study of both
the electrophysiological and biochemical correlates of
adaptation. The composition of these structures is better
characterized than that of any other primary sensory
organelle: the mass is half protein and half lipid (Fliesler
& Anderson 1983), 70% of the protein mass is rhodopsin,
17% is the G-protein transucin (Gt), and most of the
© J995 Cambridge University Press
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balance is accounted for by proteins involved in cyclic
nucleotide metabolism and protein phosphorylations
(Hamm & Bownds 1986). Rhodopsin is composed of the
protein opsin covalently linked to 11-cis retinal, and the
photochemistry of this complex has been well studied.
Thus the system allows precise quantitation of input (the
number of rhodopsin molecules hit by light). Sophisticated modelling and kinetic analysis not yet achieved in
other internal messenger systems is possible (Bownds &
Thomson 1988; Dawis 1991; Forti et al. 1989; Lamb &
Pugh 1992; Sneyd & Tranchina 1989). An outline of how
excitation works is largely in place, and interest focuses
now on adaptation.
2. What must be explained? Excitation and
adaptation
A single photon hitting one of the 3 X 109 rhodopsin
molecules present in the disk membrane system of a dark
frog ROS causes the closing of several hundred channels
in the plasma membrane. This transiently halts the inward movement of several million ions. In the frog, the
duration of the dark-adapted response is approximately
four seconds with a time to peak of 900 msec (Baylor et al.
1979a). This excitation process is very stereotyped and
reliable (Lagnado & Baylor 1992). If a step of background
illumination is turned on, the response relaxes to a
plateau level within seconds after an initial peak. This
process is usually called background adaptation. Superimposed responses to test flashes become smaller and
415
Bownds & Arshavsky: Photoreceptor adaptation
more rapid as this background light is increased over 3-5
log units. At high backgrounds, the amphibian rod response peaks at approximately 300 msec and turns off two
to three times faster (Baylor et al. 1980; Fain 1976; Nicol
& Bownds 1989). Theflashsensitivity (Sf), defined as the
change in current per photon absorbed declines in a
linear fashion as a function of the log of the background
intensity. Until recently it had been supposed that background adaptation was a characteristic of the rods of lower
vertebrates, and not displayed by mammalian rods. Yau
and his collaborators, however, have now shown that
adaptation behavior is observed in several warm-blooded
animals, including rats, rabbits, cows, and monkeys (Nakatani et al. 1991; Tamura et al. 1991).
If a significant amount of rhodopsin is bleached (greater
than ~5%), the photoreceptor s sensitivity is at first abolished. In a subsequent dark period, sensitivity begins to
recover as opsin regenerates to form rhodopsin but remains low (bleaching adaptation) as long as opsin is still
present (cf. Kahlertetal. 1990). Recent work suggests that
the underlying mechanism may be similar to that of
background adaptation (Clack & Pepperberg 1982; Cornwall & Fain 1992). The major differences between bleaching and background adaptation may be that in the former
the large amount of opsin present has residual excitatory
activity and the efficiency with which the ROS captures
photons is lowered because less rhodopsin is present. The
residual excitatory activity can be inhibited by adding
retinal or some of its analogs (Jin et al. 1993). Fain and
Lisman (1993) have suggested that residual excitatory
activity may underlie photoreceptor degeneration associated with vitamin A deficiency or rhodopsin mutations
that are constitutively active (Robinson et al. 1992).
3. The basic excitation pathway
Other articles in this review series have described reactions of the excitation cascade:
hv -• Rh* -> G* -* PDE* -» cGMP i -• gNa Ca 1
They are a variation on the ubiquitous pattern seen in
many other G-protein systems. Recent work has shown
that olfactory receptors use an analogous system, except
that cyclic AMP and/or triphosphoinositol are the relevant channel regulators (Breer & Boekhoff 1992; Firestein 1991; Ronnett & Synder 1992). The interaction of
excited rhodopsin (Rh*) with many G-protein molecules
(Gt, transducin) causes each to release GDP and bind
GTP. Gt-GTP then activates cGMP phosphodiesterase
(PDE) which hydrolyzes cGMP to 5'-GMP. The drop in
cGMP caused by PDE activation causes closing of channels held open by cGMP in the darkness, halting a
continuous entry of Na+ and Ca ++ ions. The system
inactivates as Rh* is quenched (see below) and the GTP of
Gt-GTP is hydrolyzed to GDP, stopping PDE activation.
The decrease in Ca ++ entry activates a guanylate cyclase
that enhances cGMP recovery. The excitation process
thus increases GDP and 5'-GMP concentrations, and
decreases GTP, cGMP, Na+, and Ca ++ concentrations.
All of these products are putative feedback regulators.
Most interest, however, has focused on Ca ++ and more
recently cGMP. Because the rod metabolism efficiently
buffers high energy phosphates (Groskoph et al. 1992),
levels of GTP and ATP are maintained well above the
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
binding constants of the reactions that utilize them. Thus
variations in their concentration occur only at very bright
intensities and are unlikely to be regulatory in background adaptation (Biernbaum & Bownds 1985).
Because we think it plausible that adaptation might
occur at almost any step of the photoreceptor enzymatic
cascade, this discussion outlines the activation and inactivation of the primary reactions, considering each as a
possible locus for adaptational controls. We will emphasize, where possible, measurements made on more intact
and concentrated ROS preparation at the low light levels
(between 1 and 105 Rh*/ROS/sec) at which rods normally
function and discuss technical problems that arise. It is
important to point out that the majority of biochemical
studies have used very unphysiological conditions: diluted suspensions of disrupted ROS (or purified enzymes)
and illumination bleaching a substantial fraction of the
rhodopsin present.
4. Excited rhodopsin catalyzes the activation of a
G-protein (Gt, transducin)
It is instructive to visualize rhodopsin excitation and
subsequent transduction reactions with respect to the
surfaces of single disk membranes. A typical amphibian
ROS contains a stack of approximately 2,000 disk membranes that arise as evaginations of the ciliary plasma
membrane during outer segment morphogenesis (Steinberg et al. 1980; Williams et al. 1988) and then become
self enclosed (thus there are 4,000 disk membrane surfaces). The structure is continuously renewed throughout
its lifetime. The disks occupy ~50% of the volume of the
ROS; the balance is aqueous (Korenbrot et al. 1973). The
surrounding plasma membrane contains the channels
that ultimately are gated as a consequence of bleaching
rhodopsin in the disk membranes. The ROS contains
approximately 3 X 109 rhodopsin molecules (Liebman &
Entine 1968), or roughly 106 rhodopsins/disk membrane
surface. This rhodopsin accounts for 15-20% of the disk
surface, with individual rhodopsin molecules being approximately 2 nm apart, and colliding every 1-10 (xsec.
Several recent reviews provide relevant detailed information about rhodopsin structure (Khorana 1992; Nathans
1992). The concentrations of rhodopsin, Gt, and PDE in
the ROS are approximately 6,000, 600, and 22 u,M,
respectively, calculated with respect to the aqueous volume of the ROS (Dumke et al. 1994; Hamm & Bownds
1986).
Figure 1 visualizes the relative abundance of the components. The square contains 1000 rhodopsin molecules
(the smallest dots), 100 G, (grey dots), 4 PDE molecules
(large dots) and one free cGMP molecule (the small black
dot shown between disk and plasma membrane). Phospholipids of the bilayer, ~50 for each rhodopsin molecule, are highly unsaturated and provide a fluid environment for lateral interactions of the protein components
(Fliesler & Anderson 1983). The number of channels (the
large dot in the plasma membrane) is approximately 1 for
every 10 of these frames.
When one of the rhodopsin molecules in an ROS
absorbs a photon, there is 50% probability that isomerization of the 11-cis retinal chromophore to the all-trans
configuration will occur, and lead to the formation, on a
Bownds & Arshavsky: Photoreceptor adaptation
L
Figure 1. Relative abundance of phototransduction cascade
components in rod outer segments.
millisecond time scale, of excited rhodopsin metarhodopsin II (abbreviated as Rh*) and generation of the photoresponse (Baylor etal. 1979b). Photometric measurement
of the absorption shift caused by photon absorption can
yield the number of rhodopsin molecules that form the
metarhodopsin II intermediate that interacts directly
with G t . In no other system can receptor activation be so
accurately specified. Knowing the number of excited
rhodopsin molecules permits us to calculate the gains of
subsequent steps in the excitation pathway with respect
to the initial input. We can then ask whether this gain
changes during adaptation processes.
It is unlikely that the initial excitation step is a site of
adaptation. This would be the equivalent of saying that
the quantum efficiency of bleaching changes during adaptation, and electrophysiological measurements suggest
that the probability that photon absorption will generate a
photoresponse is not altered by dim background illumination (Baylor et al. 1979b). It is possible, however, that
control of the lifetime of metarhodopsin II (Rh*) could be
a factor in adaptation and this will be considered below
with the reactions that terminate its activation.
Excited rhodopsin exposes within milliseconds a protein surface that binds to the G,-GDP, permitting the
exchange of GTP for GDP on the a subunit. The crystal
structures of both the GTP- and GDP-bound forms of the
a subunit recently have been determined (Lambright et
al. 1994; Noel et al. 1993). G ta -GTP (G*) is released from
Rh*, accompanied by dissociation of the (i-"y subunit
complex, and the process repeats many times (Fig. 2).
Interaction between Rh* (an intrinsic membrane protein) and G, occurs via translational diffusion and collision
on the disk surface (which contains 106 rhodopsin and 105
Gt molecules, Hainm & Bownds 1986). Because very little
G t -GTP formation occurs in the dark, radioactive GTPot-32P or GTP-7-35S can be used to monitor its generation
over physiological ranges of illumination that bleach 102
to 10^ rhodopsin molecules/sec. High gains (~40,000
Gf/Rh*) have been observed in electropermeabilized
ROS that retain most of their complement of G, (GrayKeller et al. 1990). In this preparation rates of 200-400
Gt*/Rh*/sec have been obtained, while measurements of
the light scattering changes which accompany G t activation have been interpreted to indicate rates as high as
1000 Gt*/Rh*/sec (Uhl 1990; Vuong et al. 1984). This
discrepancy needs to be resolved. A curious feature is that
G,-GTP can be generated not only by rhodopsin bleaching but also by mechanical disruption of ROS (Gray-Keller
et al. 1990), or freezing and thawing (Klenchin & Bownds,
unpublished data). The mechanism for this activation is
not clear.
Might the rate of G, activation be altered by adaptation
chemistry? From a functional perspective it would make
little sense at moderate background levels of light to have
the onset of the photoresponse slowed by adaptation, for a
change in light levels needs to be sensed just as rapidly as
the response to the initial onset of illumination. What is
relevant in the presence of background light is that the
amplitude of the response per absorbed photon be
smaller and that it terminate more rapidly. Thus one
would expect mechanisms that determine inactivation
times are more likely loci of adaptational control than
those that determine initial kinetics.
Two lines of evidence suggest that the initial kinetics of
Gt activation may not be sensitive to the previous history
of illumination. Kahlert et al. (1990) have shown that the
presence of bleached rhodopsin does not alter the sensitivity or gain of a light-scattering signal recorded from a
living bovine retina that is taken to monitor G, activation.
For reasons that are not understood, similar signals cannot be recorded from amphibian retinas. Earlier reports
also indicated that background illumination has very little
effect on the initial kinetics of a superimposed flash
response (Baylor & Hodgkin 1974; Lamb 1984). However,
more recently Lagnado and Baylor (1994) have shown in
recordings from truncated amphibian rods that reproducing the C a + + concentration decrease that occurs during
background illumination lowers the initial slope of the
flash response. Direct chemical measurements of the
initial kinetics of Gt* production after a dim flash, and then
after a dim flash superimposed on background illumination, might resolve the issue.
Gt, as well as PDE and several other transduction
enzymes, is covalently modified by lipid attachment.
Farnesylation of the 7 subunit of G t enhances its activation, as does further methylation (Fukada et al. 1990;
Ohguro et al. 1991; Perez-Sala et al. 1991). Neubert et al.
(1992) have characterized four different N-terminal acylations of G ta . Anant et al. (1992) have demonstrated differential prenylation in vivo of the a and (3 subunits of PDE
by farnesylation and geranylgeranylation, respectively.
Disk Membrane
Plasma
Membrane
GTP
Figure 2.
Activation of transducin by photoexcited rhodopsin.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
417
Bownds & Arshavsky: Photoreceptor adaptation
They also report in vivo farnesylation of rhodopsin kinase
(Anant & Fung 1992). It has been suggested that these
reactions might regulate enzyme activity, but it seems
unlikely that they act on the time scale of excitation and
adaptation. They are more likely to be long-term structural modifications whose purpose is to enhance membraneprotein association so that reactants are favorably poised
to interact with each other in the two dimensional surface
of the disk membrane.
The Gt cycle completes when Gf-GTP converts to G ta GDP (see below) and reassociates with Gjj r Phosphoproteins are found in both frog ROS (Components I and II,
Hamm 1990; Polans et al. 1979; Suh & Hamm 1988) and
bovine ROS (Phosducin, Lee et al. 1987; 1990a) that are
dephosphorylated after bright illumination and form
complexes with Gp 7 . If this complex formation competes
with reformation of G ta p 7 , a possible adaptation mechanism is suggested: depletion of the pool of G ta p 7 because
its reassembly has been blocked. The phosphoproteins
are present in an amount comparable with Gt. However,
their subcellular distribution is not clear. Large amounts
are found in the inner segment portions of rod receptor
cells (Lee et al. 1988; 1990b).
5. Does adaptation modulate the duration of G,
activation?
This brings us to consider the processes that terminate
the generation of Gf. Reversal of the PDE activation
caused by G* in vitro is greatly delayed by the removal of
ATP (Liebman & Pugh 1979; 1980), and the photocurrent
in whole cell clamp recordings (Sather & Detwiler 1987)
or truncated ROS (Nakatani & Yau 1988a) approaches
physiological time scales (seconds) only if ATP is present.
It is commonly assumed that the main function of ATP is
to quench Gt* generation by serving as substrate in the
phosphorylation of metarhodopsin II by rhodopsin kinase
(Fig. 3).
This light-dependent reaction leads to incorporation of
up to nine phosphates per Rh* molecule (Wilden & Kuhn
1982). The idea that the effect of ATP is mediated by Rh*
phosphorylation is confirmed by two independent lines of
evidence. First, the effect of ATP can be observed only in
the presence of rhodopsin kinase (Sitaramayya 1986; Sitaramayya & Liebman 1983a). Second, the ability of phos-
'GDP
Figure 3. Inactivation of photoexcited rhodopsin by rhodopsin kinase and arrestin.
418
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
phorylated Rh* to activate G, is reduced (Arshavsky et al.
1985; Miller et al. 1986; Wilden et al. 1986). Arshavsky et
al. (1987) have demonstrated that the rate of Gt activation
decreases 1.5 to 3.3-fold as phosphate incorporation increases from 2 to 6 phosphates/rhodopsin, and that the
inhibitory mechanism involves an increase in the time
necessary for the activation of each bound G, rather than a
reduction in the binding affinity of G t for phosphorylated
Rh*.
Rhodopsin phosphorylation is followed by binding of a
48kD protein, arrestin, a "capping" reaction that blocks
access of G, so that no further excitation can occur (Wilden et al. 1986). Palczewski et al. (1992) have demonstrated that sangivamycin (an inhibitor of rhodopsin kinase) and phytic acid (an inhibitor of arrestin binding to
phosphorylated rhodopsin) slow recovery of the photoresponse when they are dialyzed into gecko ROS. It is
interesting to note they show also that high concentrations of sangivamycin cause changes in the light response
that cannot be explained by selective inhibition of rhodopsin kinase, and suggest that other protein kinases, for
example protein kinase C, may be needed for normal rod
function. Hofmann et al. (1992) have recently shown that
recycling of rhodopsin back to the active pool requires
reduction of the retinal chromophore to retinol. Only
then does arrestin dissociate, opsin becomes dephosphorylated, and opsin combines with 11-cis retinal to
form rhodopsin again.
The shut-off of rhodopsin activation must be very reliable and irreversible, so that only one burst of activity is
set off by each photon absorption. Lagnado and Baylor
(1992) make the point that quenching of rhodopsin by
binding of arrestin to rhodopsin with one or two phosphates would not explain the constancy of the single
photon responses observed. They suggest that reproducibility of the single photon response might be due in
part to feedback control of rhodopsin's active lifetime.
Can rhodopsin phosphorylation be measured in fact to
occur before, or on the same time scale as, the turning off
of the photo response? A positive answer to this question
was obtained by Sitaramayya and Liebman (1983b), demonstrating in bovine ROS that bleaching approximately
one rhodopsin in each photoreceptor disk causes incorporation of about 20 phosphates per bleached rhodopsin
in the first two seconds after the flash. Since each Rh*
cannot incorporate more than nine phosphate groups,
this suggests that not only Rh* but also some nonactivated
rhodopsin can be phosphorylated, confirming the initial
report of Bownds et al. (1972). A slower rate of Rh*
phosphorylation, about one phosphate per bleached rhodopsin in the first 1-2 seconds after a light flash, was
documented for frog ROS by Binder et al. (1990). This
lower stoichiometry does not necessarily reflect a difference between the two species because higher bleaching
levels of about 1000 Rh* per disk were used in the frog
study. It would appear then that incorporation of one or
two phosphates per mole of rhodopsin is adequate for
photoresponse quenching. Experiments of Bennett and
Sitaramayya (1988) suggest that only one or two phosphates/Rh* may be sufficient to promote arrestin binding
and maximally quench the interaction between rhodopsin
and Gt. The residues initially phosphorylated have recently been identified as serines 338 and 343 and threonine 336 (McDowell et al. 1993; Ohguro et al. 1993; Papac
Bownds & Arshavsky: Photoreceptor adaptation
et al. 1993). The data are consistent with a role for
rhodopsin phosphorylation in quenching Gt* generation,
but a determination of the kinetic correlations between
the time course of rhodopsin phosphorylation and the
decay of G* generation has not been accomplished. An
alternative view is that the crucial event in quenching
Rh* is the binding of kinase, rather than the subsequent
phosphorylation that is catalyzed by the kinase (Pulvermuller et al. 1993). In this model the phosphorylation
would serve the function of insuring that the initial
quenching was made permanent. However, the fact that
ATP is required for quenching the cascade means that
kinase binding by itself does not insure Rh* inactivation.
Is modulation of rhodopsin phosphorylation a possible
site of adaptation? Lagnado and Baylor (1992) suggest that
calcium might influence Rh* shutoff, making it more
stereotyped because it would be determined by the fall in
calcium concentration that follows channel closure (see
below). Further, increasing the rate of Rh* phosphorylation during background illumination could potentially
cause acceleration of photoresponse recovery of the sort
observed during background adaptation. Recently Kawamura and his collaborators have provided convincing
evidence that just such a scenario may be appropriate, by
showing that decreasing C a + + concentration shortens the
duration of the photoresponse (Kawamura & Murakami
1991). (It should be noted that the physiological data on
Ca + + regulation of Rh* lifetime has thus far been obtained in broken truncated ROS whose interior is exposed
to a bathing medium.) Analysis ofphotoresponse recovery
from bright flashes in intact cells leads Pepperberg et al.
(1992; 1994) to argue that this lifetime does not change
during adaptation, but rather that a later stage is involved.
Kawamura's group has identified a protein, named
recoverin binds and imparts C a + + sensitivity to rhodopsin kinase.
The situation is not as clear with respect to cGMP as a
putative feedback regulator of rhodopsin phosphorylation. Hermolin et al. (1982) and Shuster and Farber (1984)
noted inhibition of rhodopsin phosphorylation by cGMP,
but more recently Palczewski et al. (1988b) and Binder et
al. (1989) found no effect.
Another possible role for Rh* phosphorylation in light
adaptation can be suggested: if a substantial portion of
nonbleached rhodopsin were to become phosphorylated
during dim background illumination, its excitation should
result in photoresponses with lowered amplitude and
accelerated recovery. The amplitude reduction would be
expected from the reduced ability of phosphorylated Rh*
to activate G t , while turnoff acceleration would result
from faster arrestin binding to pre-phosphorylated rhodopsin. A hint that this might take place has come from
measurements of rhodopsin phosphorylation in electropermeabilized amphibian ROS. At light levels in the
operating range of the rod, bleaching one rhodopsin
molecule can result in the incorporation of phosphate
groups into several hundred dark rhodopsin molecules
(Binder et al. 1990). The gain diminishes rapidly as each
interdiskal space begins to receive more than one photon,
and the high gain reaction is lost if outer segments are
fragmented into smaller pieces. This high gain reaction is
sensitive to calcium, being more active at 10 nM than at
\xM calcium levels (Calvert and Bownds, unpublished
observations). In permeabilized ROS, however, the highgain phosphorylation reaction saturates when it has acted
on less than 1% of the total rhodopsin present. This would
not significantly alter the probability of an ROS generating a response upon photon absorption. It: is possible,
S-modulin (M. W. 23 kDa), that confers C a + + sensitivity
however, that measurements on the living retina might
on rhodopsin phosphorylation (Kawamura 1993; Kawamura et al. 1992). This protein turns out to be the frog
homolog of bovine recoverin (Dizhoor et al. 1991), a Ca + +
binding protein originally proposed to regulate guanylate
cyclase (see below). The three-dimensional structure of
recoverin recently has been determined (Flaherty et al.
1993). Gray-Keller et al. (1993) have shown that internal
perfusion of gecko rods with gecko S-modulin as well as
recoverin delays the recovery of the photoresponse. This
correlates with the observation that at high C a + + concentration these proteins inhibit rhodopsin phosphorylation,
perhaps by acting on rhodopsin kinase. The inhibition is
relieved if Ca + + is lowered to the level caused by illumination (< 100 nM). This suggests that the lowering of
C a + + that occurs during background adaptation should
decrease the lifetime of Rh* and thus the duration of PDE
activation, causing smaller and faster responses to superimposed flashes. Both earlier (Kawamura & Bownds 1981;
Robinson et al. 1980) and more recent (Kawamura &
Murakami 1991) experiments had shown in fact such an
effect of C a + + on PDE activation. The data of Wagner et
al. (1989) also suggest C a + + regulation of rhodopsin
phosphorylation.'A light-scattering transient that they
interpret as rhodopsin deactivation in ROS suspensions
is accelerated as calcium is lowered from 1 to 0.1 u-M.
Recoverin appears to be essential for the C a + + regulation, for Palczewski et al. (1988a) have found that
C a + + does not act directly on rhodopsin kinase. Most
recently Chen and Hurley (1994) have reported that
show that the high-gain rhodopsin phosphorylation reaction alters a more significant fraction of the visual pigment
present. Such measurements have not yet been done.
The mechanism of the high-gain phosphorylation remains to be determined. Recent studies (Buczylko et al.
1991; Fowles et al. 1988; Palczewski et al. 1991) have
shown that rhodopsin kinase, on binding to bleached
rhodopsin, becomes able to phosphorylate rhodopsin
C-terminal peptide fragments. Perhaps it also phosphorylates the C-terminus of some dark rhodopsin molecules.
An alternative possibility is that another kinase acts on
rhodopsin. Newton and Williams (1991) suggest a possible analogy with the (3-adrenergic receptor system,
where both cAMP dependent kinase and (3-adrenergic
receptor kinase phosphorylate the receptor (Roth et al.
1991), with the cAMP dependent kinase more important
at low receptor occupancy and the (3-adrenergic receptor
kinase acting at higher levels of receptor occupancy. The
observation (Newton & Williams 1993) that rhodopsin is
the major substrate of protein kinase C in ROS raises the
possibility that this enzyme may play a role corresponding
to the cAMP dependent kinase of the (3-adrenergic receptor system.
Another possible mechanism leading to the accumulation of nonbleached phosphorylated rhodopsin in photoreceptor cells is an inhibition of the dephosphorylation
reaction without blocking the regeneration of bleached
rhodopsin by 11-cis-retinal. This possibility is discussed
by Biernbaum et al. (1991), who demonstrated that a dim
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
419
Bownds & Arshavsky: Photoreceptor adaptation
background illumination prevents rhodopsin dephosphorylation in a preparation of intact frog rods.
6. Excited G, activates PDE
The next step in the phototransduction cascade is the
action of Gt* on its effector, PDE. The PDE holoenzyme is
an aP"y2 tetramer. The a and (3 subunits each contain one
catalytic and one, or possibly two, noncatalytic cGMP
binding sites (Charbonneau et al. 1990; Cote & Brunnock
1993; Gillespie & Beavo 1988; 1989; Li et al. 1990). The
two identical 7 subunits serve as inhibitors of the enzyme
(Deterre et al. 1988; Fung et al. 1990; Hurley & Stryer
1982). Full activation requires the binding of one Gf
molecule to each of the two inhibitory 7 subunits. Each
frog disk face contains ~3,700 PDE molecules, anchored
by prenylation to the membrane surface. An excited Gf
contacts one of these PDE molecules rapidly enough to
activate it within milliseconds (Heck & Hoffman 1993).
This reaction is occurring by lateral diffusion on the
surface of the disk. Given that the binding constant
between PDE and Gf is approximately 100-600 nM
(Bennett & Clerc 1989), lower than the concentration of
Gt* at the disk surface (which, from above, is > 1 u,M),
essentially all of the Gt* has activated PDE within milliseconds. (In diluted fragmented ROS, a smaller fraction of
Gf is successful in activating PDE [Liebman et al. 1987].)
Pugh and Lamb (1993) point out that each of the activation
reactions in the transduction pathway can be treated as a
short first order delay stage, so that the time course of
PDE activation is a delayed ramp, with slope proportional
to light intensity; the initial delay is about 10-20 msec
(Lamb & Pugh 1992).
The PDE activation-inactivation sequence occurs in a
fundamentally different context than the Rh* — Gt sequence. In the latter case the reactants are poised for
reaction but silent in the dark ROS, awaiting the discharge
induced by illumination. PDE activation, on the other
hand, modulates an ongoing flux:
PDE
Cyclase
GTP-•cGMP-
GMP
Both biochemical and electrophysiological studies
(Dawis et al. 1988; Hodgkin & Nunn 1988) suggest flux
rates on the order of one turnover of the entire pool of
unbound cGMP per second, and Pugh and Lamb (1990)
estimate dark levels of free cytoplasmic cGMP to be 4
u,M. This means that each interdiskal space has about 650
molecules of cGMP, all turning over once a second.
Direct measurement of the dark PDE activity that
underlies this flux is complicated by the dilution of reactants that occurs when ROS are disrupted to assay the
enzyme. This disruption causes some dissociation of the
inhibitory 7 subunit of PDE and thus increases dark
background activity (cf. Arshavsky et al. 1992). Measurements performed under conditions preventing PDE^ loss
from PDE catalytic subunits indicates that the PDE
activity in the dark is at least 300-fold less than the activity
of light-activated enzyme (Arshavsky et al. 1992). Taking
this fraction of the light activity, the cGMP concentration
in the dark as constant and equal to 4 JJLM, PDE concentrations as —22 jxM (see above), Vmax (in the light) —4700
420
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
s"1 and Km as - 9 5 u,M (Bownds et al. 1992; Dumke et al.
1994) Michaelis-Menten calculations yield a dark flux
estimate for cGMP of less than 15u.M/sec. This number is
reasonably close to the 4 u,M/sec estimated in vivo (Pugh
& Lamb 1990).
The effect of the large increase in PDE activity caused
by light can be visualized by making a simple calculation
of the amount of cGMP hydrolyzed after absorption of a
single photon (i.e., 1 Rh*) in a frog rod. More precise
calculations are reported in Lamb and Pugh (1992). Let us
suppose that the rate of Gt activation is 1000 s"1 and that
this rate is constant during the time of the photoresponse.
Therefore, an average of 500 Gf molecules are present
during the rising phase of the response (— 1 sec). Since the
complete activation of a PDE molecule demands two Gf,
the average number of PDE molecules activated during
this time will be about 250 molecules. Taking Vmax of
activated PDE as about 4700 turnovers/sec and Km as 95
u-M (Dumke et al. 1994), we can calculate that each
activated PDE will hydrolyze about 200 cGMP molecules. The total amount of hydrolyzed cGMP then will be
on the order of 50,000 molecules. This corresponds to the
total cGMP content of approximately 80 interdiskal
spaces or 4% of the whole pool of unbound rod cGMP.
This is reasonably close to the length of ROS whose
conductance is suppressed following a single photon absorption (see below).
It is likely that a considerably smaller change in cGMP
would be sufficient to suppress this conductance. Because
gating of the channel by cGMP is cooperative, hydrolysis
of only a fraction of the cGMP present might be sufficient
to cause a large conductance change. Overall, it would
seem more efficient for cGMP to drop by a small amount
over a longer length of the ROS than to undergo a large
change over a small length (cf. Lamb & Pugh 1992; Rispoli
et al. 1993). Pugh and Lamb (1993) have pointed out that
these kinetic parameters can generate very different relative changes in cGMP concentration if ROS volume is
decreased, as in mammalian rods and cones. Equivalent
cascade activation in a smaller ROS causes a larger fractional change in the cGMP pool.
The PDE enzyme is so active that it depletes cGMP in
the interdiskal space very rapidly. As a result, the diffusion of cGMP into the interior of the disk stacks can
become rate limiting in conventional biochemical measurement of cGMP hydrolysis in ROS fragments. This can
have the effect of raising the apparent Km of PDE as well
as limiting its apparent maximal velocity. Recent studies
show that true values for Km and kcat of PDE (—95 u,M and
4700 sec"1) are approached only when ROS membranes
are completely disrupted into small vesicles (Dumke et
al. 1994). Techniques for directly measuring these parameters in intact cells are not currently available. Perhaps
the most accurate estimates of PDE activation kinetics in
intact ROS come from reverse calculations based on the
kinetics of the photoresponse (cf. Hodgkin & Nunn 1988;
Pugh & Lamb 1993).
7. The turnoff of activated PDE is a putative point
of light adaptation
The action of Gf, like that of other G-proteins, has been
thought to be terminated as its bound GTP is hydrolyzed
Bownds & Arshavsky: Photoreceptor adaptation
by an "intrinsic GTPase activity" (cf. Bourne et al. 1990;
1991). This activity measured in dilute suspensions has
been much too slow (Baehretal. 1982; Fung etal. 1981) to
explain the rapid turnoif of PDE in suspensions of bovine
ROS (Sitaramayya & Liebman 1983b). More recent work
has indicated that transducin GTPase under more physiological conditions is much faster than in reconstituted
systems (Arshavsky et al. 1989; 1991; Dratz et al. 1987;
Wagner et al. 1988). A number of factors appear to be
responsible for G t GTPase regulation in photoreceptors.
One is the activation of G, GTPase by PDE (Arshavsky &
Bownds 1992; Arshavsky et al. 1991; Pag<§s et al. 1992;
1993), and more specifically its 7 subunit acting as a GAP
(GTPase activating protein) (Arshavsky & Bownds 1992;
Arshavsky et al. 1994). Acceleration of G t GTPase by
PDE 7 requires the presence of ROS membranes. Antonny et al. (1993) have made the point that it is not
observed in the soluble complex of G,-GTP with P D E r A
second factor has been suggested by recent observations
that the effect of PDE 7 becomes more pronounced as
ROS membranes are concentrated (Angleson & Wensel
1994; Arshavsky et al. 1994). The nature of this second
mechanism remains unclear.
Two reports suggest that G, GTPase can be faster than
PDE turnoff. Microcalorimetric measurements on ROS
have shown GTP and light dependent rapid transients,
interpreted as reflecting the time course of G, activation
and hydrolysis (Vuong & Chabre 1990; 1991). A problem
is that the heat responses measured cannot be clearly
assigned to the hydrolysis of G,-GTP, rather than
dissociation-association reactions of Rh*-Gt or G t -PDE,
as well as some other light-dependent processes. Ting
and Ho (1991) report GTPase activity more rapid than
PDE inactivation, but do not prove that the burst of
GTPase activity observed in their experiments is associated with transducin, nor do they measure GTPase and
PDE inactivation under the same conditions. An explicit
critique of this work is provided by Antonny et al. (1993).
It remains to be seen whether mechanisms in addition
to the transducin GTPase might be involved in PDE
inactivation. The rates of G, GTPase and PDE turnoff
measured in the same suspension of bovine ROS coincide
(Angleson & Wensel 1993; Arshavsky et al. 1989). In
contrast, Erickson et al. (1992) have recently asserted that
PDE inactivation can occur in the absence of GTP hydrolysis when G t is activated by a nonhydrolyzable analog of
GTP. They propose that ROS contain a pool of PDE
inhibitor, presumably PDE -y-subunits that inhibit activated PDE prior to the G r GTPase reaction. However,
their measurements conducted in the presence of GTP,
making the G,-GTPase reaction possible, reveal faster
and more complete PDE turnoff when compared with
that observed with the nonhydrolyzable analog. The next
experiments needed are an examination of the rates of
transducin GTPase and PDE turnoff in suspensions of
permeabilized, or broken and very concentrated, ROS.
The step of PDE inactivation would seem to be a good
locus for light adaptation. A mechanism that increased the
rate of PDE turnoff would be expected to lead to a
reduction in both photoresponse duration and amplitude
during light adaptation. In fact, a regulation of PDE
turnoff by cGMP binding to noncatalytic cGMP binding
sites on PDE a and (3 subunits has been demonstrated in
recent work (Arshavsky et al. 1991; Arshavsky & Bownds
1992). When these sites are occupied, transducin GTPase
and PDE turnoff are slowed. As cGMP concentration falls
over its physiological range, from ~ 5 to below 1 u,M, the
sites empty and GTPase and PDE turnoff are accelerated
several fold.
Further work by Arshavsky et al. (1992) has provided
evidence that cGMP binding in the PDE noncatalytic
sites determines the fashion in which PDE is activated by
Gt*. If the noncatalytic sites are occupied by cGMP, G t
activates PDE and remains bound to the PDE heterotetramer. Alternatively, when the sites are empty, G*
physically removes PDE 7 from PDE a p upon activation.
These observations are summarized in the scheme (Fig.
4) shown below. Stage 1 shows PDE as aheterotetramer in
the dark photoreceptor with cGMP bound to its noncatalytic sites. Activation by Gf removes inhibition of the
catalytic sites by PDE 7 (stage 2). As long as cGMP
remains bound to the noncatalytic sites, G* remains in a
complex with PDE, and the hydrolysis of GTP that causes
PDE inactivation is slowed (upper solid arrow). In stage 3,
as illumination continues, bound cGMP dissociates and is
hydrolyzed. This dissociation might occur from both
activated and nonactivated PDE. PDE formed with the
noncatalytic sites empty (stages 3 and 4) undergoes a
different activation-inactivation sequence. G* physically
removes PDE 7 , GTPase activity becomes more rapid,
and therefore inactivation occurs faster (lower solid arrow). To complete the cycle of G t , G ta -GDP must dissociate from PDE 7 and recombine with G , P r This process
requires the mediation of Gp 7 (Yamazaki et al. 1990).
How might this process be involved in adaptation? In
the dark cGMP levels are high, the noncatalytic cGMP
sites on PDE are occupied (see Fig. 4). This slows GTPase
activity so that PDE stays on longer, making the light
response bigger and longer. When cGMP is lowered by
light, cGMP dissociates from noncatalytic sites of activated and possibly nonactivated PDE molecules. Those
PDE molecules that have lost their bound cGMP are
inactivated more rapidly upon subsequent activation,
making light responses smaller and faster. At first glance,
one might suppose that the detachment of PDE 7 shown in
Figure 4. A scheme for the putative role of PDE noncatalytic
cGMP-binding sites in the background adaptation of photoreceptors (reproduced with permission from Arshavsky et al.
1992).
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
421
Bownds & Arshavsky: Photoreceptor adaptation
stage 3 would slow inactivation of PDE, because the
reattachment of PDE 7 to PDE a p might be rate limiting.
However, direct measurements show that GTP hydrolysis, rather than PDE Y reattachment, is rate limiting
under these conditions (Arshavsky et al., in preparation).
Validation of a model of this sort requires that both the
binding constants and off kinetics of the non-catalytic
cGMP binding sites be determined. Cote et al. (1994)
have recently accomplished this, finding that activation of
frog PDE lowers the binding affinity and accelerates the
dissociation kinetics of these sites, so that the cGMP drop
occurring upon illumination favors release of bound
cGMP. The time scale for dissociation, tens of seconds,
corresponds to the period during continuous bright illumination of photoreceptors when an increase in the response recovery rate is being observed (Cervetto et al.
1984; Coles & Yamane 1975; McCarthy & Owen, personal
communication).
The mechanism may be limited to lower vertebrates,
because bovine PDE does not readily lose bound cGMP
in vitro (Gillespie & Beavo 1989). The noncatalytic cGMP
binding sites of vertebrate cone PDEs have binding
constants intermediate between the amphibian and vertebrate rod PDEs (Gillespie & Beavo 1988), and so might
potentially use this mechanism to achieve their faster
turnoff times. In both cases, it is possible that dissociation
occurs more readily for the activated enzyme.
The situation regarding PDE 7 may become even more
complicated if its recently demonstrated phosphorylation
by different protein kinases (Hayashi 1994; Hayashi et al.
1991;Tsuboietal. 1994a; 1994b; Udovichenkoetal. 1994)
is shown to change during the activation-inactivation
cycle. Tsuboi et al. (1994a; 1994b) report that a cGMP
inhibited kinase can phosphorylate either free PDE 7 or
PDE 7 in complex with G ta -GTP. The phosphorylated
forms lose their ability to bind G ta -GTP, and thus render
PDE refractory to activation. While Tsuboi et al. suggest
that this PDE 7 phosphorylation is a mechanism of rapid
PDE turnoff independent of GTPase activity of G ta , we
think it more likely that it is a means of removing some
PDE from the active pool during light adaptation. Experiments are needed to demonstrate that this phosphorylationdephosphorylation actually occurs during either flash
responses or light adaptation.
8. The drop in cytoplasmic cGMP resulting from
PDE activation causes cGMP-gated channels
to close
The last step of the phototransduction cascade is the
closure of cGMP-gated cationic channels in the ROS
plasma membrane. Because their properties are discussed in detail in several recent reviews (Kaupp & Koch
1992; McNaughton 1990; Molday & Hsu, this issue; Yau &
Baylor 1989) we will provide only a brief account. The
amphibian ROS contains approximately 5 X 105 channels,
and only about 2% of these are open in the dark-adapted
cell. Electrophysiological measurements show that absorption of a single photon leads to a 1-5% reduction in the
dark conductance of the plasma membrane, caused by
almost complete closure of channels over a length of the
ROS of not less than ljjun (Matthews 1986), or more than 6
(xm (Lamb et al. 1981). These numbers are in reasonable
correspondence with the calculations above indicating
that ~ 4 % of rod cGMP is hydrolyzed during a single
photon photoresponse.
Is the channel a site of modification during adaptation?
Recent observations of Gordon et al. (1992) suggest that
channel sensitivity to cGMP may be regulated by phosphorylation. Plasma membrane patches from frog ROS
become more sensitive to cGMP over a period of minutes
after their excision, and this sensitivity increase is delayed
if protein phosphatase inhibitors are added. Hsu and
Molday (1993) have found that the cGMP-gated channel
from bovine rods can be modulated by calmodulin. Lowering C a + + concentration over the range it falls during
illumination increases the affinity of the channel for
cGMP. Thus less cGMP might be required to reopen the
channel during recovery of the photoresponse in the
light-adapted cell than is required to keep it open in the
dark. The physiological relevance of this calmodulin effect is discussed in detail by Molday and Hsu in a companion article in this issue.
9. The drop in cytoplasmic C a + + caused by
channel closing activates a negative feedback
loop by stimulating guanylate cyclase and
cGMP recovery
C a + + ions continually enter the dark ROS through
cGMP-gated channels and are extruded by a Na + /
C a + + , K + exchanger (see Fig. 5). The decrease in cytoplasmic calcium that occurs upon channel closing has
been shown in numerous studies to be crucial in regulating recovery and adaptation of the photoresponse. Numerous reviews can be consulted on this topic (Detwiler
& Gray-Keller 1992; Kaupp & Koch 1992; Koch 1992;
Lagnado & Baylor 1992; McNaughton 1990; Pugh &
Lamb 1990; Stryer 1991).
The central electrophysiological observations are that
clamping internal C a + + concentration blocks the gain
reduction associated with adaptation (Matthews et al.
1988; Nakatani & Yau 1988b), and that buffering internal
calcium slows the recovery phase of the flash response
(Lamb et al. 1986). Biochemical studies on bovine ROS
show that guanylate cyclase activity is stimulated by
lowering C a + + from 200 nM to 50 nM (Koch & Stryer
1988). This C a + + sensitivity was first thought to be
mediated by the 26 kDa C a + + binding protein, recoverin
(Dizhoor et al. 1991; Lambrecht & Koch 1991). Recov-
TV
Na+
K+.Ca^
Figure 5.
422
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
T
Plasma membrane
Ca++ Na+
'
Regulation of guanylate cyclase by calcium ions.
Bownds & Arshavsky: Photoreceptor adaptation
erin, however, has since been shown to be a regulator of
rhodopsin phosphorylation rather than guanylate cyclase
(Hurley et al. 1993; see above). Two groups have now
isolated small protein(s) that confer C a + + sensitivity on
cyclase (Baehr et al. 1994; Dizhoor et al. 1994; Gorczyca
et al. 1994).
These observations are all consistent with the idea that
activation of cyclase caused by the lowering of calcium can
accelerate cGMP resynthesis and the restoration of dark
conductance. Several quantitative models of the photoresponse have included the calcium feedback loop and
reproduce electrophysiological recordings very closely
(Forti et al. 1989; Sneyd & Tranchina 1989; Tamura et al.
1991; Tranchina et al. 1991). These models, however, do
not exclude the possibility of further regulatory loops, and
in fact Koutalos, Nakatani, and Yau (personal communication) have established the importance of both the cyclase
feedback loop and the PDE attenuation that occurs as a
fall in Ca + + levels accelerates rhodopsin phosphorylation
and inactivation mentioned above. They used the truncated rod outer segment preparation to measure the
Ca + + -dependence of the cGMP-gated channel, the guanylate cyclase, and the steady-state PDE activity elicited
by a step of light. They were able to account for the
steady-state response and sensitivity of the intact rod as a
function of background light intensity. In terms of relative
contribution to adaptation to background light, they
found that the C a + + modulation of the cyclase is most
important at low background light intensities and modulation of light-activated PDE contributes at higher light
levels. The C a + + modulation of the channel was found to
add relatively little to light adaptation.
Several groups have used fluorescent probes (GrayKeller & Detwiler 1994; McCarthy et al. 1993; Ratto et al.
1988; Younger et al. 1992) or aequorin (Lagnado et al.
1992) to measure C a + + concentration changes that occur
on illumination. The most recent estimates judge dark
C a + + concentration to be approximately 500 nM, falling
to ~50 nM upon saturating illumination. The dark level
corresponds to ~80 C a + + ions per disk face, falling below
~ 8 upon illumination. This represents a very small fraction of the capacity of the Na + /Ca + + , K + exchanger
(McNaughton 1990). The fall occurs in two phases, the
first in less than one second and the next with a half-time
of ~ 5 seconds. Changes in cytosolic free C a + + , sensitivity and fractional current suppression follow a broadly
similar form during steady state exposure to different
background light intensities.
It is unlikely that calcium is the sole regulator of
adaptation processes. Photoresponses with normal kinetics can be measured in C a + + depleted ROS, incubated in 10 nM C a + + and calcium ionophore (Nicol et al.
1987), and there would appear to be mechanisms capable
of reducing the gain of the photoresponse and speeding its
recovery at higher background intensities in the absence
of significant changes in cytoplasmic calcium (Nicol &
Bownds 1989). The cGMP feedback mentioned above
would be one candidate for such a mechanism. Rispoli
and Detwiler (1991) have demonstrated that increasing
internal C a + + concentration speeds, rather than slows,
response recovery. In an earlier report (Rispoli & Detwiler 1989) they also noted that the recovery kinetics of
the light response were independent of the dark current
level that presumably regulates internal C a + + .
10. Can the most important sites of light
adaptation be specified?
In summary, recent experiments have provided evidence
for pathways by which the primary transduction messenger, cGMP, can exert feedback control over both its
synthesis and light-induced degradation. The lightinduced decrease in cGMP can damp further decreases
by leading to stimulation of guanylate cyclase and accelerating the inactivation of light-activated PDE:
on*
Cyclase
PDE
GTP
GMP
• cGMPA central question is whether one of the mechanisms
we have mentioned plays a predominant role in adaptation, or whether equally important multiple attenuations
occur at each step of the transduction cascade. The fact
that all of the turnoff reactions of the cascade have now
been implicated as sites of C a + + or cGMP regulation
suggests that the latter possibility is more likely. The data
suggest that light adaptation consists of enhancement of
all of the turnoff and restoration processes accompanying
the photoresponse, starting with rhodopsin phosphorylation and ending with the putative modulation of the
plasma membrane channel by C a + + or phosphorylation.
We think it likely that a sequence of reactions are recruited as background light intensities increase:
1. C a + + feedback on guanylate cyclase;
2. C a + + feedback on rhodopsin phosphorylation;
3. cGMP feedback on the lifetime of PDE;
4. phosphorylation of unbleached rhodopsin.
Three reactions appear to be most central for turnoff of
the photoresponse: rhodopsin quenching, PDE inactivation, and guanylate cyclase activation. The data presently
available do not permit us to argue strongly for one of
these being most central. The crucial balance between
the enzymes that set cGMP concentration during recovery of the photoresponse might be considered in several
ways:
Several reviews emphasize cyclase activation rather
than PDE inactivation as central in cGMP and conductance restoration after a flash of light. We think it most
likely that the function of cyclase acceleration is to avoid a
delay between PDE turnoff and cGMP concentration
restoration. Such a delay is most likely the cause of the
lengthening of the flash response that occurs when calcium buffers are injected into the ROS cytoplasm. Is
cyclase activation sufficient to contribute to adaptation
during background light? The > 300-fold activation of
PDE that occurs upon illumination (Arshavsky et al. 1992)
needs to be compensated by cyclase activation. Although
the maximum cyclase activation measured in vitro has
been < 10-fold (reviewed in Stryer 1991) this activation
(mediated by diffusible Ca + + ) could be expected to
spread much further than the disks containing activated
PDE molecules. This suggests that at lower levels of
illumination, where cyclase is activated over a significantly larger cell volume than PDE, cyclase activation
may be sufficient to be part of the mechanism that
counters PDE activation during light adaptation. This
suggestion is compatible with the finding of Koutalos,
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
423
Bownds & Arshavsky: Photoreceptor adaptation
Nakatani and Yau, mentioned above, that modulation of
cyclase activity by C a + + is most important at lower
background light intensities.
Another concern is the extent to which Rh* turnoff
versus PDE inactivation is rate limiting for terminating
cyclic GMP hydrolysis. PDE inactivation would be rate
limiting if Rh* turnoff were significantly faster than the
rate of G,-GTPase that causes termination of PDE activation. Conversely, Rh* decay would become limiting if its
rate were slower than that of the GTPase. Current biochemical data suggest that these processes are occurring
at similar times, so that each might be modulated during
light adaptation. Further progress in understanding
which of these reactions is most important requires the
development of techniques that measure them on the
time scale of the photoresponse at dim light intensities. It
would not be surprising to find controls that are redun-
424
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
dant and increase the reliability of the system. Studies on
this system are repeating the history of the discoveries of
basic catabolic and anabolic pathways in the 1950s and
1960s. After establishing a basic pathway, even more
effort is being directed toward describing the relevant
array of feedback and other regulatory controls.
ACKNOWLEDGMENT
This manuscript was prepared with support from N.I.H. grants
EY-00463 and EY-10336. We are grateful for comments on the
manuscript of Mark Gray-Keller, Peter Detwiler, MarcChabre,
and several anonymous reviewers.
NOTE
1. Arshavsky's present address is Howe Laboratories, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18, 425-428
Printed in the United States ol America
Recoverin and Ca 2+ in vertebrate
phototransduction
James B. Hurley
Department of Biochemistry and Howard Hughes Medical Institute, SL-15
University of Washington, Seattle, WA 98195
Electronic mail: jbhhh@u.washington.edu
Abstract: Recoverin is a 23 kDa Ca2+-binding protein that has been detected primarily in vertebrate photoreceptors. The role of
recoverin in phototransduction has been investigated using a variety of biochemical methods. Initial reports suggesting that recoverin
regulates photoreceptor guanylyl cyclase have not been confirmed. Instead, recoverin appears to determine the lifetime of lightstimulated phosphodiesterase activity, perhaps by regulating rhodopsin phosphorylation. Retinal recoverin is heterogeneously fatty
acylated at its ammo-terminus. The amino-terminal fatty acid appears to be involved in the interaction of recoverin with
photoreceptor membranes.
Keywords: cyclic CMP; guanylyl cyclase; phosphodiesterase; phosphorylation; phototransduction; recoverin; retina; rhodopsin
kinase
1. Introduction
Recoverin, visinin, and S-modulin were first identified as
retina-specific Ca2+-binding proteins. They are relatively
abundant in photoreceptor cells, but their functions there
remain controversial. Several highly conserved recoverin
homologues have been identified in retina and other
tissues, suggesting that this family of proteins is important
for cellular function. This target article reviews recent
studies of recoverin and related proteins with an emphasis
on their possible role in photoreceptors.
During the excitation phase of the vertebrate rod photoresponse light stimulates an enzymatic cascade that
culminates in the hydrolysis of cyclic GMP (cGMP) (Lagnado & Baylor 1992; Stryer 1991). Photoconversion of
rhodopsin to metarhodopsin II within the rod disk membranes stimulates GTP-binding to the a subunit of the
heterotrimeric G-protein, transducin. Light-activated
transducin a (Ta-GTP) dissociates from the T$y complex,
then stimulates cGMP phosphodiesterase (PDE) activity
by relieving inhibition imposed on the PDE catalytic
subunits by a small inhibitory subunit, PDE*y. The mechanism of PDE activation appears to involve formation of a
complex between Tot-GTP and PDE-y. The ensuing hydrolysis of intracellular cGMP reduces cGMP-gated cation channel activity in the rod outer segment plasma
membrane.
Several processes contribute to the recovery phase of
the photoresponse. First, photolyzed rhodopsin is phosphorylated by rhodopsin kinase. This reduces the ability
of the photolyzed rhodopsin to stimulate transducin. It
also stimulates binding of arrestin to the photolyzed
rhodopsin. Arrestin binding further quenches the ability
of the photolyzed rhodopsin to activate transducin. Activated transducin a subunits (Tot) already formed by pho-
© 1995 Cambridge University Press
0140-525X195 S9.00+.10
tolyzed rhodopsin deactivate when their bound GTP is
hydrolyzed to GDP. Ta-GDP then reassociates with TB-y
and releases the PDE-y subunit, which reinhibits PDE
activity.
Light-stimulated hydrolysis of cGMP within rod photoreceptors reduces the activity of cGMP-gated cation
channels in the photoreceptor plasma membrane. Because these channels are the major route by which Ca2+
enters the photoreceptor, the intracellular concentration
of Ca2+ falls. Lowered Ca2+ levels stimulate a photoreceptor, guanylyl cyclase, to resynthesize cGMP (Koch
& Stryer 1988). In addition, lowered Ca2+ speeds the rate
of deactivation of cGMP PDE (Kawamura & Murakami
1991). Recently, it has also been reported that Ca2+ may
reduce the affinity of the cGMP-gated plasma membrane
cation channel for cGMP (Hsu & Molday 1993). Each of
these effects of Ca2+ may help promote recovery of the
photoreceptor following photoexcitation.
The mechanisms by which Ca2+ regulates photoreceptor guanylyl cyclase and cGMP PDE are not completely
understood. However, a 23 kDa Ca2+-binding protein
specifically found in photoreceptors has been implicated
in both of these regulatory processes (Dizhoor et al. 1991;
Kawamura & Murakami 1991; Lambrecht & Koch 1991).
Initial reports suggested that one form of this protein,
recoverin, isolated from bovine retinas, is a regulator of
photoreceptor guanylyl cyclase (Dizhoor et al. 1991;
Lambrecht & Koch 1991). However, it now appears that
those initial reports were incorrect (Hurley et al. 1993).
Other observations have upheld the view that this protein
imparts Ca2+-sensitivity to the cGMP PDE (Kawamura
1993; Kawamura & Murakami 1991). In the next section, I
describe some characteristics of this protein and its homologs. The protein has been given several different names
by the laboratories that discovered it. Isolates from
425
Hurley: Vertebrate phototransduction
chicken were referred to as visinin, isolates from bovine retinas were referred to either as recoverin or
p26 and isolates from frog retinas were referred to as
S-modulin.
2. Members of the recoverin family
2.1. Visinin. Visinin was identified as a 24 kDa cone
protein from chicken retinas (Yamagata et al. 1990). Its
cDNA was isolated and used to produce visinin-specific
antibodies. These antibodies were used to localize visinin
to chicken cone photoreceptors. The nucleotide sequence of visinin cDNA was found to encode a protein
with three potential Ca2+-binding sites known as "EFhands." Ca2+-binding to visinin was confirmed using
visinin expressed in E. coli. Although the function of
visinin has not been specifically investigated, it has been
reported that visinin dialyzed into rod photoreceptors
affects photoresponse lifetime in the same way as recoverin. (Gray-Keller et al. 1993).
2.2. Recoverin. Recoverin was originally identified as a
protein with an apparent molecular weight of 26 kDa, that
bound to a column made from detergent solubilized
rhodopsin immobilized onto concanavalin A-Sepharose
(Dizhoor et al. 1991). Recoverin immunoreactivity has
been detected in rod and cone photoreceptors and in a
class of bipolar cells in the retina (Milam et al. 1992). The
amino acid sequence of recoverin was determined by
Edman degradation of proteolytic fragments (Dizhoor et
al. 1991). The sequence included Ca2+-binding sites and
strong homology with visinin. A cDNA encoding recoverin was isolated and it has been used to express recoverin
in E. coli (Ray et al. 1992). Both recoverin isolated from
bovine retinas and recoverin expressed in E. coli bind
Ca 2+ . Recently, the crystal structure of recoverin was
determined by X-ray analysis. Recoverin is a compact
molecule with a concave hydrophobic cleft between two
Ca2+-binding domains (Flaherty et al. 1993).
Initial reports suggested that recoverin is a Ca2+sensitive stimulator of guanylyl cyclase. However, more
recent findings have not confirmed this conclusion
(Hurley et al. 1993). The relationship between recoverin
and guanylyl cyclase will be described in a later section of
this paper.
2.3. S-modulin. S-modulin was identified as a protein that
binds in a Ca2+-dependent manner to frog photoreceptor
membranes (Kawamura & Murakami 1991). This property was used to purify S-modulin from frog photoreceptors. The activity of cGMP PDE in frog photoreceptors is
sensitive to Ca2+. Kawamura and Murakami found that a
factor responsible for this Ca2+-sensitivity eluted from
truncated photoreceptors when the free Ca2+ concentration was lowered to 20 nM (Kawamura & Murakami 1991).
Reconstituting truncated photoreceptors or photoreceptor homogenates with purified S-modulin restored Ca2+sensitivity to the PDE.
Recent reports (Kawamura 1993; Kawamura et al. 1993)
have clarified the effect of S-modulin and recoverin on
photoreceptor cGMP PDE. An efficient method for purifying S-modulin/recoverin allowed reconstitution experiments using concentrations of S-modulin/recoverin that
426
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
were significantly higher than were used in earlier experiments. The results demonstrated clearly that S-modulin/recoverin prolongs the lifetime of activated PDE
following a light flash. The up to four-fold effect occurred
at free Ca2+ concentrations in the submicromolar range.
Further analyses revealed that Ca2+ and S-modulin/
recoverin partially suppressed light-stimulated phosphorylation of rhodopsin.
More recently, it has been reported that recoverin
forms a Ca2+-dependent complex with rhodopsin kinase
(Chen & Hurley 1994; Subbaraya et al. 1994). There have
been no reports of recoverin interactions with other
soluble photoreceptor-specific proteins such as phosphodiesterase subunits. Although the detection of a recoverin/rhodopsin kinase complex suggests that recoverin
may regulate rhodopsin kinase directly, thesefindingsdo
not rule out other interactions of recoverin, perhaps with
membranes or membrane proteins, that might also occur
and influence rhodopsin phosphorylation.
2.4. Molecule p26 (CAR-antigen). Molecule p26 was identified in screens for proteins that reacted with antisera
from human patients with retinas degenerating from
cancer-associated retinopathy (CAR) (Polans et al. 1991;
Thirkill et al. 1992). In retinas of CAR patients, photoreceptors are degraded through an autoimmune reaction
presumably stimulated by a tumor in a nonretinal tissue.
Sera from some CAR patients showed immunoreactivity
with a retina-specific protein of apparent size 26 kDa. The
protein was purified and a partial amino acid sequence
was determined. The sequence included three potential
Ca2+-binding sites and it was found to be homologous to
chicken visinin and identical to bovine recoverin. The
mechanisms by which some tumors induce production of
antibodies that recognize recoverin have not yet been
seriously addressed. However, the existence of homologs
of recoverin in other tissues raises the possibility that
proteins expressed in some tumors could induce the
production of antibodies that cross-react with retinal
recoverin.
2.5. Recoverin homologs in other tissues. Recoverin immunoreactivity has been detected in both rods and cones
(Dizhoor etal. 1991; Milam etal. 1992). However, immunological studies using a variety of antibodies raised
against recoverin and against homologs from different
animal species have not yet shown whether or not rods
and cones express the same or different forms of recoverin
(Polans et al. 1993).
A variety of recoverin homologs including neurocalcin,
hippocalcin, and VILIP from nonretinal tissues have also
been identified. Neurocalcin has been detected in bovine
brain (Okazaki et al. 1992) and in retinal amacrine cells
and ganglion cells (Nakano et al. 1992). VILIP was detected in chicken brain and retina (Lenz et al. 1992) and
hippocalcin was detected in rat hippocampus (Kobayashi
et al. 1992). Functional studies for these recoverin homologs have not been reported.
3. Post-translational modifications of recoverin
Recoverin expressed in E. coli was found to have properties that are quite different from the properties of recov-
Hurley: Vertebrate phototransduction
erin purified from bovine retinas (Ray et al. 1992). For
example, the fluorescence emission spectrum of bovine
retinal recoverin red shifts when the concentration of
Ca 2+ is raised from 70 nM to 10 JJIM while the emission of
recombinant recoverin undergoes only a minor change in
intensity. Furthermore, the mobilities of retinal and recombinant recoverins are quite different in native gel
electrophoresis and in isoelectric focusing (Lambrecht &
Koch 1992; Ray et al. 1992).
To investigate the cause of these differences, Dizhoor
et al. (Dizhoor et al. 1992) used electro-spray mass spectrometry to examine recoverin purified from bovine retinas for post-translational modifications. The mass of
retinal recoverin was found to be 207.7 atomic mass units
larger than the mass of recombinant recoverin. This
difference is consistent with the amino-terminal methionine of recoverin being replaced by a short chain fatty
acyl residue such as a myristoyl group. This finding is
consistent with the consensus sequence for N-terminal
myristoylation present at the recoverin amino-terminus.
Closer examination of the recoverin amino-terminus by
mass spectrometric analyses of proteolytic fragments revealed four different types of ainino-termini. The N-terminal glycine of recoverin was found linked to either 12:0;
14:2 cis, cis A5A8; 14: lcis A5 or 14:0 fatty acyl residues.
The identities of the fatty acyl residues and the positions
and configurations of the double bonds were confirmed
by a variety of methods including gas phase chromatography, ozonolysis, and tandem mass spectrometry. Similar
heterogeneity was also detected at the amino-terminus of
another photoreceptor protein, the transducin a subunit
(Neubert et al. 1992). The functional significance of photoreceptor protein heterogeneity is unknown but it is
currently being investigated.
The functional significance of the fatty acyl residue on
the recoverin amino-terminus was investigated by comparing the biochemical properties of myristoylated and
nonacylated recoverin expressed in E. coli (Ray et al.
1992). Myristoylated recoverin was produced in E. coli by
coexpressing recoverin with yeast N-myristoyl transferase and myristic acid. In addition to differences in
fluorescence emission and electrophoretic mobility, nonacylated recoverin has a significantly higher affinity for
Ca 2 + than acylated recoverin. Affinities of acylated and
nonacylated recoverins for membranes were also investigated. Kawamura and Murakami (1991) had shown previously that S-modulin, a recoverin homolog from frog
photoreceptors, binds to photoreceptor membranes only
in the presence of >lu,M Ca 2+ . Ca 2+ also promotes
binding of recoverin to rod outer segment (ROS) membranes (Dizhoor et al. 1993; Zozulya & Stryer 1992).
Myristoylated recombinant recoverin was found to bind
to ROS membranes more efficiently than nonacylated
recombinant recoverin. These findings suggested that the
fatty acid on the recoverin amino-terminus may serve as a
Ca 2+ -dependent membrane anchor. To test this model,
recoverin was prepared with a [3H] myristic acid residue
at its amino-terminus. The labeled recoverin, either in its
Ca 2+ -liganded form or its Ca 2+ -free form, was then exposed to trypsin under nondenaturing conditions. The
labelled amino-terminus of Ca 2+ -free recoverin was not
cleaved by trypsin. The labelled amino-terminus of Ca 2+ recoverin was rapidly cleaved suggesting that Ca 2+ bind-
ing to recoverin induces a conformational change that
exposes the amino-terminus. Recoverin lacking a tetrapeptide from its amino-terminus failed to bind to ROS
membranes. These findings suggest that Ca 2+ binding
frees the hydrophobic acylated amino-fatty acid residue
to make it accessible for membrane interactions. The site
on the membranes with which recoverin interacts has not
been identified.
4. Recoverin and guanylyl cyclase
Guanylyl cyclase in rod outer segment homogenates is
active only at concentrations of free Ca 2+ less than approximately - 2 0 0 u,M. Koch and Stryer (1988) found that
a soluble factor required for the Ca 2+ -sensitivity of guanylyl cyclase could be eluted from rod outer segment
membranes. When recoverin was first isolated and characterized (Dizhoor et al. 1991) it was apparent that it had
properties suggesting that it might regulate guanylyl
cyclase. Preparations of purified recoverin were assayed
and were found to stimulate guanylyl cyclase in a Ca 2+ sensitive manner. These results together with findings
from another laboratory (Lambrecht & Koch 1991) led to
the conclusion that recoverin was the soluble factor that
imparted Ca 2+ -sensitivity to guanylyl cyclase. However,
the following recent findings suggest that recoverin is not
the soluble factor that stimulated guanylyl cyclase in the
original recoverin preparations (Hurley et al. 1993).
1. Reconstitution experiments using purified recombinant recoverin showed that neither myristoylated nor
nonmyristoylated recombinant recoverin stimulate guanylyl cyclase.
2. Highly purified recoverin isolated from bovine retinas by several fractionation methods does not stimulate
photoreceptor guanylyl cyclase.
3. Recoverin dissociates from photoreceptor membranes
under conditions that stimulate membrane-associated
guanylyl cyclase activity. The Ca 2+ -dependent membranebinding properties of recoverin appear to be inconsistent
with a role in guanylyl cyclase regulation.
4. Recoverin separates from other fractions containing
guanylyl cyclase stimulatory activity during several fractionation procedures. The most highly purified fractions
of guanylyl cyclase stimulatory activity contain nearly
undetectable amounts of recoverin.
Recent observations from another laboratory also suggest
that recoverin does not stimulate guanylyl cyclase activity. Gray-Keller et al. (1993) have shown that purified
recoverin dialyzed into rod cells slows recovery. These
findings are inconsistent with stimulation of guanylyl
cyclase by recoverin.
5. Summary
Recoverin is a 23 kDa Ca 2+ -binding protein specifically
found in vertebrate photoreceptor cells. The role of recoverin in phototransduction may be to increase photosensitivity by prolonging the lifetime of photolyzed rhodopsin in darkness when intracellular Ca 2+ concentrations
are high. The Ca 2+ -bound form of recoverin appears to
inhibit deactivation of photolyzed rhodopsin by phos-
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
427
Hurley: Vertebrate phototransduction
phorylation. Following illumination, lowered intracellular Ca 2 + levels may promote recovery by allowing more
rapid phosphorylation and inactivation of rhodopsin. Recoverin homologs in other tissues may impart Ca 2 + sensitivity to other signalling mechanisms in a similar
"
428
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
ACKNOWLEDGMENTS
I wish to thank all of the members of my laboratory for many
stimulating discussions. I wish to particularly thank Alexander
M. Dizhoor and Jason Chen, who are primarily responsible for
man
y °luthe f m ^ s f r o m "iy laboratory discussed in this
review. Their work was supported by grant ROl EYO6641 from
the National Eye Institute.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18, 429-440
Printed in the United States of America
Do the calmodulin-stimulated
adenylyl cyclases play a role in
neuroplasticity?
Zhengui Xia, Eui-Ju Choi, and Daniel R. Storm
Department of Pharmacology, University of Washington, Seattle, WA 98195
Electronic mall: dstorm@u.washington.edu
Christine Blazynski
Department of Biochemistry and Molecular Biophysics, Washington
University, School of Medicine, St. Louis, MO 63110
Abstract: Evidence from invertebrate systems including Aplysia and Drosophila, as well as studies carried out with mammalian
brain, suggests that Ca 2+ -sensitive adenylyl cyclases may be important for long-term synaptic changes and learning and memory.
Furthermore, some forms of long-term potentiation (LTP) in the hippocampus elevate cyclic AMP (cAMP) signals, and activation of
adenylyl cyclases and cAMP-dependent protein kinase may be required for late stages of LTP. We propose that long-term changes in
neurons and at synapses may require synergism between the cAMP and Ca 2 + signal transduction systems which regulates
transcription and synthesis of specific proteins required for long-term synaptic changes. During LTP, protein kinase C is activated
and intraccllular Ca 2 + increases. We hypothesize that the calmodulin (CaM)-regulated adenylyl cyclases may be activated during
LTP because of increases in intracellular Ca 2 + , release of free CaM from neuromodulin, activation by protein kinase C, release of
neurotransmitters, or a combination of these events. Synergistic activation of CaM-sensitive adenylyl cyclases may produce a robust
or prolonged cAMP signal required for transcriptional control. Furthermore, the coupling of the Ca 2 + and cAMP systems may
provide positive feedback regulation of Ca 2 + channels by cAMP-dependent protein kinase.
Keywords: adenylyl cyclase; calcium; calmodulin; cAMP; long-term potentiation; neuroplasticity
1. Introduction
An area of intense interest in neurobiology is the molecular mechanism underlying short-term and long-term
adaptive changes in synaptic function. Several regulatory
systems have been implicated in neuroplasticity including the cyclic AMP (cAMP) and the Ca 2+ signal transduction systems. Ca 2 + , calmodulin (CaM), CaM-sensitive
protein kinases, adenylyl cyclases have all been implicated in long-term adaptive responses in neurons and
synaptic plasticity. The purpose of this target article is to
present a hypothesis that the CaM-sensitive adenylyl
cyclases may play a crucial role in long-term adaptive
responses in brain and to present molecular models to
explain the role of these enzymes in these processes.
Evidence supporting this hypothesis is derived from
studies of the invertebrate and mammalian CaM-sensitive
adenylyl cyclases, as well as data concerning the role of
the cAMP signal transduction system for LTP in brain.
In mammalian brain, adenylyl cyclase activity is regulated by neurotransmitter and hormone receptors
coupled to the enzyme through the G regulatory proteins, Gs and Gf (reviewed by Ross & Gilman 1980), as
well as by intracellular free calcium (reviewed by Cheung
& Storm 1982). Protein phosphorylations catalyzed by the
© 199S Cambridge University Press
0140-S25X/95 S9.00+.10
cAMP-dependent protein kinase regulate several important aspects of neuronal function including ion channel
activity, gene expression, and neurotransmitter synthesis
(reviewed by Krebs & Beavo 1979; Nairn et al. 1985;
Nestler & Greengard 1983). Furthermore, cAMP has
been implicated in the regulation of synaptic plasticity
and may play an important role in mechanisms underlying learning and memory (reviewed by Dudai 1988;
Kandel & Schwartz 1982).
Elucidation of molecular mechanisms for neuroplasticity requires a multidisciplinary approach with electrophysiology as the bridging discipline between signal
transduction molecular biology and the behavioral sciences. A useful electrophysiological model for neuroplasticity is LTP which occurs in many areas of brain
including the hippocampus. The relationship between
LTP and behavior is still controversial. However, both
spatial learning in rats and LTP in the hippocampus are
blocked by N-methyl-D-aspartate (NMDA) antagonists
(Davis et al. 1992) demonstrating a possible relationship
between these two phenomena. Other evidence suggesting a correlation between LTP and learning comes from
gene disruption studies which have demonstrated that
mice lacking the alpha form of CaM kinase II (Silva et al.
1992a; 1992b) or the fyn-tyrosine kinase (Grant et al.
429
Xia et al.: Neuroplasticity
1992) are deficient in LTP and spatial learning. On the
other hand, PKC-gamma mutant mice showed impaired
LTP in the hippocampus but can perform the hidden
platform test of the Morris water task indicating that LTP
induced by conventional tetanic stimulation is not essential for this specific type of learning (Abeliovich 1993a;
1993b). These mice were, however, deficient in the transfer test of the Morris water task which may be a more
reliable measure of spatial learning.
Although many forms of LTP decay over several hours,
long-lasting LTP (L-LTP) which is produced by multiple trains of high frequency stimulation can last hours
or even days depending upon the stability of the preparation (reviewed in Matthies et al. 1990) and is sensitive
to inhibitors of transcription and translation (Frey et
al. 1988; Grecksch & Matthies 1980; Krug et al. 1984;
Squire & Davis 1981). Various forms of LTP in the hippocampus require entry of Ca2+ through NMDA receptors or voltage-gated Ca2+ channels (reviewed in
Kauer et al. 1988; Nicoll et al. 1988) and an increase in
intracellular Ca2+ can induce LTP (Malenka et al. 1988).
Consequently, Ca2+-sensitive adenylyl cyclases may
be stimulated when Ca+ channels are activated during
LTP.
2. The Aplysia adenylyl cyclase system and the
gill withdrawal reflex
One of the most interesting systems demonstrating the
possible importance of Ca2+-sensitive adenylyl cyclases
for learning and memory is the gill withdrawal reflex of
the invertebrate Aplysia (Byrne 1987; Castellucci & Kandel 1976). In this system, the conditioned stimulus, a
weak touch to the siphon, elicits a minimal defensive
response by withdrawing the gill. In contrast, the unconditioned stimulus, a shock to the tail, produces a strong
withdrawal reflex. If the conditioned stimulus is paired
with the unconditioned stimulus the animal learns to
associate touch with the shock and shows a strong withdrawal response to touch alone. The length of the memory
for this response depends upon the number of stimuli
applied (Frost et al. 1985) and can last for days when
4 or 5 noxious signals are administered (Castellucci et al.
1989). An extensive analysis of this system has led to the
conclusion that the conditioned stimulus results in an
increase in intracellular Ca2+ within the sensory neuron, and that the unconditioned stimulus results in the
release of serotonin into the same cell (Abrams et al.
1991). It has been proposed that paired activation of the
Aplysia adenylyl cyclase by Ca2+/CaM and serotonin
results in a synergistic activation of adenylyl cyclase
which may be required for long-term memory. Since
long-term facilitation can be induced by the injection of
cAMP into the presynaptic sensory neuron (Schacher et
al. 1988; Scholz & Byrne 1988) and blocked by inhibitors
of protein kinase A (Ghirardi et al. 1992), it is very likely
that cAMP protein kinase plays a central role in long-term
facilitation in Aplysia. Furthermore, long-term facilitation in this system is sensitive to inhibitors of RNA and
protein synthesis (Castellucci et al. 1989; Montarolo et al.
1986) suggesting that cAMP-mediated transcription may
be required for long-term facilitation in Aplysia (Kaang et
al. 1993).
430
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
The gene(s) for the Aplysia adenylyl cyclase(s) have not
been cloned and their relationship to the cloned mammalian adenylyl cyclases has not been determined. However, Ca2+/CaM-sensitive adenylyl cyclase activity is
present in Aplysia membranes and Yovell et al. (1992)
have demonstrated synergism for activation of Aplysia
adenylyl cyclase by Ca2+ and serotonin when brief pulses
of Ca2+ followed by serotonin were paired. Synergistic
activation of this adenylyl cyclase by Ca2+ and Gs is quite
novel in that it was only observed with sequential applications of Ca2+ and serotonin and is not observable in
steady-state assays. Although this synergism was relatively weak and its physiological significance remains to
be established, the small increment in cAMP signal may
be functionally important because of the signal amplification that is inherent in the cAMP signal transduction
pathway. Synergistic activation of the type I adenylyl
cyclases by Ca2+ and Gs in vivo is observable in steadystate assays and does not require ordered administration
of Ca2+ and activators of Gs (Wayman et al. 1994). Consequently, the Aplysia adenylyl cyclase may be quite distinct from the mammalian type I and type III CaMsensitive adenylyl cyclases.
If cAMP-regulated transcription plays a major role in
long term facilitation in Aplysia neurons, then cAMP
signals generated at synapses must either diffuse to the
cell body in order to affect transcription in the nucleus,
or a secondary signal arising from the initial synaptic
cAMP signal must reach the nucleus. This is of particular
concern in neurons because of substantial distances required for transport or diffusion of cAMP along neurites.
Recently, this question has been directly addressed in
Aplysia neurons by monitoring gradients of cAMP produced in response to serotonin stimulation of adenylyl
cyclase (Bacskai et al. 1993). Bath application of serotonin produced an extensive cAMP gradient between the
processes and the cell body which was consistent with
the diffusion of cAMP from neurite tips to the cell body
of the neuron. Nuclear translocation of the catalytic subunit of cAMP-dependent protein kinase was relatively
slow and probably only occurs after prolonged or enhanced cAMP signals. In contrast to other cAMPregulated phenomena such as regulation of metabolism
and ion channel function, which are rapid short term
responses, cAMP regulation of transcription in neurons
may require strong or persistent activation of adenylyl
cyclase activities.
3. The Drosophila rutabaga learning mutant
Other evidence that Ca2+-sensitive adenylyl cyclases
may be important for synaptic plasticity in invertebrates
has come from studies of the Drosophila learning mutant, rutabaga. Rutabaga is an X-linked recessive mutant
that is deficient in associative learning (Dudai & Zvi
1984, 1985; Livingston 1985; Livingston et al. 1984). In
contrast to wild type Drosophila, the rutabaga fly lacks
Ca2+/CaM-sensitive adenylyl cyclase activity. The gene
for an adenylyl cyclase similar to the type I adenylyl
cyclase maps within a region on the X chromosome that
includes the rut locus and a single point mutation in this
gene is sufficient to destroy all enzyme activity (Levin
et al. 1992). Feany (1990) has proposed that calcium re-
Xia et al.: Neuroplasticity
sponsiveness, rather than the overall cAMP synthesis
may be the crucial component of adenylyl cyclase activity required for associative learning in Drosophila. In
rutabaga larvae, voltage clamp analysis of neuromuscular transmission indicated deficient synaptic facilitation and post-tetanic potentiation (Zhong & Wu 1991).
The rutabaga mutant has provided convincing evidence
that the type I adenylyl cyclase is important for associative learning in invertebrates and synaptic facilitation.
4. The family of mammalian adenylyl cyclases
The existence of distinct CaM-stimulated and CaMinsensitive adenylyl cyclases in brain was first demonstrated by the separation of these two forms of the enzyme
from bovine brain using CaM-Sepharose affinity chromatography (Westcott et al. 1979). The CaM-sensitive adenylyl cyclase activity absorbed to CaM-Sepharose in the
presence of Ca 2+ and was stimulated by Ca 2+ when
reconstituted with CaM. Half-maximal stimulation of the
enzyme occurred at 80 nM free Ca 2+ . The CaMinsensitive forms of adenylyl cyclase present in brain did
not absorb to CaM-Sepharose in the presence or absence
of Ca 2 + , and were not stimulated by Ca 2+ . Furthermore,
polyclonal and monoclonal antibodies have been isolated
that distinguish between the CaM-sensitive and CaMinsensitive adenylyl cyclase in brain providing further
evidence for separate adenylyl cyclases (Mollner &
Pfeuffer 1988; Mollner et al. 1991; Rosenberg & Storm
1987a). The catalytic subunit of a CaM-sensitive adenylyl
cyclase was purified to homogeneity from bovine brain
using CaM-Sepharose and Forskolin-Sepharose affinity
chromatography (Minocherhomjee et al. 1987; Smigel et
al. 1986; Yeager et al. 1985). Characterization of the
purified CaM-sensitive adenylyl cyclase from brain indicated that it is a glycoprotein that interacts directly with
CaM (Minocherhomjee et al. 1987). Furthermore this
enzyme can couple to Gs and beta-adrenergic receptors
(May et al. 1985; Rosenberg et al. 1987b) as well as Gf and
muscarinic receptors (Dittman et al. 1994; Tota et al.
1990). The expression of the type I adenylyl cyclases in
the insect/Bacculovirus system and the development of
new strategies for its purification from these cells offers
great promise for direct characterization of the protein
(Taussig et al. 1993).
cDNA clones for the type I adenylyl cyclase have been
isolated from bovine brain (Krupinski et al. 1989) and
human brain cDNA libraries (Villacres et al. 1992). To
date, cDNA clones for eight distinct ACs have been
published (Bakalyar & Reed 1990; Cali et al. 1994; Feinstein et al. 1991; Gao & Gilman 1991; Ishikawa et al. 1992;
Krupinski et al. 1989; Yoshimura & Cooper 1992). Although these enzymes share sequence homology, they
contain hypervariable regions and exhibit different regulatory properties. I-AC (Choi et al. 1992a; Tang et al.
1991), III-AC (Choi et al. 1992b), and VIII-AC (Cali et al.
1994) are stimulated by Ca 2+ and CaM in vitro whereas
II-AC, IV-AC, V-AC and VI-AC are not. The diversity of
this enzyme system undoubtedly reflects different mechanisms for regulation of cAMP levels in animal cells, and
the variety of physiological processes that are regulated
by intracellular cAMP.
5. Regulation of the type I and III adenylyl
cyclases by calcium and CaM
5.1. TVpe III adenylyl cyclase. Modulation of adenylyl
cyclase activity by Ca 2+ has been demonstrated in several
tissues including brain and retina and it has been proposed that cAMP levels may be controlled by fluctuations
in intracellular free Ca 2 + . Most mammalian tissues contain mixtures of adenylyl cyclases, and the Ca 2+ sensitivity of specific forms of adenylyl cyclase has only recently been addressed. The type III adenylyl cyclase was
expressed in human kidney 293 cells to determine if it is
stimulated by Ca 2+ and CaM (Choi et al. 1992b). In
isolated membranes, the type III enzyme was not stimulated by Ca 2+ and CaM in the absence of other effectors.
It was, however, stimulated by Ca 2+ through CaM when
the enzyme was concomitantly activated by forskolin (Fig.
IB). The concentrations of free Ca 2+ for half-maximal
stimulation of type III adenylyl cyclases was 5.0 (JLM Ca 2+
(Fig. 2B). The sensitivity of the type III adenylyl cyclase
to Ca 2+ in vivo has not been reported.
Toscano et al. (1979) isolated a partially purified adenylyl cyclase from bovine brain that was not stimulated by
GppNHp, NaF, or Ca 2 + /CaM. Sensitivity to these effectors was restored by incubation of the adenylyl cyclase
preparation with detergent solubilized membranes from
cerebral cortex. Reconstitution of Ca 2+ /CaM sensitivity
required the presence of guanyl nucleotides. The properties of this adenylyl cyclase are similar to the type III
adenylyl cyclase and these early studies showed synergistic activation of an adenylyl cyclase by Ca 2+ and Gs.
Is Ca 2+ regulation of the type III adenylyl cyclase at
concentrations of 5 JJLM Ca 2+ physiologically significant,
and what role could it play in signal transduction pathways in olfactory sensory neurons, retina, or brain? Free
Ca 2+ in many animal cells generally varies from less than
0.1 to 10 u,M. Local Ca 2+ concentrations at the membrane surface in neurons may increase to 100 |J,M or even
higher during action potentials (Smith & Augustine 1988).
Since the type III adenylyl cyclase is only activated at the
higher end of the free Ca 2+ range, and when the enzyme
is activated by other effectors, the enzyme may allow
Ca 2+ amplification of cAMP signals. For example, the
existence of cAMP-gated ion channels in neurons suggests that initial cAMP signals, generated through receptors coupled to adenylyl cyclase, may be further amplified
by increases in intracellular Ca 2 + . Thus the type III
adenylyl cyclase is able to integrate multiple signals and
may function as a "coincidence detector" (Bourne &
Nicoll 1993).
5.2. Type I adenylyl cyclase. CaM stimulates the type I
adenylyl cyclase activity in membranes from CDM8(IAC)-transfected 293 cells at a half-maximal concentration
of approximately 20 nM (Fig. 1). Similar CaM sensitivities
have been reported for the CaM sensitive adenylyl cyclase purified from bovine brain (Minocherhomjee et al.
1987) and the type I adenylyl cyclase expressed in insect
SF9 cells (Tang et al. 1991). Half-maximal stimulation of
type I adenylyl cyclase occurred at approximately 50 nM
free Ca 2+ (Fig. 2), which is consistent with the Ca 2+
sensitivity of the CaM-stimulated adenylyl cyclase isolated from bovine brain calculated from the data of Westcott et al. (1979). These Ca 2+ dependencies were deterBEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
431
Xia et al.: Neuroplasticity
oe
E
o
a.
o
a
eu
<
A.
<
[CaM] (uM)
[CaM] (nM)
B
25
• forskolin
rO
» .X Id,
0.1
1
[CaM] (nM)
10
Figure 1. The effect of CaM on either type I (A) or type III (B)
adenylyl cyclase activities transiently expressed in human 293 cells.
Human 293 cells were transfected with control CDM8 vector,
CDM8(I-AC) to express the type I adenylyl cyclase or CDM8(III-AC)
to express the type III adenylyl cyclase. Cell membranes prepared
from transfected cells were washed with buffer A containing 1 mM
EGTA to remove endogenous CaM, and then assayed for adenylyl
cyclase activity as a function of CaM concentration. A; the effect of
CaM on control (O) or type I adenylyl cyclase activity (•). The assay
was performed in the presence of 1.9 u,M free Ca 2 + and varying
concentrations of CaM. Control activity was the adenylyl cyclase
activity in cell membranes from CDM8-transfected 293 cells. B; the
effect of CaM on type HI adenylyl cyclase activity in the absence (O) or
in the presence of 10 u,M forskolin (•). The enzyme assay was
performed in the presence of 30 |xM free Ca 2 + and varying concentrations of CaM. Adenylyl cyclase activities due to the type III enzyme
were calculated from data shown in panel C by subtracting adenylyl
cyclase activities in control cells from those of cells transfected with
CDM8(III-AC). In panel C, cell membranes from control 293 cells (X,
A) or cells transfected with CDM8(III-AC) (O, • ) were assayed for
adenylyl cyclase activity with varying concentrations of CaM and 30
(xM Ca 2 + in the absence (X, O) or presence of 10 u,M forskolin (A, • ) .
Data are from Choi et al. 1992a.
mined using EGTA/Ca 2+ buffers. Because of the uncertainties associated with calculating free Ca 2+ by this
method, these data are only estimates of the Ca 2+ dependence of the enzyme (Yovell et al. 1992).
It has been generally assumed that CaM-sensitive adenylyl cyclase can function to couple increases in intracellular free Ca 2+ to elevations in cAMP in vivo. The
purified type I adenylyl cyclase and the enzyme in membrane preparations is stimulated by Ca 2+ and CaM.
However, it was important to demonstrate that increases
in intracellular free Ca 2+ can actually stimulate the type I
adenylyl cyclase in intact cells. Therefore, we expressed
the enzyme in human 293 cells and examined the effect of
a Ca 2+ ionophore, A23187, and extracellular Ca 2+ on
intracellular cAMP (Choi et al. 1992a). Human 293 cells
were selected for these studies because their endogenous
adenylyl cyclase activity is quite low and insensitive to
extracellular Ca 2 + and the Ca 2+ ionophore A23187 (Fig.
3A). In the presence of 2 mM extracellular Ca 2+ , the
432
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
intracellular cAMP levels of control cells, transfected
with the CDM8 vector alone, were unaffected by addition
of A23187. In contrast, intracellular cAMP in 293 cells
stably expressing the type I adenylyl cyclase increased
approximately 16-fold with addition of 10 JJLM A23187
(Fig. 3A). Under these conditions, the intracellular free
Ca 2+ increased to 1.0 (xM. The increase in intracellular
cAMP stimulated by A23187 depended upon the concentration of Ca 2+ applied (Fig. 3B). Elevated cAMP was
detectable within a few minutes after addition of 10 u.M
A23187 and 2 mM Ca 2+ (Fig. 3C).
The data described above indicated that intracellular
Ca 2+ can stimulate the type I adenylyl cyclase activity in
vivo. Ca 2+ stimulation of the enzyme in vivo may be due
to direct interactions of the enzyme with Ca 2 + and CaM,
or indirect mechanism involving stimulation of the enzyme by Ca 2+ -activated protein kinases. We have made
several point mutations within the calmodulin binding
domain to determine if the Ca 2+ sensitivity of the enzyme
Xia et al.: Neuroplasticity
I
OS
e
Q.
1
1
£
a.
20
40
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80
[calcium ton] (u.M)
0.2
0.4
0.6
3
[calcium ion] (uM)
150
1
£
<
20
40
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80
[calcium ion] (|iM)
can be modified by mutagenesis (Wu et al. 1993). The
catalytic activities of the mutant enzymes were comparable to wild-type type I adenylyl cyclase. Ca 2+ and CaM
stimulation was abolished by substitution of Phe-503 with
Arg-503 (FR-I-AC). Stimulation of type I adenylyl cyclase
activity in vivo by intracellular Ca 2+ was also greatly
diminished with the Arg-503 mutant indicating that Ca 2+
stimulation of the enzyme in vivo is due primarily to
direct interactions with CaM and Ca 2+ . These data demonstrated that the Ca 2+ sensitivity of this enzyme can be
modulated by point mutagenesis within the putative
calmodulin binding domain, and indicate that the enzyme
can be directly regulated by Ca 2+ and CaM, in vivo.
Kidney 293 cells contain various receptors that can
couple directly or indirectly to adenylyl cyclases, including muscarinic receptors. Therefore, we examined the
influence of carbachol, a muscarinic agonist, on the intracellular cAMP levels of 293 cells expressing the type I
Figure 2. The effect of Ca 2 + on type I (A) or type III (B)
adenylyl cyclase activities expressed in human 293 cells. Cell
membranes from human 293 cells transfected with control
CDM8 vector, CDM8(I-AC) to express the type I adenylyl
cyclase, or CDM8(HI-AC) to express the type III adenylyl cyclase
were examined for adenylyl cyclase activity as a function of free
Ca 2 + concentration by varying concentrations of CaCl 2 in the
presence of 0.2 mM EGTA in the assay. A; the effect of Ca 2 + on
control (O) or type I adenylyl cyclase activity (•). The enzyme
assay was performed in the presence of 2.4 u,M CaM and various
concentrations (0—3.7 |J.M) of free Ca 2 + . B; the effect of Ca 2 + on
type III adenylyl cyclase activity in the absence (O) or in the
presence of 100 p-M GppNHp (•). The enzyme assay was performed in the presence of 2.4 \xM CaM and various concentrations (0-74 u,M) of free Ca 2 + . Type III adenylyl cyclase activity
was calculated froin data shown in panel C by subtracting adenylyl cyclase activities in control cells from those of cells transfected with CDM8(III-AC). In panel C, cell membranes from
control cells (A, • ) or cells transfected with CDM8(III-AC) (O,
• ) were assayed for adenylyl cyclase activity with varying concentrations of free Ca and 2.4 u,M CaM in the absence (A, O) or
presence of 100 p.M GppNHp (A, • ) . Data are from Choi et al.
1992b.
adenylyl cyclase (Choi et al. 1992a). Carbachol stimulated
intracellular cAMP levels approximately 3-fold in 293
cells stably expressing type I adenylyl cyclase, but was
without significant effect on cAMP in control cells (Fig.
4A). Maximal stimulation by carbachol occurred at approximately 100 u,M, and this increase in cAMP was
inhibited by the muscarinic antagonist, atropine. No
carbachol stimulation of intracellular cAMP was seen
without forskolin, even when IBMX was present. The
requirement for forskolin reflects the low sensitivity of
this assay for cAMP and more sensitive assays using a
CRE-beta-galactosidase reporter construct can detect
Ca 2+ -stimulated cAMP signals in 293 cells without the
presence of forskolin or phosphodiesterase inhibitors (Impey & Storm, unpublished observations).
These data illustrated that the type I adenylyl cyclase
can be regulated by muscarinic receptors in vivo either
by direct coupling through a G regulatory protein or
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
433
Xia et al.: Neuroplasticity
0.01
0.1
10
100
[A23187J (n.M)
indirectly by mobilization of intracellular Ca 2 + . The latter explanation seems most likely since carbachol had
no effect on the basal, CaM-stimulated, or forskolinstimulated activities of type I adenylyl cyclase activity in
isolated membranes. Furthermore, we analyzed the influence of carbachol on intracellular free Ca 2+ in 293 cells
using fura-2. One mM carbachol increased free Ca 2+ from
a baseline of 40 nM to 140 nM and this increase was
blocked by 100 |xM BAPTA/AM, the intracellular Ca 2+
chelator. Furthermore, 100 u-M BAPTA/AM completely
inhibited carbachol-stimulated increases in intracellular
cAMP (Fig. 4B). We concluded that the type I adenylyl
cyclase can function to couple increases in intracellular
Ca 2+ to cAMP production in whole cells, and that the
enzyme can also be indirectly stimulated through muscarinic receptors by mobilization of intracellular free
Ca 2 + .
4000
1
10
100
1000
[carbachol] (p.M)
I
800
r
5000
Time (min)
2+
Figure 3. Ca
and A23187 stimulation of intracellular
cAMP levels in human 293 cells expressing the type I adenylyl
cyclase. A; cultured 293 cells expressing the type I adenylyl
cyclase (•) or control cells transfected with the CDM8 vector
(O) were treated with varying concentrations of A23187 in the
presence of 2 mM CaCl 2 for 30 minutes. B; 293 cells expressing
the type I adenylyl cyclase (•) or control cells (O) were treated
with varying concentrations of CaCl2 for 30 minutes in the
presence of 10 (JLM A23187. C; 293 cells expressing the type I
adenylyl cyclase ( • , A) or control cells (O, X) were treated for
various periods of time with 2 mM CaCl 2 and 10 |JLM A23187 in
the presence ( • , O) or absence (A, X) of one mM IBMX.
434
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
- carbachol
carbachol
(lmM)
carbachol
BAPTA/AM
(100 nM)
Figure 4. Carbachol stimulation of intracellular cAMP levels
in human 293 cells expressing the type I adenylyl cyclase. A;
control cells ( • , Mj or human 293 cells expressing the type I
adenylyl cyclase ( • , Wj were treated with varying concentrations of carbachol for 30 minutes in the absence ( • , • ) or
presence (H, ^ ) of 1.0 p,M atropine. One mM IBMX was
present in all assays. B; pretreatment of 293 cells for one hour
with BAPTA/AM at 100 (JLM completely blocked the increase in
intracellular cAMP caused by carbachol. Data are from Choi et
al. 1992a.
Xia et al.: Neuroplasticity
6. Synergistic regulation of type I adenylyl
cyclase by Ca 2 + and isoproterenol in vivo
Evidence from several studies has indicated that adenylyl
cyclase activity from various areas of mammalian brain
may be synergistically activated by CaM/Ca 2+ and Gs, or
Gs-coupled receptors (Choi et al. 1992a; Gnegy & Treisman 1981; Natsukari et al. 1990; Toscano et al. 1979).
However, other investigators have found that CaM and G
protein stimulation of AC activities are additive and not
synergistic (Piascik et al. 1981; Salter et al. 1981; Sano
1985). This apparent discrepancy reflects the regulatory
diversity of the adenylyl cyclases, their distribution
within brain, and the different preparations used in these
studies.
Although the type I adenylyl cyclase is stimulated by
Ca 2 + in vivo, we have discovered that it is not stimulated
by Gs-coupled receptors in vivo unless it is also activated
by Ca 2+ /CaM (Wayman et al. 1994). We examined the
sensitivity of I-AC expressed in HEK-293 cells to isoproterenol or glucagon when intracellular Ca 2+ was elevated. The cells used in this study were stable transfectants expressing type I adenylyl cyclase and glucagon
receptors. The Ca 2+ ionophore A23187 stimulated the
enzyme approximately three-fold, isoproterenol did not
stimulate the enzyme, but the combination of the two
stimulated adenylyl cyclase activity 13-fold in vivo. Similarly, glucagon did not stimulate the enzyme but the
combination of A23187 and glucagon activated the enzyme 90-fold. This phenomenon was not observed with a
mutant enzyme (FR-I-AC) that is insensitive to Ca 2+ and
CaM. Therefore, I-AC may couple Ca 2+ and neurotransmitter signals to generate optimal cAMP levels, a
property of the enzyme that may be important for learning and memory in mammals. We have also demonstrated
that the expression of type I adenylyl cyclase in HEK-293
cells allows Ca 2+ to stimulate reporter gene activity
mediated through the CRE response element (Impey et
al. 1994). Simultaneous activation by Ca 2+ and isoproterenol caused synergistic stimulation of cyclic AMP
response element (CRE)-mediated transcription in
HEK-293 cells and cultured neurons.
7. Regulation of the CaM-regulated adenylyl
cyclases by protein kinase C
7.1. Activation of the type I and type III adenylyl cyclases
by activators of protein kinase C. Protein kinase C (PKC)
is activated during many forms of LTP and phorbol esters
and other activators of PKC can also affect intracellular
cAMP levels in various tissues and cultured cells. Therefore, the effect of phorbol esters on the activity of the type
I and type III adenylyl cyclases in whole cells has been
examined using stably transfected 293 cells expressing
either enzyme (Choi et al. 1993). TPA markedly enhanced
the forskolin responsiveness of the type I and type III
adenylyl cyclases expressed in kidney 293 cells. The effect
of 12-O-tetradecanoylphorbol 13 acetate (TPA) on the
activity of the CaM-sensitive adenylyl cyclases was not
mediated through increases in intracellular free calcium.
Jacobowitz et al. (1993) have also examined the sensitivities of various adenylyl cyclases to phorbol esters by
transient expression of the enzymes in 293 cells. Their
data indicated that the type II adenylyl cyclase was
particularly sensitive to phorbol esters and that types IV,
V, and VI showed modest stimulations upon phorbol
myristic acid (PMA) treatment. Since PKC is activated
during some forms of LTP, cAMP levels may be regulated
during LTP.
7.2. Does protein kinase C regulate adenylyl cyclase
activities in the brain by controlling the levels of free
CaM? The activity of the CaM-sensitive adenylyl cyclases
depends on the concentrations of free intracellular Ca 2+
and free CaM in neurons. We have described the biochemical properties of a neurospecific CaM binding protein, neuromodulin (GAP-43), that may regulate the
levels of free CaM present in neurons and consequently
may play a key role in CaM-regulated processes in neurons. The neurobiology of neuromodulin has recently
been reviewed (Benowitz & Routtenerg 1987; Liu &
Storm 1990; Skene 1989).
Neuromodulin is neurospecific and bovine brain contains approximately 60 pmol neuromodulin/ mg of membrane protein, making it the most abundant CaM-binding
protein in brain with a concentration comparable to CaM
itself (Cimler et al. 1985). Neuromodulin is a prominent
constituent of neuronal growth cone membranes, comprising up to one percent of the total growth cone membrane protein and it is transported by rapid axoplasmic
transport (Pfenninger et al. 1983; Skene et al. 1986).
Neuromodulin has been implicated in several neuromodulatory roles including axon growth and synaptic
plasticity and it has been studied extensively as a potential
mediator of synapse formation and modification. Phosphorylation of neuromodulin by PKC has been correlated
with synaptic LTP (Nelson & Routtenberg 1985).
On the basis of the affinity of neuromodulin for CaM
under physiologically relevant ionic strength (Alexander
et al. 1987) and the concentrations of these two abundant
proteins in brain, we predict that the majority of CaM will
be complexed to neuromodulin in vivo. Neuromodulin is
phosphorylated by PKC with a phosphate: protein molar
ratio of 1:1.
CaM decreases the rate of phosphorylation of neuromodulin by PKC, and phosphorylation prevents neuromodulin binding to CaM (Alexander et al. 1987). Phosphorylation of neuromodulin by PKC inhibits CaM
binding because the phosphorylation site is within the
CaM binding domain of the protein (Apel et al. 1990). In
addition, phosphoneuromodulin is an excellent substrate
for calcineurin, the CaM-stimulated phosphatase which is
particularly abundant in brain (Liu & Storm 1989). On the
basis of these observations, we have proposed that neuromodulin may bind and localize CaM at specific sites
within neurons and that PKC may regulate the levels of
free CaM available in neurons for stimulation of various
enzymes, including adenylyl cyclases and protein kinases
(Andreasen et al. 1983; Alexander et al. 1987; Lieu &
Storm 1990).
Evidence that PKC may regulate the levels of free CaM
in neurons or neuronal-like cells in vivo comes from
several different studies. For example, activation of PKC
in PC 12 cells increased the levels of free CaM, presumably because of the phosphorylation of neuromodulin or
related proteins (MacNicol & Schulman 1992). Mangels &
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
435
Xia et al.: Neuroplasticity
Gnegy (1990) demonstrated that the ratio of cytosolic to
membrane-associated CaM increases in neuroblastoma
cells when PKC is activated through muscarinic receptors
or by phorbol esters. Because these cells contain
membrane-associated neuromodulin, the redistribution
of CaM may be due to PKC phosphorylation of neuromodulin. If this hypothesis is true, then CaM may be
liberated during LTP by activation of PKC and phosphorylation of neuromodulin.
8. Tissue distribution of the type I and type II
adenylyl cyclases
8.1. Tissue distribution of the enzymes. Although the type
III adenylyl cyclase was originally cloned from an olfactory cDNA library and is greatly enriched in this tissue,
type III adenylyl cyclase mRNA is also expressed in brain,
spinal cord, adrenal medulla, adrenal cortex, heart
atrium, aorta, lung, retina, 293 cells, and PC-12 cells (Xia
et al. 1992). In contrast, the type I adenylyl cyclase is
neurospecific (Xia et al. 1993). The only bovine tissues
showing a positive signal for type I mRNA were brain,
retina, and adrenal medulla (Fig. 5). The weak signal seen
with whole adrenal was most likely due to adrenal
medulla since the cortex gave a negative signal. In addition to the expected transcript seen at approximately 11.7
kb, retina also contained a second transcript at 6.5 kb.
Several cultured cell lines including neuroblastoma cell
l l l l l
s 1 1 11
1 1 lk
ll
i
j I I ! I I If
I111
Aii
Figure 5. Northern analysis of the type I-sensitive adenylyl
cyclase using mRNA from various bovine tissues. Two micrograms of poly (A)+ selected RNA samples were electrophoresed
to a 1.2% agarose/formaldehyde gel. The blots were hybridized
with an alpha [32P]dCTP-Iabeled cDNA probe 3C that is specific
for the bovine type I adenylyl cyclase. The 0.24-9.5 kb RNA
ladder from BRL was used as the molecular weight standard.
Poly (A)+ selected RNA was isolated from various bovine tissues. Data are from Xia et al. 1993.
436
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
N1E-115, neuroglio hybridoma cell NG-108, rat glioma
36B-10 cell, and PC-12 cells were also analyzed and found
not to express mRNA for the type I adenylyl cyclase. The
restricted expression of type I adenylyl cyclase mRNA in
neural tissues contrasts sharply with most of the other
mammalian enzymes which show fairly broad distribution
in both neural and non-neuronal tissues.
8.2. Distribution of type I adenylyl cyclase mRNA in rat
retina examined by in situ hybridization. The presence of
type I adenylyl cyclase mRNA in retina is consistent with
the Ca 2+ and CaM sensitivities of adenylyl cyclase activities reported for retina (Gnegy et al. 1984). The distribution of type I adenylyl cyclase mRNA in retina was
examined in more detail by in situ hybridization. Retina
cross sections from rat, rabbit, and bovine were analyzed
with a ^S-UTP labeled bovine riboprobe specific for the
type I adenylyl cyclase (Xia et al. 1991; 1993). Messenger
RNA for the type I adenylyl cyclase was detected in the
inner segment layer of the photoreceptors (IS), and in all
three nuclear layers of the neural retina (Fig. 6). The outer
nuclear layer (ONL) which contains rods and cones, the
inner nuclear layer (INL) which contains horizontal cells,
bipolar cells, and amacrine cells, and the ganglion cell
layer (GCL) all contained mRNA for type I adenylyl
cyclase. The intensity of the labeling was strongest in the
IS (the cytoplasm of photoreceptors) and the ONL.
8.3. Distribution of type I adenylyl cyclase mRNA in rat
brain examined by In situ hybridization. The distribution
of mRNA encoding the type I adenylyl cyclase in rat brain
was also examined by in situ hybridization (Xia et al.
1991). In situ hybridizations in adult rat brain revealed
high levels of type I adenylyl cyclase mRNA in specific
areas of brain including the hippocampal formation, neocortex, entorhinal cortex, cerebellum cortex, and the
olfactory system (Figs. 7 and 8). The dentate gyms in the
hippocampal formation showed very intense labeling
which appeared to be associated with the granule cell
layer. Moderately strong labeling was also evident in
association with the pyramidal cells in CA1, CA2, and
CA3 layers of the hippocampus. The data reported in
Figure 7 shows expression of mRNA for the CaMsensitive adenylyl cyclase in granule cells of the dentate
gyrus and in pyramidal cells of the hippocampus, providing the first evidence that the enzyme is expressed in
neurons.
These data illustrate that mRNA for the type I adenylyl
cyclase is not generally distributed throughout the brain,
suggesting that it does not play a general regulatory role
(e.g., in regulation of cell metabolism) and that it may be
important for specific neuronal functions. Messenger
RNA for the type I adenylyl cyclase is highly localized to
specific regions of brain, including those areas that show
LTP and have been implicated in learning and memory.
Although these data do not define the function of the type
I CaM-sensitive adenylyl cyclase, its mRNA distribution
is consistent with the proposal that this enzyme may be
important for learning and memory.
8.4. Disruption of the gene for the type I adenylyl cyclase
leads to deficiencies in spatial learning. Recently, we
evaluated the role of the type I adenylyl cyclase for
Xia et al.: Neuroplasticity
Mutant and wild type mice were analyzed for spatial
learning by the Morris water-maze task, a set of assays
that has been used to examine spatial learning in other
mutant mice deficient in specific genes. Both sets of
animals showed decreased escape latencies with training,
and there were no statistically significant differences in
the ability of the mutant and wild type mice to find the
visible or hidden platform. However, escape latencies in
the hidden platform task are a poor indicator of spatial
learning and even rodents with hippocampal lesions that
affect other forms of spatial learning can learn to find the
hidden platform in the Morris water-maze task (Davis et
al. 1992; Morris et al. 1982; 1986; Morris 1990). A better
indicator of spatial learning is the transfer test in which
the animal is trained to find the hidden platform at a
specific site in the pool. The platform is then removed,
and the number of times that a mouse swims across the
target area or the time in the target quadrant is quantitated. There were significant and reproducible differences in transfer test behavior between the mutant and
wild type mice. Wild type mice crossed the target area 6
± 0 . 4 times during a 60 sec trial whereas the mutant mice
crossed only 4 ± 0.4 times (p < .002). The difference in
transfer ability was also evident when the time in various
quadrants was analyzed. Only wild type mice showed a
bias for quadrant A. They spent 42% ± 3.0 of their time in
quadrant A searching for the platform. The mutant mice
showed no significant preference for quadrant A (27% ±
2.0) indicating an impaired ability in this specific task (p <
.001). These data illustrate that the mutant mice have a
significant and lasting place navigational impairment that
was dissociated from visual, motivational, or motor requirements of the test. We conclude that I-AC may play
an important role for signal transduction pathways underlying some forms of learning and memory.
Figure 6. Distribution of the type I Ca2+/CaM-sensitive adenylyl cyclase mRNA within various layers of bovine neural retina
examined by in situ hybridizations. Cross sections of bovine
eyecups hybridized with 35S-UTP labeled bovine type I adenylyl cyclase specific riboprobe 3C were treated with NTB2
emulsion for two weeks, and counterstained with cresyl violet
acetate. A; light microscope photomicrograph; B; phase contrast
photomicrograph; C; dark field photomicrograph. Abbreviations: IS, inner segment layer of the photoreceptor cells; ONL,
the outer nuclear layer which contains nuclei of rods and cones;
INL, the inner nuclear layer which contains cell bodies of
horizontal cells, bipolar cells, and ainacrine cells; CCL, the
ganglion cell layer. Data are from Xia et al. 1993.
learning and memory by disruption of the gene for the
enzyme in mice (Wu & Storm, unpublished observations). Brain coronal sections showed no detectable anatomical differences in the hippocampus, neocortex, or
cerebellum between wild type and mutant mice. There
were also no differences in the arrangement of cell body
layers of the hippocampus or cerebellum. The mutant
mice had normal motor coordination, suckling behavior,
weight gain, and reproduced with litter sizes comparable
to wild type mice. An analysis of Ca 2+ -sensitive adenylyl
cyclase activity in membranes from the cerebellum, neocortex, hippocampus, and brain stem revealed decreases
of 62%, 38%, 46%, and 6%, respectively.
9. Role of adenylyl cyclases and cAMP in longterm potentiation
The general hypothesis under consideration in this paper
suggests that the Ca 2+ -sensitive adenylyl cyclases
may play an important role in neuroplasticity and participate in signal transduction pathways underlying longterm adaptive responses in neurons such as LTP. Because
LTP results in postsynaptic increases in Ca 2+ and various
areas of the hippocampus including CA1, CA3, and the
dentate gyrus contain type I adenylyl cyclase (Xia et al.
1991), one might expect that activation of NMDA receptors or other Ca 2+ channels during LTP may elevate
cAMP in these regions. Indeed, activation of NMDA
receptors gives increased cAMP in area CA1 of the hippocampus (Chetkovich et al. 1991). Furthermore, LTP in
the dentate gyrus (Stanton & Sarvey 1985a) and the CA1
(Chetkovich & Sweatt 1993) have both been reported to
produce increases in cAMP. Although the cAMP increase
caused by LTP in the CA1 was very small (20-25%), it may
be difficult to detect these increases against a background
of cells in the preparation which are not potentiated.
There is also evidence that adenylyl cyclases, cAMP,
and cAMP-dependent protein kinases may play an important role in some forms of LTP, particularly in the hippocampus. For example, stimulation of adenylyl cyclase
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
437
Xia et al.: Neuroplasticity
Riboprobe
Control
Cx
IG
SHi
4t:J-
Pir
Tu
Oliaonucleotide Probe
'*• (
Figure 7. In situ hybridizations for type I adenylyl cyclase in middle rat brain. Rat brain sections were hybridized with either ^Slabeled antisense riboprobe 3C (A and C) or oligonucleotide probe ZX4 (£). The specificity of hybridization was demonstrated by
incubation of the respective 35S-labeled probe with a 1000-fold molar excess of the unlabeled probe (B, D, F). Exposure time: 3 days
(A-D), and 7 days (E-F). Abbreviations: Cx, neocortex; DG, dentate gyrus; Hi, hippocampus; IG, indusium griseum; Pir, piriform
cortex; SHi, septohippocampal nucleus; Tu, olfactory tubercle. Data are from Xia et al. 1991.
activity in the dentate gyrus by norepinephrine produces
LTP (Hopkins & Johnston 1988; Stanton & Sarvey 1985b).
The early phase of LTP in the CA1 persists only 1 to 2
hours and is initiated by a single train of high-frequency
stimulation, whereas the late phase requires three or
more trains of high-frequency stimulation and lasts up to
10 hours. Since D : dopamine antagonists block L-LTP
and Dj receptors are coupled to stimulation of adenylyl
cyclase (Frey et al. 1991) it has been proposed that cAMPstimulated protein kinase may play a pivotal role in
L-LTP. In fact, dibutyryl cAMP induces increases in
synaptic efficacy in the CA1 region of the hippocampus
(Slack & Pockett 1991) and L-LTP in the CA1 is blocked
by Rp-cAMPS, an inhibitor of cAMP-dependent protein
kinase (Frey et al. 1993). Furthermore, Sp-cAMPS,
which activates cAMP-dependent kinase, produces
L-LTP in the CA1.
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
10. Biochemical model for the role of the CaMregulated adenylyl cyclases in neuroplasticity
The type I adenylyl cyclase is a neural specific adenylyl
cyclase (Xia et al. 1993) with a highly restricted expression
in mammalian brain which includes the dentate gyrus,
CA1, CA2, and CA3 regions of the hippocampus (Xia et
al. 1991). Furthermore, the type III and VIII adenylyl
cyclases are also expressed in the hippocampus (Cali et al.
1994; Glatt & Snyder 1993). What is the function of these
enzymes in neurons, and what possible role(s) might they
have in signal transduction systems important for neuroplasticity?
During LTP, PKC is activated (reviewed by Linden &
Routtenberg 1989), intracellular Ca 2 + increases, and neuromodulin is phosphorylated by PKC (Routtenberg 1985).
We hypothesize that phosphorylation of neuromodulin at
Xia et al.: Neuroplasticity
Cb
BS
B
Figure 8. /n situ hybridizations for type I adenylyl cyclase in cerebellum. Rat brain cerebellum sections were hybridized with 35Slabeled antisense riboprobe 3C (A and B). Specificity of hybridization was demonstrated by incubation of the respective 3SS-labeled
probe with a 1000-fold molar excess of unlabeled probe (C). Exposure time: 3 days (A, B) and 7 days (C). Abbreviations: Cb,
cerebellum; BS, brain stem. Data are from Xia et al. 1991.
specific sites in neurons may regulate the concentrations
of free CaM available to activate the CaM-regulated
adenylyl cyclases and other enzymes, including CaM
kinases and NO synthetase (Fig. 9).
Long-term changes in synaptic function may be due, at
least in part, to cAMP control of transcription through
cAMP-responsive DNA elements such as CRE (reviewed
by Mitchell & Tjian 1989). We hypothesize that synergistic stimulation of adenylyl cyclases by Ca 2+ and neurotransmitters or PKC may produce exceptionally strong or
prolonged cAMP signals required for stimulation of transcription. Stimulation of transcription by cAMP, which
requires the nuclear translocation of PKA (Hagiwara et al.
1993; Nigg et al. 1985), requires higher or more persistent
cAMP signals than other cAMP-regulated events, particularly in neurons. For example, stimulation of PKA nuclear translocation in Aplysia neurons (Bacskai et al. 1993)
and serotonin stimulation of transcription through CRE
(Kaang et al. 1993) are relatively slow processes that require
multiple doses of serotonin for AC activation. Robust
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
439
Xia et al.: Neuroplasticity
Neurotransmitter
POTENT1ATION
NO Synthase
\
N 0
Cytoskeleton
I
Remodeling of
the synapse
CaM Kinase u
+
Neurotransmitter
release
Retrograde
messenger
Phosphorylation of
transcription factors
Protein
biosynthesis
Long term
synaptlc change
Figure 9. Hypothetical model for the role of neuromodulin and the type I adenylyl cyclase in neuroplasticity. it is hypothesized
that neuromodulin binds and concentrates CaM at specific sites in neurons and that the levels of free CaM are regulated by
phosphorylation of neuromodulin by protein kinase C (PKC). During LTP, PKC is activated and Ca 2 + is mobilized. We propose that
the CaM-sensitive adenylyl cyclases may be activated during LTP by Ca 2 + /CaM, neurotransmitters, and/or activation of PKC.
Activation of the CaM-sensitive adenylyl cyclase(s) results in coupling of the Ca 2 + and cAMP regulatory systems during LTP.
Simultaneous or ordered activation of the Ca 2 + and cAMP regulatory systems may be important for amplified cAMP signals required
for transcription, synergism between Ca 2 + and cAMP activated kinases, and/or positive feedback regulation of Ca 2 + channels by
cAMP-dependent kinase. Abbreviations: CaM, calmodulin; NM, neuromodulin (GAP-43); PKC, protein kinase C; NM-P, neuromodulin phosphorylated on Ser-41; PKA, cAMP-dependent protein kinase.
cAMP signals may be required for transcriptional control
in neurons because a significant cAMP gradient must be
established from the synapse to the cell body. Elevated
cAMP signals arising from synergistic activation of the
type I adenylyl cyclase by Ca 2+ and neurotransmitters (or
other signals) may therefore play an important role in
synaptic plasticity. Alternatively, the coupling of the Ca 2+
and cAMP systems may result in simultaneous or sequentially ordered activation of the Ca 2+ and cAMP stimulated
protein kinases, or provide positive feedback regulation of
440
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Ca 2+ channels by cAMP-dependent protein kinase. All of
these mechanisms are dependent upon the unique property of the CaM-sensitive adenylyl cyclases to integrate
multiple signals for modification of synaptic function.
ACKNOWLEDGMENT
This research was supported by NIH grant NS 20498.
Choi was supported by a Washington Heart Association
postdoctoral fellowship and Xia was supported by a Keck
neuroscience fellowship.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18, 441-451
Printed in the United States of America
The cGMP-gated channel of
photoreceptor cells: Its structural
properties and role in
phototransduction
Robert S. Molday
Department of Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, B.C., Canada V6T 723
Yi-Te Hsu
Department of Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, B.C., Canada V6T 1Z3
Electronic mail: molday@unixg.ubc.ca
Abstract: The cyclic GMP-gated channel responds to changes in free intracellular cGMP, and as a result, it plays a central role in the
phototransduction process in rod and cone photoreceptor cells. Recent biochemical, immunochemical, and molecular biology
studies indicate that this channel consists of a complex of two distinct subunits and one or more associated proteins. Primary structural
analysis indicates that the a and (3 subunits contain a cGMP-binding domain, an even number of membrane-spanning segments, a
voltage sensor motif and a pore region. The latter two features are found in voltage-gated channels and suggest that these two classes
of channels have evolved from the same ancestral channel protein. A working model for the membrane topography of the channel
subunits is proposed based on immunogold labeling studies and sequence analysis. Recent studies also indicate that calmodulin binds
to the 240 kDa protein of the channel complex and modulates the sensitivity of the channel for cGMP in a Ca2+-dependent manner.
The molecular properties of the channel complex and the possible role of Ca2+-calmodulin modulation of the channel during
photoactivation and photorecovery are discussed in relation to the current mechanism of phototransduction in photoreceptor cells.
Keywords: calmodulin; cGMP-gated channel; photorecovery; phototransduction; rod photoreceptor cells
1. Introduction
The cyclic GMP-gated channel of vertebrate rod photoreceptor cells plays a central role in the phototransduction process. In the dark a relatively high concentration of
cGMP in the rod outer segment (ROS) maintains a significant number of channels in their open state by the direct,
reversible binding of cGMP to the channels (Fesenko et
al. 1985; Yau & Baylor 1989; Yau & Nakatani 1985a). This
allows for a steady influx of Na + and Ca 2+ into the outer
segment and maintains the cell in a partially depolarized
state. The influx of Ca 2+ through the channel is balanced by
an efflux of Ca 2+ by the Na + /Ca 2+ -K+ exchanger in the ROS
plasma membrane, thereby maintaining the intracellular
level of Ca 2+ at a relatively constant level (Yau & Nakatani
1984a). Low intracellular Na + concentration is maintained
by the balanced extrusion of Na + by Na + -K + ATPase localized in the plasma membrane of the rod inner segment.
Photobleaching of rhodopsin in the ROS disk membrane leads to an activation of the visual enzyme cascade
system (Chabre & Deterre 1989; Stryer 1986; 1991) and a
reduction in the concentration of cGMP by a phosphodiesterase catalyzed reaction (Fig. 1). This results in the
closure of the cGMP-gated channels and a transient
hyperpolarization of the cell. The closure of the channels
© 1995 Cambridge University Press
0U0-525XI95S9.0O+.10
to Ca 2+ results in a reduction in intracellular Ca 2+ because
the Na + /Ca 2 + -K + exchanger continues to extrude Ca 2+
from the outer segment. The reduction in Ca 2+ has been
shown to be important in the recovery of the rod outer
segment to its dark state and in light adaptation (Kaupp &
Koch 1992; Koch & Stryer 1988; Matthews et al. 1988; Yau
& Nakatani 1985b). This feedback mechanism is suggested
to occur through the interaction of Ca 2+ -binding proteins
with various proteins involved in the phototransduction
process, and in particular, guanylate cyclase (Koch &
Stryer 1988). Cone photoreceptors appear to have a similar visual cascade system and a related cGMP-gated channel (Bonigk et al. 1993; Haynes & Yau 1985; Picones and
Korenbrot 1992; Watanabe & Murakami 1991).
The physiological properties of the cGMP-gated channel of ROS have been extensively studied and recently
reviewed (Kaupp 1991; Yau & Baylor 1989). Generally,
cGM P directly and cooperatively binds to and activates the
channel with a K1/2 of 10-50 u,M and a Hill coefficient (n) of
1.7-3.5. Analogues of cGMP with substituents at position
8, suchas8-bromocyclicGMP, are also effective activators
of the channel in the (JLM range (Zimmerman et al. 1985).
Although the channel is cation selective, it exhibits a low
degree of specificity for various monovalent and divalent
cations (Furman & Tanaka 1990; Hodgkin et al. 1985;
441
Molclay & Hsu: Photoreceptor cells
Exchanger
- 4 No'
cGMP-gated Channel
Phosphodiesterase
I GTP
Light
Ca"
Disk Membrane
Plasma Membrane
Figure 1. A diagram showing the basic reactions of the visual transduction pathway. In the dark, elevated cGMP concentrations
maintain a relevant number of cGMP-gated channels in their open state. This allows the influx of Na+ and Ca2+ into the outer
segment and maintains the photoreceptor cell in a partially depolarized state. Photobleaching of rhodopsin, involving the
photoisoinerization of 1 l-cis retinal to its all-trans isomer, results in the formation of activated (meta II) rhodopsin which catalyzes the
exchange of bound GDP for GTP on transducin. Transducin will in turn activate phosphodiesterase which catalyzes the hydrolysis of
cGMP to 5'-GMP. The decrease in free cGMP concentration will cause the channel to close as cGMP dissociates from the channel.
Closure of the channel to the influx of Na+ and Ca2+ will result in a transient hyperpolarization of the cell. The recovery of the rod
outer segment to its dark state occurs through (1) the inactivation of rhodopsin by a rhodopsin kinase (RK) catalyzed phosphorylation
reaction and the binding of arrestin (Ar); (2) inactivation of transducin by hydrolysis of bound GTP to GDP; (3) inhibition of
phosphodiesterase by the rebinding of the inhibitory subunits to the catalytic subunits of phosphodiesterase; (4) resyn thesis of cGMP
from GTP by guanylate cyclase (GC); and (5) the reopening of the cGMP-gated channel as cGMP levels increase.
Schnetkamp 1990; Yau & Nakatani 1984b). The rod channel is
permeable not only to the physiological ions, Na + , Ca 2+ , and
Mg 2+ , but also to other monovalent and divalent cations
including Li+, K+, Cs+, Rb + , Mn 2+ , andBa 2+ . Under physiological conditions the channel is relatively insensitive to
voltage changes. Divalent cations have been shown to block
the flow of ions through the channel, thereby reducing the
effective conductance of the channel (Yau & Baylor 1989).
L-cis diltiazem has been shown to be a stereoselective inhibitor of channel activity in the micromolar concentration
range (Koch & Kaupp 1985; Stern et al. 1986). The channel
has been localized to the plasma membrane of ROS and has
a density of 200-500 channels per |xm2 (Bodoia & Detwiler
1985; Cook et al. 1989; Zimmerman & Baylor 1986).
Recently, biochemical, immunochemical, and molecular biology studies have provided insight into the molecular properties of the cGMP-gated channel of rod and cone
photoreceptors. In this target article, issues related to the
molecular composition and structural features of the cGMPgated channel of photoreceptor cells are addressed, and the
regulation of the cGMP-gated channel by ealmodulin is
discussed in terms of its possible role in phototransduction.
442
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
2. Molecular composition of the cGMP-gated
channel
2.1. Identification and purification of the a-subunit. In the
latter half of the 1980s several laboratories had identified
different polypeptides as candidates for the cGMP-gated
channel subunit of ROS. Cook et al. (1987) isolated a 63
kDa polypeptide from CHAPS detergent-solubilized
bovine ROS by ion exchange and red-dye affinity chromatography. This preparation exhibited cGMP-dependent
ion flux activity when reconstituted into liposomes, but,
in contrast to ROS membranes, the channel activity was
not inhibited by micromolar concentrations of l-cis diltiazem. At about the same time, Matesic and Liebman
(1987) described the partial purification of a 39 kDa
polypeptide from cholate-treated bovine ROS. This 39
kDa protein, which was reported to exhibit immunochemical crossreactivity with rhodopsin, could be photoaffinity labeled with 8'-azido-cGMP and reconstituted
into vesicles for measurements of cGMP-dependent
channel activity. On this basis, they concluded that
the ROS channel was composed of 39 kDa subunits.
Molday & Hsu: Photoreceptor cells
PMs 5E11
PMc 1D1
1.0 -
[cGMP] (pM)
Figure 2. Immunoaffinity purification and functional reconstitution of the cGMP-gated channel complex of bovine rod outer
segments (ROS). (a) SDS gels of ROS membranes (lane a) and the
iminunoaffinity-purified channel (lane b) stained with Coomassie
Blue (CB) or transferred to Immobilon membranes and labeled
with monoclonal antibodies PMs 5E11 against the 240 kDa
channel protein and PMc 1D1 against the a-subunit of the
channel, (b) cCMP-dependent Ca2+ efflux from liposomes reconstituted with the immunoaffinity purified channel. The sigmoidal
curve was calculated usinga Km of33 (iM and a H ill coefficient of3.3
for cGMP. For these studies, CHAPS detergent-solubilized ROS
membranes were added to a PMc 6E7 anti-channel monoclonal
antibody-Sepharose column and eluted with a N-terminal peptide corresponding to the epitope for this antibody (Molday et
al. 1991). The purified channel complex was reconstituted into
lipid vesicles by the procedure of Cook et al. (1987).
Shinozawa et al. (1987) suggested that a 250 kDa protein
constituted the cGMP-gated channel subunit on the basis
of photoaffinity labeling of frog ROS membranes with 3 HcGMP and channel activity measurements in planar lipid
bilayers. Finally, Clack and Stein (1988) reported that
purified opsin preparations exhibited cGMP-activated
single-channel activity, suggesting that the photopigment
protein rhodopsin itself may function as the cGMP-gated
channel.
Controversy surrounding the identity of this channel
subunit was initially resolved when a series of immunochemical studies established the 63 kDa polypeptide as
a major subunit of the channel. This subunit is now
referred to as the a-subunit. A monoclonal antibody
which specifically labeled the 63 kDa channel subunit in
both ROS membranes and purified channel preparations
(Cook et al. 1989) was shown to quantitatively immunoprecipitate both cGMP-gated channel activity and a
complex consisting of a 63 kDa and a 240 kDa polypeptides (Molday et al. 1990). In contrast, an antirhodopsin
monoclonal antibody which selectively immunoprecipitated rhodopsin did not immunoprecipitate cGMPdependent channel activity. More recently, a monoclonal
antibody generated against a peptide corresponding to
the N-terminal segment of the 63 kDa polypeptide
(Molday et al. 1991) has been used to immunoaffinitypurify the channel complex from detergent-solubilized
ROS in a functionally active form. As shown in Figure 2a,
the immunoaffinity-purified channel consists of the 63
kDa a-subunit of the channel and an associated 240 kDa
polypeptide. The 240 kDa polypeptide had been previously observed by Cook et al. (1987) in some of their
earlier channel preparations. The immunoaffinity-purified
channel complex, when reconstituted into liposomes,
exhibits cGMP-dependent channel activity. The K1/2 of 33
(xm and Hill coefficient of 3.3 for cGMP (Fig. 2b) are in
general agreement with the values obtained for red-dye
purified channel preparation as reported by Cook et al.
(1987). More recently, Brown et al. (1993) have labeled
the 63 kDa a-subunit with 8-p-azidophenacylthio-cGMP,
a photoaffinity derivative of cGMP, thus confirming the
presence of a cGMP binding site.
A cGMP-Sepharose column has been used by Hurwitz
and Holcombe (1991) as an affinity matrix to purify the
channel from detergent-solubilized ROS preparations.
This channel preparation was reported to consist of an 80
kDa polypeptide which was sensitive to 1-cis diltiazem.
Since this polypeptide binds a monoclonal antibody
against the 63 kDa channel subunit, it is likely that this 80
kDa polypeptide is related to the 63 kDa a-subunit of the
channel. The higher apparent molecular weight as determined by SDS gel electrophoresis may be due to anomalies in the migration of this channel subunit under different electrophoresis conditions. Alternatively, it may be
the result of the isolation of the unprocessed form of the
channel subunit. The observation that the channel isolated by cGMP-Sepharose affinity chromatography is inhibited by 1-cis diltiazem has been interpreted by Hurwitz and Holcombe (1991) to indicate that 1-cis diltiazem
sensitivity is a property of the full length 80 kDa channel
subunit, and not the truncated 63 kDa form. However,
recent studies by Chen et al. (1993) indicate that the 1-cis
diltiazem sensitivity of the channel as found in native ROS
is not obtained when the full-length 80 kDa a-subunit is
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
443
Molday & Hsu: Photoreceptor cells
expressed by itself, but instead requires the coexpression
of the a and 3 subunits (see below). This suggests that the
channel sensitivity to 1-cis diltiazem is inherent in the
3-subunit or the interaction of the 3 subunit with the a
subunit. The differences in apparent molecular weights
and 1-cis diltiazem sensitivities for the different isolated
channel preparations still remain to be clarified. More
recently, calmodulin-Sepharose chromatography has
been used to obtain a highly enriched channel preparation (Hsu & Molday 1993). The channel isolated by this
method was found to contain the 63 kDa and 240 kDa
polypeptides as the major components and several other
calmodulin binding proteins as minor contaminants.
These studies taken together indicate that the
a-subunit of apparent molecular mass (Mr) 63 kDa is a
major component of the native channel complex. Previous reports indicating that a 39 kDa polypeptide and
rhodopsin exhibit cGMP-dependent channel activity appear to be a result of contamination of these preparations
identified a second subunit (P-subunit) by screening a
human cDNA library with a partial clone of the a-subunit
under low stringency conditions. Sequence analysis of
this 3-subunit indicates that it exhibits a 30% overall
sequence identity to the a-subunit and 50% identity
within the cGMP binding domain. Alternative splicing
results in two transcripts which differ in size. The shorter
transcript codes for a polypeptide of 623 amino acids with
a calculated molecular weight of 70,843 and the longer
transcript codes for a polypeptide of 909 amino acids
having a molecular weight of 102,330. The two polypeptides are identical in sequence except for an extended
N-terminal segment of 286 amino acids for the latter.
Patch-clamp studies of human kidney 293 cells transfected with the 3-subunit cDNA indicate that neither
form of the 3-subunits when expressed alone is functionally active (Chen et al. 1993). However, when coexpressed with the a-subunit, cGMP-gated channel activity
can be measured that more closely resembles the activity
with this channel complex. An earlier report of Shinozawa
found in the ROS membranes. In particular, coexpression
et al. (1987) suggesting that the channel subunit is a 250
kDa polypeptide may be related to a recent finding that
the channel contains a second subunit referred to as the
3-subunit (see below).
of the a and 3-subunits results in rapid bursts of channel
openings or a flickering response and u,M sensitivity of
the channel to 1-cis diltiazem as found for the channel in
ROS membranes. In contrast, the expressed'a-subunit,
by itself, displays a more stable opening and closing
behavior and is over an order of magnitude less sensitive
to 1-cis diltiazem (Chen et al. 1993; Kaupp et al. 1989).
Immunocytochemical studies have confirmed that the
3-subunit is present in human rod outer segments (Chen
et al. 1993). An antibody raised against a peptide corresponding to the C-terminus of the 3-subunit was observed to label the outer segment layer of human rod
photoreceptor cells, but not cone photoreceptor cells.
However, an antibody generated against a peptide segment near the N-terminus of the polypeptide encoded by
the longer 3 transcript and not present in the polypeptide
encoded by the shorter transcript did not label photoreceptor outer segments. On the basis of these studies,
Chen et al. (1993) have suggested that the 3-subunit in
ROS is encoded by the shorter transcript. Direct studies
showing that the shorter transcript is in fact the predominant form expressed in ROS, however, have not been
carried out, leaving some question as to whether the long
or short form of the 3-subunit is preferentially expressed
in rod photoreceptors. Other issues that need to be
addressed include the detection and identification of the
3-subunit in both isolated ROS membranes and isolated
channel preparations and the stoichiometric relationship
between the a and 3-subunits.
2.2. Cloning and expression of the a-subunit. Direct
evidence indicating that the 63 kDa polypeptide is a
major subunit of the cyclic GMP-gated channel was obtained from the cloning and expression studies of Kaupp
et al. (1989). Oligonucleotide probes prepared from tryptic peptide sequences of the 63 kDa polypeptide were
used to screen a bovine retinal cDNA library. A fulllength cDNA clone was obtained which encoded a 79.6
kDa polypeptide containing up to six putative transmembrane segments and a cGMP binding domain. Injection of Xenopus oocytes with mRNA derived from the
cloned cDNA resulted in the functional expression of a
cGMP-gated channel having many of the electrophysiological properties found for the channel in ROS
membranes (Kaupp 1991; Kaupp et al. 1989). The expressed channel has been reported to be cooperatively
activated by cGMP with a K1/2 of 52 |xM and a Hill
coefficient of 1.75, to display a cation selectivity similar to
that found in ROS, and to exhibit a single channel conductance of 20 pS. The expressed channel, however, is
considerably less sensitive to 1-cis diltiazem than the
native channel found in ROS. The a-subunit expressed in
both monkey kidney COS-1 cells and Xenopus oocytes
has an apparent Mr of 78 kDa indicating that the full
length polypeptide is expressed in these cells (Molday et
al. 1991). Dhallan et al. (1992) have cloned and functionally expressed the cDNA for the a-subunit of the
human ROS channel in embryonic kidney 293 cells.
2.3. Identification and characterization of the p-subunit.
The cGMP-gated channel has been generally considered
to consist of four or five subunits (Kaupp 1991), each of
which contains a cGMP binding site. This is based on the
finding that cGMP cooperatively activates the channel
with a Hill coefficient of 2-3.5. The Hill coefficient gives a
minimum number of cGMP molecules required for activation of the channel. Until recently, it had been assumed
that the cGMP-gated channel consisted of identical
a-subunits. Chen et al. (1993), however, have recently
444
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
2.4. The 240 kDa channel-associated polypeptide. In addition to the 63 kDa a-subunit, cGMP-gated channel
preparations from bovine ROS also contain another prominent polypeptide having an apparent Mr of 240 k by SDS
gel electrophoresis under reducing conditions (Fig. 2a).
Several monoclonal antibodies have been generated
against the 240 kDa polypeptide (Molday et al. 1990;
Wong & Molday 1986). One antibody was found to crossreact with red blood cell spectrin and brain fodrin. On the
basis of this crossreactivity, its size, and its ability to bind
calmodulin (see below), it had been initially suggested
that the 240 kDa polypeptide may be a member of the
spectrin family of cytoskeletal proteins. However, recent
rotary shadowing studies have indicated that the 240 kDa
Molday & Hsu: Photoreceptor cells
polypeptide does not have the extended rod-shaped
structure observed for spectrin (L. Molday & R. Molday,
unpublished results). Furthermore, initial peptide sequence studies indicate that the 240 kDa polypeptide is
not related to spectrin, but instead consists of two components, one of which is the (3-subunit of the channel
(M. Illing, A. Williams & R. Molday, unpublished results). The finding that the P-subunit is contained within
the 240 kDa polypeptide explains the reported findings
that cGMP and 8-p-azidophenacylthio-cGMP photoaffinity label a high Mr component (240-250 kDa) in frog
and bovine ROS and channel preparations (Shinozawa et
al. 1987; Brown et al. 1993).
On the basis of these recent studies the cGMP-gated
channel appears to consist of at least two subunits. The
a-subunit appears to be the major functional subunit
since cGMP-gated activity can be obtained by expression
of this subunit alone; the (J-subunit interacts with the
a-subunit in rod photoreceptors to alter some of the
properties of the channel and regulate the cGMPdependent activity of the channel (see below). The channel complex also contains at least one additional polypeptide which, together with the B-subunit, constitutes the
240 kDa polypeptide observed by SDS gel electrophoresis in purified channel preparations. Additional
studies are needed to further identify, characterize, and
quantify these components and to define their role in the
structure and function of the channel in rod and cone
photoreceptor membranes.
3. Primary structural features of the cGMP-gated
channel subunits
3.1. Structure of the at-subunit. The primary structure of
the bovine cGMP-gated channel a-subunit was first determined by cloning and sequence analysis of its cDNA
(Kaupp et al. 1989). The full-length cDNA encodes a
polypeptide chain of 690 amino acids with a calculated Mr
of 79,601. A stretch of 80-100 amino acids close to the
C-terminus of the channel (Fig. 3a) has been identified as
the cGMP binding domain on the basis of its sequence
similarity to the tandem cGMP binding domains of
bovine lung cGMP-dependent protein kinase and to
other cyclic nucleotide binding proteins (Kaupp et al.
1989; Kaupp 1991), and on the basis of site-directed
mutagenesis studies (Altenhofen et al. 1991). The N-terminal region contains a hydrophilic stretch of about 60
amino acids with a lysine-rich segment. Hydropathy plots
suggest the existence of six relatively hydrophobic segments designated H1-H6, which are of sufficient length to
span the lipid bilayer. With the exception of H4 and H5
the hydrophobicity index of these segments is generally
lower than that observed for transmembrane segments of
well-characterized membrane proteins. Accordingly, it
remains to be experimentally determined if all or only
some of these segments span the membrane. Five consensus sequences (Asn-X-Thr or Asn-X-Ser) for N-linked
glycosylation are present at asparagine residues 90, 91,
177, 327, and 423. Lectin binding and endoglycosidase
studies have confirmed that the a-subunit is glycosylated
at a single site (Wohlfart et al. 1989).
The a subunit of the rod cGMP-gated channel has been
cloned from several species. The mouse and human
sequences are about 85% identical to bovine (Dhallan et
al. 1992; Pittler et al. 1992), whereas the chicken rod
subunit is 75.7% identical (Bonigk et al. 1993). The
chicken cone a-subunit has also been cloned and shown to
be 65% identical to the bovine rod subunit (Bonigk et al.
1993). The cone a-subunit has the same structural features as the rod channel and similar electrophysiological
properties when expressed in Xenopus oocytes.
The molecular weight of the bovine channel a-subunit
calculated from the cDNA-derived protein sequence
(79.6 kDa) is considerably larger than the apparent molecular weight of 63 kDa observed by SDS-PAGE. This
difference is not due solely to anomalous migration of the
channel subunit on SDS gels, because the channel asubunit expressed in mammalian cells and Xenopus oocytes migrates with an apparent Mr of 78 k (Molday et al.
1991). The lower molecular weight of the channel subunit
in ROS is primarily due to the absence of the' first 92
amino acids as shown by N-terminal sequence analysis.
This truncated form of the channel consisting of 598
amino acids has a calculated molecular weight of 69.4 k.
The difference in molecular weight of the a-subunit as
determined by SDS gel electrophoresis (63 kDa) and that
calculated on the basis of the amino acid composition of
the truncated channel (69.4 kDa) is attributed to the
inaccuracy of molecular weight determinations of membrane glycoproteins by SDS gel electrophoresis (Molday
et al. 1991). Immunochemical labeling studies for light
microscopy and electron microscopy employing a monoclonal antibody specific for the N-terminus of the 63 kDa
a-subunit has confirmed that this shortened form of the
channel is the major form present in ROS and is not a
result of nonspecific proteolysis during the preparation of
ROS membranes. It is likely that a photoreceptor-cellspecific posttranslational cleavage reaction results in the
truncated form of the channel subunit found in ROS,
although this remains to be determined experimentally.
The a-subunit of other mammalian species has also been
observed to migrate on SDS gels with an apparent Mr of
63 k, suggesting that truncation of the a-subunit is a
general characteristic of the mammalian rod channel
(Molday et al. 1991). Comparison of the rod and cone
a-subunits in chicken retinal cell extracts with that of the
a-subunits transiently expressed in COS-1 kidney cells
indicates that a similar cleavage reaction occurs in chicken
rod and cone photoreceptor cells (Bonigk et al. 1993). The
cleavage site between serine 92 and serine 93 in the
bovine sequence N-N-S-S-N-K-E (N-Asn, S-Ser, K-Lys,
E-Glu) is conserved in the human and mouse channel
a-subunit (Fig. 3b). Related although nonidentical sequences are found in the chicken rod and cone channel
subunits; it remains to be determined if this segment is
also the site of cleavage for the chicken channel rod and
cone a-subunits.
Sequence analysis of the rod and cone channel
a-subunit has also revealed two structural features in
voltage-gated channels (Fig. 3b; Bonigk et al. 1993). A
voltage sensor motif related to the S-4 segment of the
voltage-gated shaker K+ channel is found in photoreceptor, as well as olfactory, cyclic nucleotide-gated channel
subunits (Goulding et al. 1992; Jan & Jan 1990). This motif
consists of a repeating sequence of a positively charged
Arg (R) or Lys (K) residue separated by two predominantly hydrophobic amino acids. Voltage-gated channel
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
445
Molday & Hsu: Photoreceptor cells
CYCLIC NUCLEOTIDE BINDING DOMAIN
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: >
:
8 N N T N E D
(53-59)
Chicken Cone (a)
s s NK B
(90-96)
(145-151)
Figure 3. Sequence alignment of various structural domains of cyclic nucleotide-gated channels from different species, (a)
Alignment of the potential cyclic nucleotide binding sites of the bovine rod a-subunit (Kaupp et al. 1989); human and mouse rod
a-subunit (Pittler et al. 1992); the chicken rod and cone a-subunits (Bonigk et al. 1993); the bovine olfactory channel subunit (Ludwig
et al. 1990); and the rod (3-subunit (Chen et al. 1993). A high degree of sequence identity within the nucleotide binding domain is
observed, (b) Sequence alignment of the voltage-sensor-like motifs (S4), pore regions, and putative N-terminal cleavage sites.
subunits generally have seven repeating sequences,
whereas the nucleotide-gated channels have four or fewer
(Fig. 3b). It has been suggested that in voltage-gated
channels this sequence spans the membrane bilayer. A
change in the voltage across the membrane is thought to
induce a conformational change in this segment which in
turn affects the ion translocating properties of the channel. In the photoreceptor and olfactory cyclic nucleotidegated channel this voltage-sensor-like motif is more limited and contains several negatively charged residues.
446
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
These negatively-charged residues or ineffective coupling
of this segment to the pore region may be responsible for
the insensitivity of these cyclic nucleotide-gated channels
to physiological changes in voltage (Kaupp 1991; Yau &
Baylor 1989).
Photoreceptor and olfactory channel subunits also contain a segment that has considerable sequence similarity
to the pore region of the voltage-gated K+ channel
(Bonigk et al. 1993; Goulding et al. 1992; Heginbotham et
al. 1992). For voltage-gated channels, this region consists
Molday & Hsu: Photoreceptor cells
of a stretch of about 21 amino acids, which is considered to
extend into the lipid bilayer as two antiparallel (3 strands
connected by a hairpin turn (Durell & Guy 1992; Yellen et
al. 1991). Toxin binding studies and site-directed mutagenesis studies of the K + channel have provided support
for the functional role of this segment in the translocation
of ions across membranes. Yool and Schwartz (1991) have
reported that mutations in this pore region of the K +
alters the ion selectivity of the channel without affecting
the gating properties. Heginbotham et al. (1992) have also
shown that deletion of two amino acids (Tyr-Gly-) in the
pore region of the K + channel that are absent in cyclic
nucleotide-gated channels results in a loss of K + selectivity and introduction of a divalent ion blockage of the
channel as found in cGMP-gated channels. In the case of
cyclic nucleotide-gated channels, exchange of the pore
sequence of the rod a-subunit with the pore sequence of
the olfactory subunit has been shown to impart ionconducting properties of the olfactory channel onto the
rod channel (Goulding et al. 1993). Site-directed mutagenesis studies have also defined the residue in the pore
region which is responsible for the observed divalent
cation block of conductance (Root & McKinnon 1993). In
these studies mutation of glutamic acid 363 to a glutamine
residue has been shown to effectively eliminate the blockage of the channel conductance by external Mg 2+ and
Ca 2 + . These studies support the role of this segment of
the channel as the primary structural domain involved in
ion translocation through the channel.
3.2. Structure of the p-subunit. The P-subunit of the rod
channel has many of the structural features of the
a-subunit (Chen etal. 1993). In particular this subunit has
a modified voltage-sensor-like motif and a pore region in
positions similar to that of the a-subunit (Fig. 3b). The
putative pore region of the p-subunit, however, unlike
the pore region of the a-subunit, does not have a negatively charged residue. Hydropathy plots also show the
presence of up to six hydrophobic segments, although
some of these are less hydrophobic than corresponding
segments of the a-subunit. Unlike the a-subunit, the
P-subunit does not contain a consensus sequence for
N-linked glycosylation, suggesting that this subunit is
either not glycosylated or is O-glycosylated on serine or
threonine residues.
cated form of the a-subunit as found in bovine ROS
membranes, there are three potential glycosylation sites
at positions 177, 327, and 423. Position 177 is within the
HI hydrophobic segment and is not considered as a likely
site for glycosylation. Immunocytochemical and biochemical studies using antibodies generated against synthetic peptides encompassing glycosylation sites at 327
and 423 have confirmed that asparagine 327 contains the
N-glycosylation site and is localized on the extracellular
surface of the ROS plasma membrane.
On the basis of labeling studies and sequence analysis,
we have proposed a working model for the organization of
the a-subunit of the channel within the membrane as
shown in Figure 4. In this model the N-terminal and
C-terminal segments are oriented on the cytoplasmic side
and the glycosylation site at Asn 327 is exposed on the
extracellular surface of the ROS plasma membrane. The
voltage sensor motif S4 is found in voltage-gated channels
and hydrophobic segments H1-H5 are viewed as transmembrane segments. The pore region located between
H4 and H5 is viewed as extending into the membrane in
an antiparallel P conformation as suggested for the K +
channel (Durell & Guy 1992). The cGMP binding domain
is located near the C-terminus and likely interacts with
the pore region to control the ion translocation properties
of the channel. This model has many general structural
features common to voltage-gated channels and suggests
that the two families of ion channels may have evolved
from the same ancestral channel. Similar models have
been developed independently by other groups on the
basis of sequence similarities of the cyclic nucleotidegated channels to voltage-gated channels (Goulding et al.
1992; Heginbotham et al. 1992).
The P-subunit is envisioned to have a similar topography to that of the a-subunit with both a voltage-sensormotif and a pore region (Chen et al. 1993). The pore
Pore
Extracellular
CHO
Intracellular
4. Proposed topological model for the channel
subunits in the membrane
Immunocytochemical labeling studies have led to some
insight into the organization of the a-subunit of the
channel in ROS membranes. Monoclonal antibodies directed against epitopes near the N-terminus and the
C-terminus have been observed to label the cytoplasmic
surface of the ROS plasma membrane (Cook et al. 1989;
Molday et al. 1991; 1992). These studies indicate that the
N and C termini of the a-subunit of the native channel are
localized on the cytoplasmic side of the ROS plasma
membrane and support the view that this subunit contains an even number of membrane-spanning segments.
The identity and orientation of the glycosylation site of
the a-subunit has been determined using peptidedirected antibodies (Wohlfart et al. 1992). In the trun-
.- -
-NH,
•-'-NH,
Figure 4. Working model for the organization of the
a-subunit of bovine rod cGMP-gated channel within the lipid
bilayer. The first 92 amino acids (dashed line) as predicted from
cDNA sequence analysis are absent in the channel in ROS
membranes possibly by a posttranslational cleavage reaction.
Segments labeled H1-H5 and the S4 voltage-sensor-like motif
are considered as transmembrane segments and the segment
between H4 and H5 is the pore region. Immunogold labeling
studies have established the N and C termini on the cytoplasmic
side (Cook et al. 1989; Molday et al. 1991) and asparagine
Asn-327 containing a N-linked oligosaccharide chain on the
extracellular side (Wohlfart et al. 1992). The cGMP binding
domain is located near the C-terminus. The P-subunit is suggested to have a similar topographical organization.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
447
Molday & Hsu: Photoreceptor cells
regions of both the a- and 3-subunits would likely line the
central cavity of the channel and be responsible for many
of the ion translocating properties of the channel. There
are, however, some sequence differences in the a- and
P-subunits. Of particular interest is the absence of a negatively charged glutamic acid residue in the (3-subunit.
This may affect the binding affinity of divalent cations to
the channel and alter divalent cation blockage properties
found when the a-subunit alone is expressed for
structure-function studies (Root & MacKinnon 1993).
(Hsu & Molday 1993). When detergent solubilized ROS
membrane proteins are passed through a calmodulinSepharose column in the presence of Ca 2+ and are subsequently eluted in Ca 2+ -free buffer containing EDTA, the
cGMP-gated channel complex consisting of the 63 kDa
a-subunit and the 240 kDa protein is observed as the
major component as visualized by SDS gel electrophoresis. Western blots of the calmodulin-purified and
immunoaffinity-purified channel complex labeled with
125
I-calmodulin indicate that calmodulin binds to the 240
kDa polypeptide, but not to the 63 kDa a-subunit.
5. The rod cGMP-gated channel is a member of a
family of nucleotide-gated ion channels
6.2. Calmodulin modulation of cGMP-gated channel activity in ROS membranes. The effect of calmodulin on cGMPgated channel ion flux activity was measured in ROS
vesicles loaded with the Ca 2+ -sensitive dye Arsenazo III
(Hsu & Molday 1993). In the absence of calmodulin,
the channel in ROS membrane vesicles was observed to
be cooperatively activated by cGMP with a Km of 19 |xM
and a Hill coefficient of 3.7 for cGMP (Fig. 5). In the
presence of Ca 2+ -calmodulin the dose-response curve
for cGMP was shifted to the right so that the Km increased to 33 (JLM, but the Hill coefficient and the maximum rate (Vmax) of ion influx essentially remained unchanged. Although this shift in Km in the presence of
Ca 2+ -calmodulin is modest, the presence of calmodulin
can result in a 5-6-fold decrease in channel activity at
relatively low cGMP (~12 (JLM) as may be found under
physiological conditions, because the cooperativity is
high. This change in the apparent Km likely reflects an
effect of Ca 2+ -calmodulin on the binding affinity of the
channel for cGMP and not on ion translocation properties
of the channel. This is supported by the finding that the
calmodulin effect is observed for different cations and
does not affect the maximum velocity of ion influx (Hsu &
Molday 1993). Caretta et al. (1988) had earlier reported a
Two major classes of ion channels include the voltagegated channels and the ligand-gated channels (Jan & Jan
1989; Miller 1989; 1991). Voltage-gated channels respond
to changes in voltage across a membrane and are represented by K + selective channels, Na + selective channels,
and Ca 2+ selective channels. Ligand-gated channels re-
spond to the binding of extracellular ligands and are
represented by such complexes as the acetylcholine receptor, GABA receptor, and glycine receptor. Cyclic
nucleotide-gated channels constitute another family of
channels that respond to intracellular cyclic nucleotides.
In addition to cGMP-gated channels of rod and cone
photoreceptor cells (Bonigketal. 1993;Kauppetal. 1989;
Pittler et al. 1992), the olfactory neurons contain channels
that respond to cAMP as well as cGMP (Nakamura &
Gold 1987). The a-subunit of olfactory channels has been
cloned from bovine (Ludwig et al. 1990), rat (Dhallan et
al. 1990), and catfish (Goulding et al. 1992) olfactory
neurons. They share a high degree of sequence identity
(~57%) to the a-subunit of rod and cone photoreceptor
channels and contain a cyclic nucleotide binding domain,
a voltage-sensor-like motif, and a pore region (Figs. 3a
and 3b). It is likely that the olfactory channel, like the rod
and cone channels, contains more than one subunit,
although this remains to be determined.
Recent molecular biology and electrophysiology studies
indicate that cyclic nucleotide gated channels are also
present in other cells and tissues including pineal (Dryer
& Henderson 1991), heart (Biel et al. 1993), kidney
(Ahmad et al. 1990; Light et al. 1990), and bipolar cells
(Nawy & Jahr 1990). Initial cloning studies indicate that
the corresponding a-subunits from different tissues show
a high degree of sequence similarity to the rod, cone, or
olfactory channel a-subunit (Ahmad et al. 1992; Biel et al.
1993; Hundal et al. 1993). Detailed studies are needed to
define the subunit composition, structure, location, and
role of these channels in cell function.
6. Interaction of calmodulin with the cGMP-gated
channel
6.1. Binding of calmodulin to the channel. Earlier studies
had indicated that ROS contain significant amounts of the
calcium binding protein calmodulin (Kohnken et al.
1981), but the identity of the target proteins for calmodulin.in ROS was not determined. Recently, we have
shown by calmodulin affinity chromatography and Western blotting that the cGMP-gated channel complex is a
major calmodulin binding protein of ROS membranes
448
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
cGMP Concentration
Figure 5. The effect of calmodulin on the activation of the rod
channel by cGMP. Calcium influx assays were carried out using
ROS membrane vesicles containing Arsenazo III dye either in
the absence (•) or presence (•) of calmodulin. Calmodulin is
shown to increase the Km of the channel for cCMP from 19 |AM
to 33 u.M without affecting its maximum velocity or cooperativity for cGMP. The solid lines were drawn from a sigmoidal
binding isotherm using the indicated Km and a Hill coefRcient
of 3.6 (Hsu & Molday 1993).
Molday & Hsu: Photoreceptor cells
similar effect of Ca 2+ on the binding of fluorescentlabeled cGMP to ROS membranes. It is likely that endogenous calmodulin was present in the ROS preparations
used in this study and was responsible for this observed
Ca 2+ -dependent decrease in cGMP-binding.
Calmodulin modulation of the channel activity has
been found to be dependent on Ca 2+ (Hsu & Molday
1993). In the absence of calmodulin, Ca 2+ has no effect on
channel activity as measured by ion flux assays. However,
in the presence of calmodulin, Ca 2+ suppresses the channel activity over a Ca 2+ concentration range of 50-300
nM. The calmodulin effect is also inhibited by excess
masstoparen, an inhibitor of calmodulin (Hsu & Molday,
unpublished results). These studies indicate that at the
level of isolated ROS membranes, Ca 2+ -calmodulin alters
the sensitivity of the channel for cGMP. Modulation of
the affinity of the channel for cGMP by Ca 2+ calmodulin
is analogous to modulation of the affinity of hemoglobin
for oxygen by changes in pH as defined by the Bohr effect.
Recently, patch clamp studies have indicated that the
sensitivity of the olfactory channel for cyclic nucleotides is
also modulated by Ca 2+ (Kramer & Siegelbaum 1992).
The calcium binding protein that interacts with the olfactory channel has not yet been identified, however. It is
possible that calmodulin may be involved in this modulation of the olfactory channel. A light-activated channel
encoded by the trp gene in Drosophila photoreceptors
has also been shown to contain a calmodulin binding
domain (Hardie & Minke 1992). Its role in invertebrate
phototransduction has not yet been defined, however. It
remains to be determined if the Ca 2+ -calmodulin modulation is a feature of many cation channels and in particu-
lar other members of the family of nucleotide-gated
channels. It also remains to be determined if other endogeneous Ca 2+ -binding proteins interact with the channel
and if the channel is regulated by other mechanisms such
as phosphorylation, as suggested in the studies of Gordon
et al. (1992).
7. Possible role of Ca2+-calmodulin regulation of
the channel in phototransduction
If Ca 2+ -calmodulin binds to and modulates the channel
activity in intact photoreceptors under physiological conditions as suggested by the in vitro studies, then one may
envision a mechanism in which the channel switches
between a low affinity and high affinity state during
photoactivation and recovery (Fig. 6). In the dark, free
cGMP concentration of 4-10 p,M maintains a significant
number of channels in their open state, allowing for the
influx of Na + and Ca 2 + into the outer segment. The
balanced efflux of Ca 2 + by the Na + /Ca 2 + -K + exchanger
maintains Ca 2+ levels at about 300 nM. Under these
conditions, Ca 2+ -calmodulin would bind to the channel
and maintain it in its low-affinity state (Fig. 7a). Photobleaching of rhodopsin and activation of phosphodiesterase via the visual cascade system results in a
decrease in cGMP and a closure of the cGMP-gated
channels (Fig. 7b). This will result in a lowering of intracellular Ca 2+ concentration below 100 nM as Ca 2+ is
extruded through the Na + /Ca 2 + -K + exchanger. Reduced
Ca 2+ concentration will cause Ca 2+ to dissociate from
calmodulin and cause the channel to switch from its low-
Co"
K
cGMP
cGMP
tea"
Hi
K
cGMP
• •Co*
High C a ( > 300 nM) (Dark)
.2+ (<100nM) (Ught)
LowCa"
Low cGMP affinity (High K')
High cGMP affinity (Law K)
2*.
Figure 6. Schematic model depicting the interaction of calmodulin with the channel complex. In the presence of high intracellular
Ca 2 + (~300 nM) as found in dark-adapted rod photoreceptors, Ca 2+ -calmodulin binds to the 240 kDa protein of the channel complex
and maintains the channel in a low-affinity state (high K') for cGMP. Under conditions of low intracellular Ca 2 + (< 100 nM), as found
after photoexcitation, Ca 2 + and possibly calmodulin dissociate from the channel complex and cause the channel to switch to its highaffinity state (low K) for cGMP. Calmodulin binds to either the P-subunit of the channel or to unidentified polypeptide(s) (X) making
up the 240 kDa polypeptide. In this model, the channel is viewed as a complex consisting of three a-subunits (one subunit is omitted
for simplicity) and two B-subunits. The actual number of subunits and associated polypeptides, however, remains to be determined.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
449
Molday & Hsu: Photoreceptor cells
Ca2+K
2
tCa '
PDE
tcGMP
(Inactive state)
2+
Ccf' - ' '
Ught
PDE-
Rho*
M
Ugh AflWty State
GC (basaD — • GC
W ~ (active)
cGMPt
GC (basal)
Low Afflntty State
Figure 7. Possible role for calmodulin modulation of the channel during the visual transduction process, (top) In the dark, an
elevated level of cGMP maintains a significant number of cGMP-gated channels in their open state and allows the influx
of Na+ and Ca2+ into the outer segment. The balanced influx of Ca2+ through the channel with the efflux of Ca2+ through the
Na + /Ca 2+ -K + exchanger results in a relatively high level of Ca2+ (~300 nM) within the outer segment. Under these conditions
calmodulin associates with the channel complex and maintains the channel in its low-affinity state for cGMP. The channel in its lowaffinity state will respond to a decrease in the level of cGMP during photoexcitation of the outer segment, (bottom) Photobleaching of
rhodopsin (Rho*) results in the activation of phosphodiesterase (PDE) and a decrease in the level of cGMP ©. This causes the closure
of the cGMP-gated channel ® and a decrease in intracellular Ca2+ (D due to the continuous extrusion of Ca2+ by Na + /Ca 2+ -K +
exchanger. This drop in Ca2+ will cause calmodulin to dissociate from the channel complex and shift the channel from its low-affinity
state to its high-affinity state for cGMP. The low level of Ca2+ will also activate guanylate cyclase through a Ca2+-dependent
guanylate-cyclase-activating protein. The combined effect of resynthesizing cGMP © and making the channel more sensitive to
cGMP levels would facilitate the recovery of the outer segment to its dark level. As the channels reopen, the Ca2+ level in the outer
segment is restored <D. This results in the rebinding of calmodulin to the channel and conversion of the channel to its low-affinity state.
Guanylate cyclase is also restored to its basal level of activity.
450
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Molday & Hsu: Photoreceptor cells
affinity state to its high-affinity state (Fig. 7 bottom). This
will enable the channel to open at lower cGMP concentrations. Reduced intracellular Ca 2+ levels will also activate
guanylate cyclase through a guanylate-cyclase-activating
protein (Koch & Stryer 1988). These two processes, activation of guanylate cyclase and conversion of the channel
from a low to a high-affinity state for cGMP, will facilitate
photorecovery to dark levels (Fig. 7 bottom). An increased intracellular Ca 2+ concentration resulting from
reopening of the channels will return guanylate cyclase to
its basal level of activity and the channel to its low-affinity
state by rebinding Ca 2+ -calmodulin (Fig. 7 top).
The presence of several Ca 2+ -binding proteins in ROS
suggests that changes in Ca 2+ levels during photoactivation and recovery may affect several reactions. These
include regulation of guanylate cyclase activity by a putative guanylate cyclase activator protein (Koch & Stryer
1988), modulation of the affinity of the channel for cGMP
by calmodulin (Hsu & Molday 1993) and regulation of the
light activation of phosphodiesterase (PDE) through the
effect of S-modulin (recoverin) on rhodopsin phosphorylation (Kawamura 1993).
8. Summary
The cGMP-gated channel of rod and cone photoreceptor
cells is a member of a family of cyclic-nucleotide-gated
channels found in many different cells. Primary structural
analysis, site-directed mutagenetic, biochemical, and immunochemical studies have indicated that the channel
consists of two major siibunits and one or more associated
proteins. The a- and ($-subunits have a cGMP-binding
domain near the C-terminus, an even number of transmembrane segments (possibly as many as six), a voltagesensor-like motif, and a pore region. The latter two
features are found in voltage-gated channels and suggest
that the nucleotide-gated channels and the voltage-gated
channels have evolved from the same ancestral channel.
In rod and cone photoreceptor cells the a-subunit undergoes a posttranslational cleavage reaction involving the
removal of a segment at the N-terminus. The ($-subunit
appears to be associated with another polypeptide which
together constitute a 240 kDa polypeptide observed in
purified channel preparations by SDS gel electrophoresis. The basis for these modifications is not yet
known. Calmodulin has also been shown to bind to the
240 kDa polypeptide and modulate the activity of the rod
photoreceptor channel in a Ca 2+ -dependent manner in in
vitro systems. This Ca 2+ -dependent interaction of the
channel with calmodulin alters the affinity of the channel
for cGMP. Together with other Ca 2+ -dependent processes, this Ca 2+ -dependent regulation of the channel is
suggested to play a role in the recovery of the photoreceptor cell following photoactivation. Although significant
progress has been made over the past several years on
analysis of the molecular properties of cyclic-nucleotidegated channels, much remains to be understood about the
molecular composition, structure, regulation, and localization of these channels in various cell systems and the role
of these channels in cell processes.
ACKNOWLEDGMENT
This work was supported by grants from NIH (EY-02422), MRC
(MT 9588), and the RP Foundation of Canada.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
451
BEHAVIORAL AND BRAIN SCIENCES (1995) 18, 452-467
Printed in the United States ot America
Correlation of phenotype with
genotype in inherited retinal
degeneration
Stephen P. Daiger, Lori S. Sullivan, and
Joseph A. Rodriguez
Human Genetics Center, School of Public Health, The University of Texas
Health Science Center, Houston, TX 77030
Electronic mail: daiger@gsbs21.uth
Abstract: Diseases causing inherited retinal degeneration in humans, such as retinitis pigmentosa and macular dystrophy, are
genetically heterogeneous and clinically diverse. More than 40 genes causing retinal degeneration have been mapped to specific
chromosomal sites; of these, at least 10 have been cloned and characterized. Mutations in two proteins, rhodopsin and peripherin/RDS, account for approximately 35% of all cases of autosomal dominant retinitis pigmentosa and a lesser fraction of other retinal
conditions. This target article reviews the genes and mutations causing retinal degeneration and proposes mechanisms whereby
specific mutations lead to particular clinical consequences, that is, the relationship between genotype and phenotype. Retinitis
pigmentosa and macular dystrophy are genetically heterogeneous diseases that cause retinal degeneration in humans and often result
in severe visual impairment or blindness. Although many of the genes causing these diseases have not been identified, three
photoreceptor-specific proteins have been implicated: rhodopsin, peripherin/RDS, and the P-subunit of rod phosphodiesterase.
Mutations in the genes for these three proteins can cause either dominant retinitis pigmentosa, recessive retinitis pigmentosa,
dominant congenital stationary night blindness, or dominant macular degeneration. Why this multiplicity of clinical phenotypes?
Our target article summarizes the genetic and biochemical background to this question and proposes a number of possible
explanations. Discussion focuses mainly on 73 distinct disease-causing mutations of rhodopsin. We feel that rhodopsin and other
photoreceptor proteins can serve as model systems for unraveling the connection between genotype and phenotype, not only for
inherited retinal diseases but for other degenerative disorders as well.
Keywords: human genetic diseases; inherited retinal degeneration; macular dystrophy; peripherin/RDS; phosphodiesterase
P-subunit; retinitis pigmentosa; rhodopsin; rod and cone photoreceptor cells
1. Introduction
Within the past decade rapid progress has been made in
identifying genes and mutations causing many forms of
inherited retinal degeneration in humans and other animals. However, we are still ignorant of the biological
mechanisms that underlie many of these diseases. The
connection between genotype, that is, specific mutations,
and phenotype, such as retinal degeneration, is often
obscure or unknown. Although much has been learned
about human genes, less than 2% of the human genome
has been sequenced and fewer than 1% of human genes
have been fully characterized (Burks et al. 1992). Also,
gene products function within complex cellular environments, and the relationship between proteins, their substrates, organelles, and other cellular components is
poorly understood. Furthermore, we know even less
about the function of proteins as they become degraded
with time. Finally, proteins may function differently in
different tissues, or their function may change in the same
tissue at different developmental stages.
The vertebrate retina has served as a model system for
investigating embryogenesis, cellular ultrastructure, signal transduction, signal transmission and processing, and
452
cellular turnover and death. We believe that the retina,
the mammalian retina in particular, also serves as a useful
model for investigating genotype-phenotype relationships in inherited degenerative diseases and disorders.
Many retinal genes have been cloned, and many specific
mutations of photoreceptor-specific genes are known to
cause retinal degeneration in humans. Such mutations
can contribute to our understanding of both abnormal and
normal visual processes.
There are more than 200 inherited diseases that lead to
retinal degeneration in humans (probably an underestimate). At least 10% of inherited diseases involve the
retina directly or indirectly (McKusick 1992). Over the
pastfiveyears more than 40 genes causing retinal degeneration in humans have been mapped, mostly by linkage
methods. Seven of these genes have been cloned. Thus
there are a number of opportunities to correlate genotype
with phenotype of the resulting disease. Our discussion
focuses on the genes for rhodopsin, peripherin/RDS,1
and the rod phosphodiesterase P-subunit (PDEB) genes that are expressed specifically in photoreceptors.
Rhodopsin is the rod photoreceptor visual pigment located in disc membranes and in the plasma membrane of
rod outer segments (ROS). Rhodopsin constitutes approx© 7995 Cambridge University Press
0140-525X195 S9.00+.10
Daiger et al.: Inherited retinal degeneration
imately 90% of the protein in ROS. Peripherin/RDS is a
protein of uncertain function that is expressed in both
rods and cones. It is found on the periphery of photoreceptor discs, where it may play a structural role. PDEB
is a catalytic subunit of phosphodiesterase, the protein in
the visual transduction cascade that hydrolyses cGMP to
5'-GMP when disinhibited by activated transducin (the
rod-specific G-protein). The phosphodiesterase subunit
that maps to 4p appears to be rod-specific, although
expression in other retinal cells cannot be excluded. Of
primary interest here are mutations in genes for rhodopsin, peripherin/RDS, and PDEB that result in phenotypes that cause progressive retinal degeneration, such
as autosomal dominant retinitis pigmentosa, autosomal
recessive retinitis pigmentosa, and autosomal dominant
macular dystrophy.2 Several recent reviews provide additional details (Berson 1993; Heckenlively 1988; Humphries et al.1992; 1993; Musarella et al. 1992).
Recently, mutations in the gene for rod cGMP-gated
channel (CNCG) have been suggested as another cause
of arRP (autosomal recessive retinitis pigmentosa; McGee
et al. 1994). This supports the expectation that many of
the 40 or more genes causing inherited retinal degeneration are likely to be photoreceptor-specific.
2. Background
2.1. Diseases causing Inherited retinal degeneration
Diseases causing inherited retinal degeneration in humans can be classified broadly into those that first affect
peripheral vision and the peripheral retina, such as retinitis pigmentosa, and those that primarily affect central
vision and the macula, such as macular dystrophy. Although the macula, especially the fovea, has the highest
concentration of cones and the peripheral retina is dominated by rods, factors contributing to whether a given
degeneration is expressed as a peripheral disease or a
macular disease are more complicated than simply the
density of a particular type of photoreceptor.
The clinical symptoms of retinitis pigmentosa include
night blindness and loss of peripheral vision (Heckenlively 1988). With time, visual impairment progresses
toward the center of the retina causing "tunnel-vision."
The disease is usually accompanied by pigmentary deposition, although the appearance of pigment is thought to
be a consequence of the disease and not its cause. Early
symptoms usually occur within the first or second decades
of life. For many patients visual fields continue to constrict over a period of years or decades, eventually resulting in blindness. Retinal degeneration is usually bilateral
and is often preceded and accompanied by characteristic
changes in the electroretinogram (ERG).
Retinitis pigmentosa can be subdivided into several
genetic categories: autosomal dominant (adRP), autosomal recessive (arRP) X-linked (xlRP) or syndromic. The
overall incidence of retinitis pigmentosa is about 1/4000
without apparent ethnic or racial distinctions (Fishman
1978; Halloran 1985). AdRP (autosomal dominant retinitis
pigmentosa) accounts for about 20% of cases; arRP for
15%; XlRP for 10%; and syndromic forms, such as the
recessive disease Usher syndrome (which combines congenital deafness with retinitis pigmentosa), for 20%. The
remaining 35% of cases are isolated or sporadic. Thus the
mode of inheritance cannot be determined for all patients, but it is assumed that all cases have a genetic basis.
There are also a number of clinical categories for
retinitis pigmentosa. Limiting discussion to autosomal
dominant forms of the disease, adRP families and patients
can be classified by a number of clinical criteria such as
age of onset of night blindness and visual impairment,
rate of progression, whether pigmentary deposits are
diffuse, regional, or sectorial, and whether ERG responses are diminished (or absent) from rods, from cones,
or from both. These classes have been condensed into two
broad categories. Type 1 retinitis pigmentosa is characterized by rapid progression and diffuse, severe pigmentation; type 2 retinitis pigmentosa has a slower progression and more regional, less severe pigmentation (Massof
& Finkelstein 1981). In an alternate system of classification (Lyness et al. 1985), these two types correspond
roughly to type D or "diffuse" and type R or "regional"
retinitis pigmentosa. Some investigators propose "sectorial" retinitis pigmentosa as a.third classification, as well
as additional types (Fishman et al. 1985; Heckenlively
1988).
Classification of macular degeneration is more tenuous
(Fishman 1990). Macular degeneration can have either a
genetic basis or it may be an acquired disease. Approximately 10% of Americans over the age of 50 are afflicted
with age-related macular degeneration (Bressler et al.
1988), an acquired form of disease. The inherited forms of
macular degeneration are much less common but usually
more severe. Inherited macular degeneration is characterized by early development of macular abnormalities
such as yellowish deposits and atrophic or pigmented
lesions, followed by progressive loss of central vision. The
macular lesions may appear early in life and often precede
the loss of vision by many years. Like retinitis pigmentosa, macular degeneration is usually bilateral. Unlike
retinitis pigmentosa, only the central retina is affected.
The relative incidence and importance of inherited
versus noninherited factors is unclear. In addition, genetic factors are likely to play a role in acquired forms of
macular degeneration. The high frequency of acquired
forms makes it very difficult to determine the incidence of
inherited forms. However, several distinct, heritable
forms of the disease such as autosomal dominant North
Carolina macular dystrophy, autosomal dominant Best
macular dystrophy, and autosomal recessive Stargardt
disease have been mapped. Genes causing the inherited
forms of macular degeneration are very promising candidates for genetic factors that predispose to age-related
macular degeneration.
There are many other inherited diseases that cause
retinal degeneration in humans. Among these are gyrate
atrophy, Norrie disease, choroideremia and various conerod dystrophies. In addition there are numerous inherited, systemic diseases, such as Bardet-Biedl, CharcotMarie-Tooth, and Refsum disease, which include retinal
degeneration among a multiplicity of other symptoms.
2.2. Mapped and cloned genes causing Inherited retinal
degeneration
Table 1 lists mapped and cloned genes causing retinal
degeneration and related diseases in humans. Most of the
diseases have been mapped to specific chromosomal sites
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
453
Daiger et al.: Inherited retinal degeneration
Table 1. Cloned and/or mapped human genes causing inherited retinal diseases" (in chromosomal order)
Symbol''
McKusick no.
Location
Diseases'1, protein
(STDG1)
248200
Ip21-pl3
USH2
RP12
RHO
PDEB
276901
180380
180072
iq
Iq31-q32.1
3q21-q24
4pl6.3
CNCG
RP7
123825
179605
4pl4-ql3
6p21.2-cen
MCDR1
RCD1
RP9
DCMD
RP10
BCP
RP1
VMD1
OAT
USH1C
ROM1
136550
180020
180104
153880
180105
190900
180100
153840
258870
276904
180721
6ql3-ql6
6q25-q26
7p
7pl5-p21
7q
7q31.3-32
8qll-q21
8q24
10q26
Ilpl5-pl3
VMD2
EVR1
VRN1
USH1B
RMCH
USH1A
(RP13)
CORD1
CORD2
RP11
SFD
RS
RP6
DMD
RP3
COD1
PRD
NDP; XLFEVR
153700
133780
193235
276903
216900
276900
600059
120970
Ilql3
Ilql3-q23
Ilql3
Ilql4
136900
312700
312612
310200
312610
304020
312500
310600
18q21-q21.3
19ql3.1-ql3.2
19ql3.4
22ql3-qter
Xp22.2-p22.1
Xp21.3-p21.2
Xp21.3-p21.1
Xp21.1
Xp21.1-pll.2
Xpll.3
Xpll.4-pll.3
CSNB1
RP2
AIED
CHM
RCP
310500
312600
300600
303100
303900
Xpll.4-pll.23
Xpll.4-pll.23
Xpll.4-q21
Xq21.1-q21.3
Xq28
GCP
303800
Xq28
recessive juvenile Stargardt's disease (fundus flavimaculatus)
recessive Usher syndrome, type 2
recessive RP
dominant RP, recessive RP and others; rhodopsin
recessive RP and dominant CSNB; mouse rd, mouse r
and Irish Setter rcdl; cGMP phosphodiesterase P
recessive RP; rod cGMP-gated channel
dominant RP, dominant MD, digenic RP with ROM1
and others; mouse rds; peripherin-RDS
dominant North Carolina MD
dominant retinal-cone dystrophy 1
dominant RP
dominant cystoid MD
dominant RP
dominant tritanopia; blue cone pigment
dominant RP
dominant atypical vitelliform MD
dominant gyrate atrophy; ornithine aminotransferase
recessive Usher syndrome, Acadian
"digenic" RP with RDS, possible dominant RP; rod
outer segment membrane protein 1
dominant Best MD
dominant familial exudative vitreoretinopathy
dominant neovascular inflammatory vitreoretinopathy
recessive Usher syndrome, type 1
recessive rod monochromacy
recessive Usher syndrome, French
dominant RP (RP12 in OMIM)
dominant cone-rod dystrophy 1
dominant cone-rod dystrophy (2)
dominant RP, locus distinct from CORD2
dominant Sorby's fundus dystrophy
X-linked retinoschisis
X-linked RP
Oregon eye disease; may be dystrophin
X-linked RP
X-linked cone dystrophy 1
primary retinal dysplasia
Norrie disease, familial exudative vitreoretinopathy;
Norrie disease protein
X-linked CSNB
X-linked RP
Aland island eye disease
choroideremia; geranylgeranyl transferase A
protanopia and rare retinal dystrophy in blue cone
monochromacy; red cone pigment
deuteranopia and rare retinal dystrophy in blue cone
monochromacy; green cone pigment
Ilql3
14
14q32
17p
"References are in GDB and OMIM, or in the text.
^Symbols in parentheses are pending.
C
'RP = retinitis pigmentosa. MD = macular dystrophy. CSNB = congenital stationary night blindness.
Source:
454
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Daiger et al.: Inherited retinal degeneration
by linkage testing. For genes listed as "cloned," the
causative gene has been isolated, sequenced, and characterized to a limited extent at least. The table is not
exhaustive in that most syndromic and systemic causes of
degeneration are not included. The list is certain to be
outdated soon, because new disease genes are being
mapped and cloned at a rapid pace. The table lists the
GDB-approved symbol, if known, and the McKusick
number. The McKusick number is the code assigned in
OMIM (see n. 1). We use the GDB or OMIM symbols
because they are the most commonly accepted terms, but
we recognize that different symbols may be used with
equal validity (McKusick 1992; Pearson et al. 1992).
2.2.1. Mapped genes causing retinitis pigmentosa and
macular degeneration. The first mapped gene for retinitis
pigmentosa was RP2, a gene for xlRP that maps to Xpll
(Bhattacharya et al. 1984). Subsequently, one form of
adRP was mapped to 3q (McWilliams et al. 1989). Shortly
thereafter the 3q form of adRP was shown to be caused
by mutations in rhodopsin (Dryja et al. 1990a; Farrar et
al. 1990b). Since then additional genes causing adRP
have been mapped to 6p, 7p, 7q, 8q, 17p, and 19q (AlMaghtheh, Inglehearn et al. 1994; Blanton et al. 1991;
Farrar et al. 1991a; Greenberg et al. 1994; Inglehearn et
al. 1993; Jordan et al. 1993). Of these, the gene on 6p has
been identified as the gene for peripherin/RDS; the
others have not been cloned as yet. Two additional genes
for xlRP have also been reported (Muscarella et al. 1990;
Ott et al. 1990). Finally, recessive retinitis pigmentosa
may be caused by mutations either in genes for rhodopsin, PDEB (which maps to 4p), or CNCG (which also
maps to 4p - McGee et al. 1994; McLaughlin et al. 1993;
Rosenfeld et al. 1992). Recently, another form of arRP,
RP12, was mapped to lp, but the causative gene is not
known (van Soest et al. 1994).
The first mapped gene for autosomal dominant macular
dystrophy (adMD) was VMD1, which was assigned to 8q
(Ferrell et al. 1983). Thereafter, the gene for North
Carolina macular dystrophy was mapped to 6q (Small et
al. 1992) and the gene for Best macular dystrophy was
mapped to l l q (Stone et al. 1992). Additional genes
causing adMD have been mapped to 6p and 7p (Jordan et
al. 1992; Kremer et al. 1994). One of these has been
identified as the gene for peripherin/RDS (Jordan et al.
1992; Kajiwara et al. 1991); the others have not been
cloned as yet.
In summary at least 3 genes can cause xlRP (RP2, RP3,
and RP6), 7 can cause adRP (rhodopsin, peripherin/RDS,
RP1, RP9, RP10, RP11, and RP13), 4 can cause arRP
(rhodopsin, PDEB, CNCG, and RP12), and 5 can cause
adMD (peripherin/RDS, MCDR1, DCMD, VMD1, and
VMD2). In addition, genes for Usher syndrome have
been mapped to lq, l i p , l l q , and 14q (Kaplan et al. 1991;
Kimberling et al. 1990; Kimberling et al. 1992; Lewis et
al. 1990; Smith etal. 1992). As indicated in Table 1, many
other retinal conditions have been mapped. For most
disease categories it is important to emphasize that there
are affected families whose disease gene is excluded from
all known loci (e.g., Kumar-Singh et al. 1993).
2.2.2. Cloned genes causing retinal degeneration and
related conditions. From Table 1 and the preceding section we know that mutations in the genes for rhodopsin,
peripherin/RDS, and PDEB can cause either adRP or
arRP. About 30% of adRP cases are caused by rhodopsin
gene mutations and 5% by mutations in the peripherin/RDS gene (Dryja 1992; Kajiwara et al. 1991). Mutations in the PDEB gene may account for 1% to 5% of arRP
cases (McLaughlin et al. 1993); the percent of arRP cases
caused by rhodopsin is unknown. It is known that mutations in the peripherin/RDS gene also can cause adMD.
Furthermore, mutations in rhodopsin and, possibly,
PDEB can cause dominant congenital stationary night
blindness (adCSNB) (Dryja et al. 1993; Rao et al. 1994;
Sieving et al. 1992).
There is an additional form of retinitis pigmentosa with
a mode of inheritance distinct from dominant or recessive: digenic RP (Kajiwara et al. 1994). Degeneration in
these patients is the result of a compound of one mutation
in peripherin/RDS and one in rod outer segment membrane protein 1 (ROM1); neither mutation alone in a
heterozygote causes disease. One implication of this finding is that both peripherin/RDS and ROM1 should be
screened for mutations in patients with retinitis pigmentosa for which the mode of inheritance is unclear.
The genes for peripherin/RDS and PDEB were first
found to be the cause of retinal degeneration in mice
before their connection to human diseases was demonstrated. The recessive mouse disease retinal degeneration slow (rds) is caused by a 10 kb (kilobase) insertion in
the gene for peripherin/RDS (Connell et al. 1991; Travis
et al. 1991). The recessive disease retinal degeneration
(rd) is caused by insertion of a retrovirus gene fragment
(Bowes et al. 1990; 1993) or by a nonsense mutation
(Pittler & Baeher 1991) in the gene for PDEB. The mouse
rd nonsense mutation is identical to the mouse r mutation
described more than 70 years ago (Pittler et al. 1993).
Finally, the autosomal recessive disease rod/cone dysplasia 1 (rcdt) found in the Irish Setter is also caused by a
nonsense mutation in PDEB (Farber et al. 1992; Suber et
al. 1993).
What these findings demonstrate most clearly is the
exceptional heterogeneity of inherited retinal degeneration. Mutations in different genes can cause the same
disease (genetic heterogeneity); different mutations
within the same gene can cause different diseases and
different clinical subtypes (allelic heterogeneity); or the
same mutation can have different clinical consequences in
different individuals (clinical heterogeneity). Thus, it is
not possible to deduce the underlying causative gene or
mutation based on the clinical phenotype of a patient or
family. Eventually psychophysical or electrophysiological
findings may distinguish one disease gene from another
but this is not possible yet.
2.3. Retinal biology relevant to inherited degeneration
Rhodopsin is synthesized in rod inner segments, posttranslationally modified by the addition of oligosaccharides and fatty acids, assembled into vesicles, transported to the connecting cilium, and inserted into nascent
discs. Rhodopsin has 7 transmembrane domains, and is a
member of the G-coupled receptor superfamily (Hargrave & McDowell 1992a; 1992b; Nathans 1992; Stryer
1986). (See Figure la.) The amino-terminus of rhodopsin
is on the intradiscal or lumenal side of disc membranes,
and the carboxyl-terminus is on the cytoplasmic side.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
455
Daiger et al.: Inherited retinal degeneration
cytoplasmic
t
r
a
n
s
m
e
Er
a
n
e
Figure la.
Human rhodopsin
Dark-adapted rhodopsin contains 11-cts retinal as its
photosensitive chromophore, attached by a protonated
Schiffbase to a lysine at codon 296. Light isomerizes the
chromophore to an a\\-trans configuration and activates
rhodopsin into an equilibrium between metarhodopsin I
and metarhodopsin II. Metarhodopsin II binds transiently to transducin. While transducin is bound to rhodopsin, the ot-subunit of the G-protein exchanges a bound
GDP for GTP. The a-subunit of transducin with bound
GTP dissociates from the 0- and 8-subunits and subsequently binds to one of the two inhibitory 8-subunits of
phosphodiesterase. The disinhibited membrane-bound
a- and (3-subunits are then activated to hydrolyze cGMP
to 5'-GMP. Lowering the cytosolic concentration of
cGMP reduces the conductance of a cGMP-gated channel
in the plasma membrane, which hyperpolarizes the cell
and reduces the rate of transmitter release from the rod
synapse. Inactivating this cascade requires phosphorylation of metarhodopsin II by rhodopsin kinase, blockage of
further interaction of metarhodopsin II with transducin
by binding of arrestin (also called S-antigen) to phosphorylated rhodopsin, release and recycling of all-trans
retinal, regeneration of cGMP from GTP by guanylate
cyclase and other related but poorly defined steps. Therefore, although many of the proteins (and their genes)
involved in phototransduction are well characterized,
there are certain to be other genes and proteins involved
that are yet unknown.
456
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Similar mechanisms and homologous proteins are involved in phototransduction within cones, but the details
are different and different genes code for these proteins.
Specifically, there are distinct cone opsins, transducin
subunits, phosphodiesterase subunits, and cGMP-gated
channels (Blatt et al. 1988; Levine et al. 1990; Peng et al.
1992). Thus, rod and cone degeneration may be caused by
functionally similar proteins that are encoded by different
genes.
3. General considerations
How can mutations in the genes for three photoreceptor
proteins - rhodopsin, peripherin/RDS, and PDEB account for several different diseases including adRP,
arRP, adMD, and adCSNB? To address this question,
some generalizations are in order.
First, dominant diseases are often the result of positiveacting mutations, particularly in structural proteins,
whereas recessive mutations are often the result of nullfunction mutations, usually in enzymes.
Second, degenerative retinal diseases may be related
to each other in several ways. Over most of the retina,
rods and cones are closely interdigitated in a dense, twodimensional cellular matrix. They share many metabolites and cofactors and have at least some genes in
common. Thus gene mutations responsible for retinitis
pigmentosa and macular degeneration may affect com-
Daiger et al.: Inherited retinal degeneration
mon metabolic pathways and result in similar end effects
upon photoreceptor physiology. In addition, autosomal
dominant congenital stationary night blindness (adCSNB)
is part of a continuum of rod dysfunction that may result
from either a diminution of rod function or a loss of the
rods themselves.
Third, it is important to recognize inherent biases in
the available data. The known associations between diseases and genes derive from linkage testing in families
and from mutation screening in selected patients. Because many possible genes may cause similar diseases,
absence of linkage in a particular family does not mean
that the excluded gene cannot cause the disease in another family. Further, some families are too small to
establish linkage, and recessive diseases in particular
present special problems for detecting linkage because of
heterogeneity, and other factors. Moreover, mutation
testing has its own bias, namely, the selection of patients
to be tested: if we have not guessed the correct gene to
test then we will not find the underlying mutation. Also,
the problem of many genes causing similar disorders
means that an uncommon molecular cause may be missed
in a modest sample of patients.
Finally, there are technical difficulties with mutation
screening. Several laboratories, our own included, have
tested collections of patients for mutations in rhodopsin
and peripherin/RDS (Dryja et al. 1991; Gannon et al.
1993; Ingleheam et al. 1992; Kajiwara et al. 1991;
Rodriguez et al. 1993b; Sheffield et al. 1991). Less extensive surveys of PDEB and other photoreceptor proteins
have also been conducted (Jacobson & Bascom 1993;
McGee et al. 1992; Ringens et al. 1990). Most mutation
screening is usually done using single-strand conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), GC-clamped DGGE, or heteroduplex
analysis. At best, each of these methods is only 90%
effective, thus some mutations are missed. In addition,
since multiple polymerase chain reactions (PCR) are involved, the larger the gene the more cumbersome the
test. For example, rhodopsin with 5 exons and peripherin/RDS with 3 exons are amenable to PCR analysis, but
PDEB with 22 exons is considerably more challenging
(Kajiwara et al. 1991; McLaughlin et al. 1993; Rodriguez
et al. 1993a). Thus, because present methods for detecting mutations are very laborious, no current research
group has tested a multiplicity of genes in a wide range of
patients. We hope that as new techniques are realized,
such as analysis of illegitimate transcripts and improvements in DNA sequencing, these limitations will be
resolved.
3.1. Available data
Despite the inherent limitations of linkage testing and
mutation screening, a large number of disease-causing
mutations have been reported. At least 73 mutations in
the rhodopsin gene have been described. These are listed
in Table 2, including unpublished mutations from our
laboratory. At least 23 peripherin/RDS mutations and 5
PDEB mutations, plus many benign variants, have been
described (Humphries et al. 1993; McLaughlin et al.
1993). These mutations constitute the raw material for
beginning the elucidation of the connection between
genotype and phenotype. In this review we focus on the
rhodopsin mutations, because more rhodopsin-related
diseases have been reported, because better clinical data
are available, and because rhodopsin is better characterized than peripherin/RDS or PDEB.
3.2. Why do these proteins cause these diseases?
3.2.1. Rhodopsin. Rhodopsin is a membrane-spanning
protein that comprises the bulk of ROS protein. Thus
mutations that change its tertiary structure within the
membrane should have a dominant effect since, in this
context, rhodopsin has a structural role. Yet rhodopsin
also has a catalytic role in phototransduction so that, in
this context, mutations may be recessive. Still other
mutations may simply perturb phototransduction without
cell damage, resulting in congenital stationary night
blindness in which the perturbation may act dominantly
or recessively. We will show examples of each of these
possibilities.
Do mutations in cone opsins cause cone degeneration?
Abnormalities in cone opsins are generally associated
with color blindness, not degeneration (Nathans 1992;
Nathans et al. 1992). X-linked deuteranopia is caused by
absence of green function, X-linked protanopia is caused
by absence of red function, and autosomal dominant
tritanopia is caused by absence of blue function. X-linked
blue cone monochromacy results from absence of both
red and green function (Nathans et al. 1993). The molecular basis of these diseases is well established (Nathans
1992). The red and green cone opsins are X-linked; blue
cone opsin maps to 7q. Deuteranopia, protanopia, and
tritanopia can be caused by missense mutations, nonsense
mutations, deletions, or rearrangements in each applicable gene. Tritanopia can be caused by multiple mutations,
by rearrangements of the red and green opsins, or by
deletion of a locus control region for these genes. However, none of the known mutations causes cone degeneration. Retinal degeneration is occasionally seen with blue
cone monochromacy (mutations unknown) but never with
dominant tritanopia. It may be that the density of each
cone type taken separately is too low to cause recognizable degeneration, especially the blue cones which are
absent from the central retina. Also, developmental pathways during embryogenesis may simply exclude production of cones with an aberrant opsin, compensating for the
deficit with additional normal cones. In any case, this
remains a challenging mystery.
3.2.2. Peripherin/RDS. Peripherin/RDS, like rhodopsin,
is a membrane-spanning protein found in photoreceptor
discs (Arikawa et al. 1992; Connell et al. 1991; Travis etal.
1991). Unlike rhodopsin, it is expressed both in rods and
in cones. Peripherin/RDS has four transmembrane domains, so both the amino-terminus and carboxylterminus are on one side of the disc, the cytoplasmic side.
Very little is known about the function of peripherin/RDS. In the disc membrane it forms a homodimer
and is associated with another membrane-spanning protein. ROM1, which is about 30% similar in sequence to
peripherin/RDS (Bascom et al. 1992). These two proteins
are found predominantly around the rim of discs in rods
where, it is speculated, they contribute to the formation
and maintenance of the disc structure.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
457
Daiger et al.: Inherited retinal degeneration
Table 2. Rhodopsin mutations in humans
Codon
no.
Codon
change
Codon location or function; sequence conservation"
4
ACA-> AAA
1st intradiscal
Thr4Lys (T4K)
15
AAT-> AGT
Asnl5Ser
(N15S)
17
A C G ^ > ATG
1st intradiscal, glycosylated; conserved in
vertebrates
1st intradiscal
23
C C C - >CAC
23
CCC -» CTC
28
Mutation 6
Clinical consequences; clinical phenotype; comment 0
References
A. Disease-causing missense mutations
dominant RP; affects glycosylation of Asn-2 (possibly not incorporated
into membrane)
dominant RP; type 2, regional
Bunge et al. 1993
Thrl7Met
(T17M)
dominant RP; type 2, regional, retinal neovascularization in Japanese;
class Ha, multiple families, C -» T in CpG
1st intradiscal; conserved in vertebrates
Pro23His
(P23H)
Pro23Leu
(P23L)
C A G - • CAC
1st intradiscal; conserved in vertebrates
1st intradiscal
dominant RP; type 2, regional, variable severity
(some with normal function); class Ha, 10% of
USA Caucasians
dominant RP; class Ha
Bunge et al. 1993;
Dryja et al. 1991;
Fishman et al.
1992b; Fujiki et al.
1992; Hayakawa et
al. 1993; Sheffield et
al. 1991
Berson et al. 1991a;
Dryja et al. 1990b;
Heckenlively et al.
1990; Stone et al.
1991
Dryja et al. 1991
dominant RP
Bunge et al. 1993
40
C T G - > CGG
1st transmembrane
GIn28His
(Q28H)
Leu40Arg
(L40R)
dominant RP; early onset,
severe
45
T T T - * CTT
1st transmembrane
dominant RP; class I
46
C T G - • CGG
1st transmembrane
51
G G C - » GCC
1st transmembrane
51
GGC -> CGC
1st transmembrane
51
GGC
GTC
1st transmembrane
53
C C C -»CGC
1st transmembrane
58
A C G - > AGG
1st transmembrane
Phe45Leu
(F45L)
Leu46Arg
(L46R)
GIy51Ala
(G51A)
GIy51Arg
(G51R)
Gly51Val
(G51V)
Pro53Arg
(P53R)
Thr58Arg
(T58R)
Al-Maghtheh et al.
1994; Kim et al.
1993
Sung et al. 1991a
87
G T C - > GAC
2nd transmembrane
dominant RP; class lib
89
G G T - »GAT
2nd transmembrane
90
G G C - •* GAC
2nd transmembrane
VaI87Asp
(V87D)
Gly89Asp
(G89D)
Gly90Asp
(G90D)
-H»
Kranich et al. 1993;
Sullivan et al. 1993a
dominant RP; type 1, rapid
progression
probable dominant RP
Rodriguez et al. 1993a
dominant RP
Dryja et al. 1992
dominant RP; class I
Dryja et al. 1991
dominant RP; class lib
Inglehearn et al. 1992
dominant RP; type 2, regional; class lib, multiple
families, reduced transducin activation
Bunge et al. 1993;
Dryja et al. 1990a;
Fishman et al. 1991;
Min et al. 1993;
Moore et al. 1992;
Richards et al. 1991
Sung et al. 1991a
dominant RP; class lib
dominant congenital stationary night blindness;
constitutively activates
transducin w/o chromophore
Macke et al. 1993
Dryja et al. 1991;
Sung et al. 1991a
Rao et al. 1994; Sieving et al. 1992
(continued)
458
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Daiger et al.: Inherited retinal degeneration
Table 2. (Continued)
Codon location or function; sequence conservation"
Codon
no.
Codon
change
106
CGG -» AGC
1st intradiscal loop;
conserved in vertebrates
GlylO6Arg
(G106R)
dominant RP; mild, regional; class lib, multiple
families
106
GGC - * TGG
GlylO6Trp
(G106W)
dominant RP; class lib
110
TGC -> TAC
CysllOTyr
(C110Y)
dominant RP
Dryja et al. 1992
125
CTG -> CGG
1st intradiscal loop;
conserved in vertebrates
1st intradiscal loop,
disulf. w/187; conserved in G-coupled
receptors
3rd transmembrane
Fishman et al. 1992;
Inglehearn et al.
1992; Macke et al.
1993
Sung et al. 1991a
dominant RP; class lib
Dryja et al. 1991
135
CGG -* GGG
Leul25Arg
(L125R)
Argl35Gly
(R135G)
dominant RP; class lib
Bunge et al. 1993;
Macke et al. 1993
135
CGC -» CTG
Argl35Leu
(R135L)
Andr6asson et al. 1992
135
CCG -» CTT
135
CGC -» CCG
dominant RP; rapid progression, severe; Swedish
family, distinct from
Argl35Leu (CGC -»
CTT) below
dominant RP; severe; class
lib, absent transducin
activation; unusual GG
—> TT mutation
dominant RP
135
CCC -> TGG
Argl35Trp
(R135W)
dominant RP; severe; class
lib, absent transducin
activation
Sung et al. 1991a
140
TGT -» TCT
Cysl40Ser
(C140S)
dominant RP
Macke et al. 1993
167
TGC -> CGC
dominant RP; class lib
Dryja et al. 1991
171
CCA -» CTA
Cysl67Arg
(C167R)
Prol71Leu
(P171L)
dominant RP; class Ha
Dryja et al. 1991
171
CCA -» TCA
Prol71Ser
(P171S)
dominant RP
Stone et al. 1993;
Vaithinathan et al.
1993
178
TAC -» TGC
Tyrl78Cys
(Y178C)
Bell et al. 1992; Farrar
et al. 1991b
180
CCC -» CCC
dominant RP; type 1, diffuse, early onset; class
Ha, multiple families
from British Isles
dominant RP
3rd transm., transducin activation;
conserved in
G-coupled receptors
3rd transm., transducin activation;
conserved in
G-coupled receptors
3rd transm., transducin activation;
conserved in
G-coupled receptors
3rd transm., transducin activation;
conserved in
G-coupled receptors
3rd transm., transducin activation;
conserved in
G-coupled receptors
2nd cyto. loop, transducin interaction;
conserved in vertebrates
4th transmembrane
4th transmembrane;
conserved in vertebrates and invertebrates
4th transmembrane;
conserved in vertebrates and invertebrates
2nd intradiscal loop
2nd intradiscal loop;
conserved in vertebrates and invertebrates
Mutation 6
Argl35Leu
(R135L)
Argl35Pro
(R135P)
Prol80Ala
(P180A)
Clinical consequences; clinical phenotype; comment c
References
Jacobson et al. 1991;
Min et al. 1993;
Sung et al. 1991a
Rodriguez et al. 1993a
Rodriguez et al.,
unpublished
(continued)
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
459
Daiger et al.: Inherited retinal degeneration
Table 2. (Continued)
Codon
no.
Codon
change
Codon location or function; sequence conservation"
181
GAG -» AAG
182
GGC -» AGC
186
TCG -^ CCG
187
TGT -> TAT
188
GGA -+ AGA
188
GGA -> GAA
190
Mutation^
Clinical consequences; clinical phenotype; comment0
2nd intradiscal loop
Glul81Lys
(E181K)
dominant RP; type 1; class
Ha, multiple families
2nd intradiscal loop;
conserved in vertebrates and invertebrates
2nd intradiscal loop;
conserved in vertebrates
2nd intradiscal loop,
disulfide w/110;
cons, in vet. and invet.
Glyl82Ser
(G182S)
dominant RP; mild, regional; class Ila
Bunge et al. 1993;
Dryja et.al. 1991;
Rodriguez et al. unpub.; Saga et al.
1993
Fishman et al. 1992b;
Sheffield et al. 1991
Serl86Pro
(S186P)
dominant RP; class Ila
Dryja et al. 1991
Cysl87Tyr
(C187Y)
Nathans et al. 1993;
Scott et al. 1993
Glyl88Arg
(G188R)
Glyl88Glu
(G188E)
dominant RP; class Ila
Macke et al. 1993
GAC -» AAC
2nd intradiscal loop;
conserved in vertebrates
2nd interdiscal loop;
conserved in vertebrates
2nd intradiscal loop
dominant RP; severe; polymorphic C203R mutation
at homologous site in
green opsin causes inactivation but not degeneration
dominant RP; class Ila
Aspl90Asn
(D190N)
dominant RP; mild, regional; class Ila, multiple
families
190
GAC -» GGC
2nd intradiscal loop
dominant RP; class Ila
190
GAC -»• TAC
2nd intradiscal loop
207
ATG -+ AGG
5th transmembrane
209
GTG -» ATG
5th transmembrane
211
CAC -»• CGC
5th transmembrane,
strongly stabilizes
inetarhodopsin II
Aspl90Gly
(D190G)
Aspl90Tyr
(D190Y)
Met207Arg
(M207R)
Val209Met
(V209M)
His211Arg
(H211R)
Bunge et al. 1993;
Dryja et al. 1991;
Keen et al. 1991;
Rodriguez et al.
1993b
Dryja et al. 1991;
Sung et al. 1991a
Fishman et al. 1992c
211
CAC -> CCC
His211Pro
(H211P)
dominant RP; class Ila
216
ATG - * AAG
Met216Lys
(M216K)
dominant RP
220
TTT^TGT
5th transmembrane,
strongly stabilizes
inetarhodopsin II
5th transmembrane;
Leu found in all
other opsins
5th transmembrane
dominant RP
Bunge et al. 1993
222
TGC -» CGC
5th transmembrane
dominant RP
Bunge et al. 1993
267
CCC -» CTC
6th transmembrane;
conserved in vertebrates
Phe220Cys
(F220C)
Cys222Arg
(C222R)
Pro267Leu
(P267L)
dominant RP; mild, regional; class Ha, P264S
mutation in homologous
site in blue opsin causes
dominant tritanopia
Fishman et al. 1992c;
Sheffield et al. 1991;
Weitz et al. 1992b
dominant RP; severe, diffuse
dominant RP; type 1, severe; first Irish family
probable dominant RP
dominant RP; type 1-type
2; class Ila, multiple
families
References
Bunge et al. 1993;
Dryja et al. 1991
Farrar et al. 1992
Macke et al. 1993
Macke et al. 1993;
Reig et al. 1994;
Rodriguez et al.
1993a; Weitz &
Nathans 1992a
Keen et al. 1991;
Weitz & Nathans
1992a
Al-Maghtheh et al.
1994a
(continued)
460
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Daiger et al.: Inherited retinal degeneration
Table 2. (Continued)
Codon
no.
Codon
change
Codon location or function; sequence conservation"
292
CCC -> CAG
7th transmembrane
Ala292Glu
(A292E)
296
AAC -» CAG
Lys296Glu
(K296E)
296
AAG -> ATC
328
CTG -+ CCG
7tli transmembrane,
11-cis retinal attachment; cons, in vet.
& invet.
7th transmembrane,
11-cts retinal attachment; cons, in vet.
& invet.
last cytoplasmic region
341
GAG -» AAG
342
ACG -» ATG
345
GTG -» TTG
or CTG
345
CTG -» ATC
347
CCG -» GCG
347
CCG -» CGG
347
CCC -> CAC
347
CCC -»• CTG
347
CCG -» TCG
347
CCC -» ACC
last cytoplasmic region; conserved in
vertebrates
last cytoplasmic region, phosphorylated
last cytoplasmic region; conserved in
vertebrates
last cytoplasmic region; conserved in
vertebrates
last cyto. region; penultimate proline
conserved in vertebrates
last cyto. region; penultimate proline
conserved in vertebrates
last cyto. region; penultimate proline
conserved in vertebrates
last cyto. region; penultimate proline
conserved in vertebrates
last cyto. region; penultimate proline
conserved in vertebrates
last cyto. region; penultimate proline
conserved in vertebrates
Mutation 6
Clinical consequences; clinical phenotype; comment 0
References
dominant congenital stationary night blindness;
constitutively activates
transducin w/o chromophore
dominant RP; type D, severe; constitutively activates transducin
Dryja et al. 1993
dominant RP; profound
early rod loss, central
cone involvement minimal
dominant RP
Sullivan et al. 1993
dominant RP; mild
Scott et al. 1993
Thr342Met
(T342M)
dominant RP; C —* T in
CpG
Stone et al. 1993
Val345Leu
(V345L)
dominant RP
Vaithinathan et al.
1993
Val345Met
(V345M)
dominant RP; moderate,
variable severity; class I
Pro347Ala
(P347A)
dominant RP
Berson et al. 1991c;
Bunge et al. 1993;
Dryja et al. 1991
Stone et al. 1993
Pro347Arg
(P347R)
dominant RP; type 1, moderate; multiple families
Pro347Gln
(P347Q)
dominant RP
Pro347Leu
(P347L)
dominant RP; type 1, diffuse, early onset; class I,
multiple families including European and Japanese; C —* T in CpG
Pro347Ser
(P347S)
dominant RP; type 1;
class I
Apfelstedt-Sylla et al.
1992; Berson et al.
1991b; Bunge et al.
1993; Dryja et al.
1990; Fujiki et al.
1992; Hotta et al.
1992; Nakazawa et
al. 1991; Orth et al.
1991; Shiono et al.
1992a
Bunge et al. 1993;
Dryja et al. 1990a
Pro347Thr
(P347T)
dominant RP
Rodriguez et al. 1993b
Lys296Met
(K296M)
Leu328Pro
(L328P)
Glu341Lys
(E341K)
Hunge et al. 1993;
Keen et al. 1991
Rodriguez et al. 1993b
Bunge et al. 1993; Gal
et al. 1991;
Neimeyer et al.
1992
Vaithinathan et al.
1993
(continued)
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
461
Daiger et al.: Inherited retinal degeneration
Table 2. (Continued)
Codon
no.
Codon
change
Codon location or function; sequence conservation"
64
C A G ^ TAG
1st cytoplasmic loop
249
GAG-> TAG
remainder of protein;
conserved in vertebrates
344
C A C - * TAG
last cytoplasmic region
through carboxyterminus
Mutation 6
Clinical consequences; clinical phenotype; comment 0
References
B. Disease-causing premature stop mutations
Gln64stop
(Q64X)
Glu249stop
(E249X)
Gln344stop
(Q344X)
dominant RP; mild, some
with normal retina
recessive RP; remainder
missing starting with 6th
transmemb. domain;
heterozygotes at lower
limit of ERG response to
dim blue flash; early
night blindness
dominant RP; mild, some
with normal retina; class
I; deletes terminal GlnVal-Ala-Pro-Ala
Jacobson et al. 1994;
Macke et al. 1993
Rosenfeld et al. 1992
Jacobson et al. 1991;
Jacobson et al. 1994;
Sung et al. 1991a
C. Disease-causing insertions, deletions and splice-site mutations
68-71
—
1st cytoplasmic loop
A68-71
(496dell2)
255/256
ATC -> —
6th transmembrane
AIle255/256
(1057del3)
264
TGC - * —
ACys264
(1084del3)
dominant RP
(312313)
—
6th transmembrane;
conserved in vertebrates
last cytoplasmic region
through carboxyterminus
1230 + 1G
(312313)
last cytoplasmic region
through carboxyterminus
1231 - 1G
-> A
(312313)
last cytoplasmic region
through carboxyterminus
1231-23del
30insl50
dominant RP; highly variable expression (Macke et
al. 1993), some with normal retina or unaffected
(Rosenfeld et al. 1992); intron 4 donor splice site G
—» T eliminating terminal
36 amino acids
dominant RP; type 2, mild;
intron 4 acceptor splice
site G —* A, effect on
protein unknown
dominant RP; type 2, regional; 30 bp deletion of
3' intron 4 (23 bp) and 5'
expon 5 (7 bp) plus 150
bp insertion
dominant RP; type 1-type
2; 17 bp out of frame deletion, new stop at codon
346
dominant RP; mild, late onset, diffuse; 1 bp deletion
replaces terminal 9 AA's
with 19 new AA's (preserves terminal Pro-Ala)
dominant RP; mild, slow
progression; deletion of
stop codon may lengthen
to 386 amino acids
332
GAG -+ G—
last cytoplasmic region, phosphorylated
A332stop
(1289dell7)
340
ACG -» A-G
last cytoplasmic region, phosphorylated
A340
(1313del C)
340-348
—
last cytoplasmic region
through carboxyterminus, phosphorylated
A340-348
(1312del24)
dominant RP; mild; class
Ila;, deletes Leu-ArgThr-Pro, does not bind
11-cts retinal
dominant RP; type 1, severe, diffuse; class Ila,
multiple families
Keen et al. 1991; Min
et al. 1993
Artlich et al. 1992;
Bunge et al. 1993;
Inglehearn et al.
1991
Vaithinathan et al.
1993
Jacobson et al. 1994;
Macke et al. 1993;
Rosenfeld et al.
1992
Rodriguez et al. unpub.
Al-Maghtheh et al.
1994a; Kim et al.
1993
Rodriguez et al. 1993b
Horn et al. 1992
Restagno et al. 1992
(continued)
462
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Daiger et al.: Inherited retinal degeneration
Table 2. (Continued)
Codon
no.
Codon
change
341-343
Codon location or function; sequence conservation"
last cytoplasmic region, phosphorylated
Clinical consequences; clinical phenotype; comment 1
Mutation*
A341-343
(1315del8)
dominant RP; mild; 8 basepair deletion replaces
terminal 8 AA's with 9
new AA's (preserves terminal Pro-Ala)
References
Horn et al. 1992;
Bunge et al. 1993
D. Silent substitutions, benign variants and polymorphisms
—
—
5' noncoding region
296A - • G
104
C T C - * ATC
VallO4Ile
(V104I)
intron 1
—
1st intradiscal loop;
conserved in vertebrates
—
120
GGC -> CCT
3rd transmembrane
146
T T C - » TTT
160
ACC -» ACT
2nd cytoplasmic loop,
transducin interaction
4th transmembrane
173
CCC -* GCT
4th transmembrane
186
TCG^-TCA
244
CAG - ^ CAA
248
AAG -» AAA
297
AGC -* AGT
2nd intradiscal loop;
conserved in vertebrates
3rd cytoplasmic, transducin interaction;
conserved in vertebrates
3rd cytoplasmic, transducin interaction
7th transmembrane
—
—
—
—
intron 4
3' noncoding region
normal variant—
polymorphic with 14%
frequency
normal variant
Sung et al. 1991c
Weber & May 1989
Glyl20Gly
(654C -> T)
Polymorphic microsatellite
variation in intron 1
normal variant—silent substitution
Phel46Phe
(732C -> T)
normal variant—silent substitution
Thrl60Thr
(774C -» T)
Reig et al. 1994; Sung
et al. 1991a; Macke
et al. 1993
Alal73Ala
(813C -» T)
Serl86Ser
(852G -> A)
normal variant—infrequent
silent substitution; ACC
-> ACA (T160T) found in
a Spanish family
normal variant—silent substitution; C -» T in CpG
normal variant—silent substitution
Gln244Gln
(1026G - » A)
normal variant—silent substitution
Rosenfeld et al. 1992
Lys248Lys
(1038G -* A)
Ser297Ser
(1185C -H> T)
normal variant—infrequent
silent substitution
normal variant—silent substitution; C —> T in CpG
Sung et al. 1991a
1231-23G -» A
1338 + 46C
-» A
normal variant—infrequent
normal variant-polymorphic
with 13% frequency
[CA]n
Macke et al. 1993
Bunge et al. 1993;
Dryja et al. 1991;
Macke et al. 1993
Macke et al. 1993
Bunge et al. 1993;
Dryja et al. 1991
Rosenfeld et al. 1992
Rosenfeld et al. 1992;
Saga et al. 1993;
Macke et al. 1993
Sung et al. 1991a
Sung et al. 1991a
"Based on Fryxell and Meyerowitz (1991) with 6 vertebrate opsins (human red, green and blue; human, pig ovine, and bovine
rhodopsin), 2 Drosophila opsins, and 10 additional, homologous G-coupled protein receptors.
''Nomenclature based on Beaudet and Tsui (1993), numbering nucleotides from the 5' start of the cDNA (including 294 untranslated
nucleotides) (GenBank Accession K02281).
'Class refers to rhodopsin cDNA transfection experiments in human embryonic kidney cells by Sung et al. (1991b; 1993). Class I =
wild typo characteristics; class Ha = distinctly abnormal and remains in endoplasmic reticulum; class lib = distinctly abnormal but
some localizes in plasma membrane.
Diseases caused by mutations in peripherin/RDS run
the gamut of dominant retinal degeneration, including
adRP, adMD, retinitis punctate albescens, fundus flavimaculatus, and various pattern dystrophies (Kajiwara et al.
1993; Keen et al. 1994; Nichols et al. 1993; Wells et al.
1993). Furthermore, one mutation, a 3 bp deletion of a
lysine at codon 153 or 154, causes retinitis pigmentosa,
pattern macular dystrophy, and Stargardt dystrophy in
different members of the same family (Weleber et al. 1993).
In the absence of a clear understanding of the functional domains of peripherin/RDS it would be naive to try
to explain these findings mechanistically. Since the gene
for peripherin/RDS is expressed both in rods and in
cones, some domains may be important for rod function,
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
463
Daiger et al.: Inherited retinal degeneration
some for cone function, and some for both. ROM1 is not
expressed in cones, so one distinguishing domain may be
the R0M1 binding site. (Mutations in patients with "digenic" disease may point to sites of interaction between
peripherin/RDS and R0M1; McGee et al. 1994.) It is
reasonable to speculate that destabilization of the disc
membrane would be very damaging to the photoreceptor.
Why some mutations are highly variable in their expression is currently unknown, though this suggests that
genetic background and environmental factors play important roles. At present, the limited number of known,
disease-causing mutations is insufficient to make meaningful correlations. However, the accumulating number
of pedigrees with mutations in the peripherin/RDS gene
may prove instructive.
3.2.3. Phosphodiesterase p-subunit(PDEB). Mutations in
PDEB are associated with arRP and adCSNB (Gal et al.
1994; McLaughlin et al. 1993). Reduced or absent PDEB
should retard hydrolysis of cGMP and make photoreceptors less sensitive to light. Thus, dominant CSNB is a
logical outcome. Recessive degeneration, though, is less
expected. The reported PDEB mutations causing arRP
are compound heterozygotes of three possible nonsense
mutations, one out-of-frame deletion, and one missense
mutation (McLaughlin et al. 1993). These mutations cause
typical retinitis pigmentosa with early onset and no detectable rod ERG in adulthood. They are likely to be nullfunction mutations that, among other consequences,
would elevate cGMP levels.
3.2.4. Why do these mutations lead to cell death? For
many photoreceptor mutations the initiating cause of
retinal degeneration can be inferred, such as membrane
instability, aberrant activation of phototransduction,
or increased cGMP levels. Studies of retinal degeneration
in animals suggest that increased cGMP may be toxic to
photoreceptors (Lolley et al. 1977; Ulshafer et al. 1980).
The connection between increased cGMP and cell death
is not known, but increased cellular permeability, direct
cytotoxicity, or unbalanced Ca 2 + /Na + flows are possibilities (Hargrave & McDowell 1992b). Why these various
conditions lead to degeneration is unclear. One possibility is a simple necrotic process, wherein the cell ceases
its metabolic activity, its membranes decompose, and
phagocytic activity - either by the retinal pigment epithelium (RPE) or by infiltrating macrophages - removes the cellular debris. An alternative is an enhanced,
catabolic interaction between the RPE and the ROS,
possibly exacerbated by a feedback loop triggered by
accumulating disc debris. However, recent evidence implicates apoptosis, a form of programmed cell death, as
the mechanism responsible for photoreceptor cell death
in rodent models of retinal degeneration. In apoptosis,
genetic switches in the cell initiate a process that turns off
normal cellular activity and causes condensation of nuclei, DNA degradation, and cell death. The cardinal
features of apoptosis have been observed in the rd mouse,
the rds mouse, and a transgenic mouse with the rhodopsin Pro347His mutation (Chang et al. 1993; Lolley et al.
1994). Thus, it seems likely that some forms of retinal
degeneration in humans may result from apoptosis. However, these observations do not preclude alternative
mechanisms in other forms of degeneration. Further464
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
more, they beg the question of what triggers apoptosis in
the first place.
4. Clinical consequences of rhodopsin mutations
Table 2 summarizes data on human rhodopsin mutations
from many sources (references are in Table 2 unless
otherwise noted). The table gives the codon, nucleotide
change, and amino acid change for each mutation. In
addition, functional properties, protein domain, 3 and
evolutionary conservation of affected amino acids are
given (Fryxell & Myerowitz 1991; Hargrave & O'Brien
1991; Nathans et al. 1993). Also listed is the type of disease
observed, clinical features of the disease, if reported, and
comments. Included in the comments is the biochemical
class (I, Ha, or lib) based on transfection-expression
experiments by Sung and colleagues (Sung & Schneider
et al. 1991; 1993).
The table is grouped into disease-causing missense
mutations; nonsense mutations; deletions, insertions, or
splice-site mutations; and benign variants. There are 73
reported disease-causing mutations of the rhodopsin
gene, including 63 missense mutations, 3 nonsense mutations, and 7 rearrangements. Of these, 70 cause dominant
retinal degeneration, 2 cause adCSNB, and 1 causes
recessive retinal degeneration (only homozygotes or compound heterozygotes are affected). The locations of the
missense mutations are diagrammed in Figure lb. This
organization emphasizes the missense mutations, because simple amino acid substitutions are easiest to interpret in terms of functional consequences. Thus, most of
the following discussion is based on the 63 missense mutations, although the other mutations are also considered.
Important functional domains in rhodopsin include the
intradiscal, transmembrane, and cytoplasmic domains.
Amino acids with critical functions include the acetylated
amino-terminus, glycosylated asparagines at codons 2 and
15, cystines at codons 110 and 187, which form a disulfide
bound (Karnik & Khorana 1990), the glutamate at codon
113, which provides a counterion for the Schiff base
linkage to codon 296, lipid-binding cystines at codons 322
and 323, which attach to the cytoplasmic side of the
membrane, and phosphorylation sites at codons 334, 336,
338, 340, 342, and 343 (Figure la; Hargrave & O'Brien
1991).
Mutations in the rhodopsin gene have also been classified by the severity of the disease produced, if reported.
There are many ways to classify retinitis pigmentosa: by
type, by progression, by fundus findings, by ERG findings, and so on. Several classification schemes have been
proposed (Fishman et al. 1985; Heckenlively 1988; Lyness et al. 1985; Massof & Finkelstein 1981). Unfortunately, different patients have been classified in different ways. Lacking uniform classification criteria (an
undertaking we strongly advocate), we have adopted the
simplest possible clinical categories. Thus in what follows
we equate "mild" adRP with type 2 or R (late onset and
slow progression), and "severe" adRP with type 1 or D
(early onset and rapid progression). "Moderate" is intermediate. We also consider adCSNB and arRP as inherently mild. We do not incorporate ERG profiles or funduscopic findings though, needless to say, they would be
relevant to a more sophisticated analysis. Based on these
Daiger et al.: Inherited retinal degeneration
cytoplasmic
Figure lb.
Missense mutations
criteria, a total of 40 rhodopsin mutations can be classified, admittedly a small sample.
4.1. General considerations
4.1.1. Population distribution. The majority of mutations
in Table 2 are unique, occurring in one patient or one
family only. A few mutations occur in multiple, unrelated
families. Among the latter are the Thrl7Met, Thr58Arg,
and Pro347Leu mutations found in .both Europe and
Japan; the Pro23His mutation found in nearly 10% of
American Caucasian adRP patients (but not in Europeans;
Farrar et al. 1990a); the Thrl78Cys and AIle255/256
mutations found in the British Isles; and the GlylO6Arg,
GlulSlLys, Aspl90Asn, His211Arg, and Pro347Arg mutations found in European and American families.
Do these multiple pedigrees represent multiple mutational events or descent from a common ancestor (founder
effect)? A single chromosomal haplotype is in complete
linkage disequilibrium with the Pro23His mutation, providing strong evidence for founder effect (Dryja et al.
1991). In addition, those mutations confined to a limited
geographical range, such as the Tyrl58Cys and AIle255/
256 mutations, are probably the result of founder effect.
By contrast, molecular evidence suggests that the
Thr58Arg and Pro347Leu mutations arose on multiple
occasions. Also, it seems likely that the mutations found in
both Europe and Japan arose independently. The main
effect of this uncertainty is to imply that Table 2 should
include multiple entries for some of the mutations. Unfortunately the multifamily mutations are not: helpful in
population screening because, taken in aggregate, they
represent only a small percent of all reported rhodopsin
mutations.
4.1.2. General distribution and types of mutations. Based
on Table 2, the ratio of transitions to transversions is
43:32, which is not significantly different from the expected ratio of 2:1 in mammalian genes (Nei 1987). Although a few C—»T transitions in CpG dinucleotides are
observed, the number is not exceptional (Barker et al.
1984). Most of the amino acid substitutions change the
charge, hydrophobicity, or size of the wild-type residue,
but this also is not exceptional, since most random
changes would affect one or more of these properties.
However, there are 18 evolutionarily conserved sites in
human rhodopsin (Fryxell & Meyerowitz 1991); 29% of
the 63 missense mutations occur at conserved sites,
whereas only 5% are expected by change alone. Thus,
substitutions at conserved sites are significantly more
likely to cause retinal degeneration.
There are two other striking anomalies in the distribution of mutations - first, in the number of codons with
multiple, independent mutations and, second, in the
distribution of mutations within protein domains. For the
pedigrees with rhodopsin-based retinal degeneration
studied to date, 305 of the 348 amino acids in human
rhodopsin have zero mutations, 32 have one mutation
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
465
Daiger et al.: Inherited retinal degeneration
each, 6 have two, 3 have three, 1 has five (arginine at
codon 135), and 1 has six mutations (proline at codon 347).
The expected random distribution would be 290 amino
acids with zero mutations, 53 with one, 5 with two, 0.30
with three, and fewer than 10~6 with five or more
(Chakraborty 1993). Thus, codons 135 and 347 are highly
unusual, particularly considering the multiple, independent families with a Pro347Leu mutation. Note that
although both codons contain a CpG dimer, this is an
insufficient explanation, since many other codons contain
the same dimer.
At a superficial level the mutations seem uniformly
distributed between the intradiscal, transmembrane, and
cytoplasmic regions of the protein. About 50% of rhodopsin amino acids are within the membrane and 25% are on
each side. The observed distribution of mutations is
19:32:12, which is not significantly different from 1:2:1.
However, nearly all the cytoplasmic mutations are in the"
eight terminal amino acids; there is a paucity of mutations
in the four cytoplasmic loops. The cytoplasmic loops
include the binding sites for the a-subunit of transducin
(Konig et al. 1989; Min et al. 1993) and arrestin. We
speculate that mutations may be as frequent here as
elsewhere in the molecule, but that they do not cause
adRP.
4.1.3. General distribution of clinical types. There are no
striking anomalies in the distribution of mild, moderate,
or severe mutations. Six intradiscal mutations are mild
and four are severe; two transmembrane mutations are
mild (four counting adCSNB) and eight are severe; and
two cytoplasmic mutations are mild and two are severe.
Thus, protein domain alone does not determine severity
of disease.
It is interesting that many of the "mild" forms of adRP
caused by rhodopsin mutations show a sectorial phenotype
in some patients. These include the Thrl7Met, Pro23His,
Thr58Arg, GlylO6Arg, Glyl82Ser, and Pro267Leu mutations. Given that clinicians will differ in attributing this
phenotype to a particular patient, the evidence suggests
that sectorial adRP is not a separate genetic category.
4.1.4. Distribution of biochemical classes. Sung and colleagues (Sung & Schneider et al. 1991; 1993) have used
site-directed mutagenesis to distinguish broad classes of
rhodopsin mutations. They constructed expression vectors containing the human rhodopsin sequence with
specific mutations and used these to transfect human
embryonic kidney cells. They then evaluated synthesis,
transport, opsin binding capability, and immunologic
properties of the mutant rhodopsins. In total, they tested
21 human mutant types. Based on their findings they
classified the mutants into three classes, class I with wildtype properties, class Ha with aberrant properties and
complete failure to leave the site of synthesis, and class
lib with aberrant properties but partial transport to the
plasma membrane. Class I mutants clustered within the
first transmembrane domain and at the extreme carboxylterminus. Class II mutants clustered within the other
transmembrane and extracellular domains (Sung et al.
1993). There are clinical data on 18 of the 20 mutations
tested. Nine of the mutations produce mild disease, 7
produce severe disease, and 2 produce moderate disease.
Surprisingly, there is no simple correlation between se466
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
verity and class: the ratios of mild-to-severe for class I are
1:2, for class Ila, 6:3, and for class lib, 2:2.
4.2. The connection between phenotype and genotype
of specific rhodopsin mutations
Within the limits of the data cited above, specific rhodopsin genotypes can be correlated with broad phenotypic
categories. Different mutations cause retinal degeneration for distinctly different reasons. However, there is an
organizing principal: the clinical phenotype is a consequence of where and when the mutation affects the
function of rhodopsin.
4.2.1. Mutations producing no protein or unstable protein
will be mild (or recessive) in heterozygotes. This may
seem counterintuitive, since it is often assumed that a
defective protein will accumulate at the point of synthesis
and "poison" the cell. However, the cellular system for
degradation and clearance of aberrant proteins is highly
efficient (Klausner & Sitia 1990). Furthermore, a 50%
diminution in rhodopsin levels may be mildly deleterious
at worst. This category includes mutations producing no
message or unstable message, mutations producing truncated or otherwise aberrant proteins, or mutations producing proteins that are not transported to or inserted
into the disc membrane because of incorrect folding or
incorrect post-translational modification. Examples include early stop mutations, such as Gln64stop and
Glu249stop, and substantial deletions, such as A68-71 and
1231-23del30insl50 that produce mild disease (or recessive disease in the case of Glu249stop). The two splice-site
mutations, 1230 + lG-»Tand 1231-1G-»A, produce very
mild disease and are highly variable in expression. This
may be because the splice-site mutations are spliced
differently in different individuals, that is, they are
"leaky" mutations. Finally, the Asnl5Ser mutation would
preclude glycosylation of this highly conserved site, a
potentially major defect. However, the phenotype of this
mutation is also mild, suggesting that this change may
simply interfere with targeting or insertion into the disc
(Hargrave & O'Brien 1991).
4.2.2. Heterozygous mutations producing proteins that
are inserted into but destabilize the membrane will be
severe. Since the discs are geometrically precise structures with complex functional requirements and rapid
turnover, and since rhodopsin constitutes the bulk of the
disc protein, destabilizing mutations are expected to be
severe. We speculate that this is why the Leu40Arg,
Leu46Arg, Argl35Leu, Argl35Trp, and Met207Arg mutations are severe. The Argl35 mutations are particularly
noteworthy, because this is a highly conserved site at the
margin of the membrane. Several mutations at this site
cause severe adRP. In addition, the Argl35Leu and
Argl35Trp mutations are class lib. If results from embryonic kidney cells can be extrapolated to intact photoreceptors, this implies that some protein gets to the disc
membrane. Furthermore, this amino acid may have a
critical role in rod function beyond simple membrane
integrity, since Argl35Leu and Argl35Trp mutants fail to
activate transducin (Fahmy & Sakmar 1993; Min et al.
1993). However, as we consider subsequently, this effect
alone is not likely to cause severe dominant disease.
Daiger et al.: Inherited retinal degeneration
The Cysl87Tyr mutation eliminates the disulfide bond
with CysllO and produces severe disease, possibly by
destabilizing the membrane. The A255/256IIe deletion,
another transmembrane mutation, also produces severe
disease. On the other hand, some transmembrane mutations, such as Thr58Arg, only produce mild disease,
suggesting that mutations of transmembrane regions may
not necessarily perturb the membrane in significant
ways.
4.2.3. Heterozygous mutations that fail to incorporate
11-c/s retinal or disrupt the Schiff base counterion will
constitutively activate transducin and produce severe
disease. Artificial mutations that change the lysine at
codon 296 or the glutamate at codon 113 result in constitutive activation of transducin (Robinson et al. 1992). No
human pedigrees expressing a Glull3 mutation have been
identified. However, the Lys296Glu and Lys296Met mutations that produce very severe, dominant degeneration,
Opsins with substitutions at the Lys296 position fail to
bind 11-cis retinal and are constitutively active. Thus,
these mutations are likely to induce high levels of cGMP
with toxic consequences (Lolley et al. 1977; Ulshafer et al.
1980).
a heterozygote may be diminished sensitivity to dim light
or adCSNB (or recessive CSNB), but not significant degeneration. Finally, heterozygous mutations affecting
phosphorylation sites may affect recovery from photobleaching but not cell survival.
Possible examples of mild mutations that meet these
criteria are Thrl7Met, Pro23His, GlylO6Arg, and Glu341Lys. The Thr58Arg mutation is an example of a mild
phenotype associated with diminished transducin activation (Min et al. 1993). Such examples may help to explain
the paucity of disease pedigrees caused by dominant
mutations within the cytoplasmic loops.
One intriguing observation is the importance of the
penultimate proline, Pro347, in the carboxyl-terminal
cytoplasmic domain (Humphries et al. 1993). The two
terminal amino acids (Pro-Ala) are highly conserved in
mammals. This is the most common site of mutations
causing adRP, and the disease phenotype is severe. By
contrast, two mutations that delete these amino acids,
1313delC and 1515del8, have a mild phenotype. However, by coincidence, the terminal Pro-Ala residues are
preserved in each of these mutations. Unfortunately for
the argument, this is not the case for the 1289dell7 and
1312del24 mutations, which also produce mild disease.
4.2.4. Heterozygous mutations that bind 11-c/s retinal
normally but also activate transducin in the absence of
chromophore will be mild or stationary. The revealing
examples in this case are the two mutations that cause
adCSNB, Gly90Asp and Ala292Glu. Both mutant opsins
bind chromophore are inactive in the dark, and are
activated normally by light (Dryja et al. 1993; Rao et al.
1994). However, in contrast to wild-type opsin, these
proteins can also activate transducin in the absence of
chromophore. Thus, unlike the Lys296 mutations, which
are permanently activated, these proteins cause inappropriate activation of transducin when all-trans retinal
leaves opsin during the regeneration cycle. This minor
abnormality is sufficient to diminish sensitivity to dim
light but is not sufficient to damage the photoreceptor.
Significantly, the Lys296, Glull3, and Gly90 sites all fall
in close proximity within the three-dimensional structure
of rhodopsin (Baldwin 1993), suggesting that the glycine
participates in chromophore stabilization.
4.2.6. We do not know enough about rhodopsin to guess
which mutations might interfere with disc assembly, progression along the ROS, shedding, or phagocytosis; nor
can we know their clinical consequences. We do not
know what role rhodopsin plays, if any, in disc morphogenesis, axial displacement, shedding, and phagocytosis
by the RPE. We also do not know whether defects in these
processes might be related to human retinal degeneration. In any case, we know so little about these processes
that guessing may not be productive. It is our expectation
that new disease-causing mutations will reveal previously
unrecognized functional sites in rhodopsin that are involved in disc membrane renewal.
4.2.5. Heterozygous mutations in amino acids outside of
the membrane are likely to be mild or recessive unless
indirectly involved in membrane stability or chromophore
binding. We speculate that the following general principles apply to such mutations. First, mutations of some of
the amino-terminal amino acids will affect targeting of
rhodopsin to the disc and will have mild consequences
(sect. 4.2.1). Second, some of the amino acids in the
intradiscal and cytoplasmic loops will be essential for
proper protein folding. If mutations in these sites prevent
insertion of the protein into the membrane, the consequences will be mild (sect. 4.2.1). Third, heterozygous
mutations in the intradiscal loops are not likely to affect
phototransduction directly unless they disturb chromophore binding or isomerization (sects. 4.2.3; 4.2.4).
Fourth, mutations in the cytoplasmic loops will affect
transducin binding or activation (Franke et al. 1988;
Konig et al. 1989). The consequences of such mutations in
NOTES
1. Since more than one mammalian protein is named peripherin, "peripherin/RDS" is used to identify the photoreceptor
protein (Travis 1991).
2. Gene symbols, such as PDEB, are assigned by the Nomenclature Committee of the Genome Data Base (GDB) and
are recorded in GDB or the OnLine McKusick Catalog (OMIM)
(McKusick 1992; Pearson et al. 1992). GDB and OMIM are
accessible by Internet or modem. At the request of the Nomenclature Committee, gene symbols in humans are all upper case
letters (lower case italics in nonhumans; personal communication). Symbols for diseases with multiple causes, such as autosomal dominant retinitis pigmentosa, i.e., "adRP," include both
upper- and lower-case letters to distinguish them from genes
(Beaudet & Tsui 1993).
3. Determining the exact location of transmembrane boundaries is somewhat speculative (Dryja et al. 1991; Fryxell &
Meyerowitz 1991; Nathans 1992). We base our boundaries on
Nathans (1992).
ACKNOWLEDGMENTS
Supported by grants from the RP Foundation Fighting Blindness and the George Gund Foundation, and NIH NRSA Grant
EY06467 to L. S. S., and a Fellowship from the Schissler Foundation to J.A.R.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
467
Commentary!Controversies in Neuroscience III
Open Peer Commentary
Commentary submitted by the qualified professional readership of this
journal will be considered for publication in a later issue as Continuing
Commentary on this article. Integrative overviews and syntheses are
especially encouraged.
Calcium/calmodulin-sensitive adenylyl
cyclase as an example of a molecular
associative integrator
Thomas W. Abrams
Department of Neuroscience, University of Pennsylvania, Goddard Labs,
Philadelphia, PA 19104-6018. tabrams@mail.sas.upenn.edu
Abstract: Evidence suggests that the Ca 2+ /calmodulin-sensitive adenylyl cyclase may play a key role in neural plasticity and learning in
Aplysia, Drosophila, and mammals. This dually-regulated enzyme has
been proposed as a possible site of stimulus convergence during associative learning. This commentary discusses the evidence that is required to
demonstrate that a protein in a second messenger cascade actually
functions as a molecular site of associative integration. It also addresses
the issue of how a dually-regulated protein could contribute to the
temporal pairing requirements ofclassical conditioning: that relationship
between stimuli display both temporal contiguity and predictability.
[XIA ET AL.] It is widely accepted that modulatory changes in
neurons, as in other cells, are frequently mediated by second
messenger cascades involving covalent modifications of existing
proteins via phosphorylation and dephosphorylation (Greengard 1979). Moreover, cellular studies suggest that neuronal
changes produced by learning may be mediated by these intracellular signaling cascades (e.g., Kandel & Schwartz 1982;
Malenka et al. 1989; Malinow et al. 1989; Silva et al. 1992). An
intriguing possibility is that during associative learning, duallyregulated proteins within these intracellular signaling cascades
may serve as the sites at which inputs from multiple stimuli or
behaviors converge (Abrams & Kandel 1988; Bourne & Nicoll
1993). According to this hypothesis, these proteins serve an
associative role: when two stimuli are paired with the appropriate temporal relationship, the protein responds to the pairing by
triggering a second messenger cascade that initiates persistent
alteration of neuronal properties.
As discussed by XIA ET AL., Ca 2+ /CaM-sensitive adenylyl
cyclase in Aplysia was the first such example of a molecular
site of associative integration shown to contribute to neural
plasticity. Based on cellular studies, Abrams and colleagues
(Abrams 1985; Abrams & Kandel 1988; Abrams et al. 1984;
Kandel et al. 1983) and Occor et al. (1985) proposed that during
classical conditioning in Aplysia, the dually-regulated Ca 2 + /
CaM-sensitive adenylyl cyclase serves as a site of associative
integration that detects the pairing of the cellular signals from
the conditioned and unconditioned stimuli. They found that
optimal stimulation of cAMP synthesis required temporal pairing of activity and Ca 2 + influx in the presynaptic sensory
neuron, which are triggered by the conditioned stimulus (CS),
and release of modulatory transmitter, which is triggered by the
unconditioned stimulus (US); cAMP, in turn, initiates synaptic
facilitation in the sensory neurons of the CS pathway (Ghirardi
et al. 1992; Goldsmith & Abrams 1991). The second example of a
molecular mechanism of associative integration that has been
well characterized in the context of associative neural plasticity
is the NMDA-type glutamate receptor in hippocampus (Collingridge 1987; Madison et al. 1991).
critical to distinguish between an actual role as an associative
integrator, where the protein serves as a molecular site of
associative signal convergence, and a role as an essential relay
step (or amplification step) in a signal transduction cascade. In
many experimental systems, it is difficult to demonstrate that a
protein is functioning to integrate paired stimuli, even when in
vitro experiments demonstrate the protein can be synergistically activated by two signals. For example, most studies
that utilize inhibitors of an enzyme or receptor in a second
messenger cascade (Davis et al. 1992) or studies of mutants
lacking the relevant protein cannot distinguish between an
associative and a relay role. Neither the studies of the rutabaga mutant in Drosophila, which lacks Ca 2+ /CaM-sensitive
adenylyl cyclase (Dudai & Zvi 1984; Levin et al. 1992; Livingstone et al. 1984), nor the experiments of XIA ET AL. on a mouse
mutant in which the type I adenylyl cyclase gene has been
disrupted, demonstrate that the affected enzyme is playing an
associative role (as has been suggested in Aplysia); it is possible
that stimulation of cAMP synthesis is simply an essential step in
learning, or even that a basal level of cAMP-dependent phosphorylation is permissive for neural plasticity. In order to demonstrate an associative function, it is necessary to conduct
cellular studies measuring the response of the enzyme or receptor to paired or unpaired signals. Nevertheless, it is exciting that
a similar Ca 2+ /CaM-sensitive adenylyl cyclase may play acritical
role in learning in three separate phyla, suggesting that molecular mechanisms for learning are highly conserved through
evolution.
2. Detecting relationships between stimuli: temporal contiguity. Because a species of adenylyl cyclase is activated synergistically by two stimuli, it could, as XIA ET AL. point out,
function as what has been termed a "molecular-coincidence
detector" (Bourne & Nicoll 1993; Hille 1992). However, the
optimal timing of paired stimuli for proteins that perform
associative integration may not be always be simultaneous. This
is the case particularly for proteins that mediate the learning of
predictive relationships between paired stimuli. In the conditioning of the defensive withdrawal reflex of Aplysia, as in many
Pavlovian paradigms, training is more effective if the CS precedes the onset of the US by a short interval than if stimulus
onset is simultaneous; moreover, associative learning disappears
if the US begins first, even if the US continues through the CS.
Using a perfused membrane cyclase assay that permits transient
exposures to stimulating ligands, Yovell and Abrams (1992)
found that forward pairing of Ca 2 + and serotonin (with Ca 2 +
beginning first) gave more powerful cyclase activation than
backward pairing (with serotonin before Ca 2 + ); i.e., adenylyl
cyclase activation was more effective when the signal representing the CS began before the signal representing the US. Thus,
the cyclase displayed a sequence preference analogous to that of
the conditioning that the CS precede the US during stimulus
pairing. Recently, Abrams and Galun (1993) found that CaMmediated stimulation of Ca 2+ /CaM-sensitive adenylyl cyclase
was relatively delayed, and began after a brief transient initial
period of direct inhibition of cyclase by Ca 2 + . This delayed Ca 2 +
stimulation may contribute to the requirement that the CS (and
Ca 2 + influx) begin before the US (and transmitter binding). The
NMDA receptor also shows temporal asymmetry in the dependence of its activation on the relative timing between paired
signals (Gustafsson et al. 1987).
3. Detecting relationships between stimuli: Contingency and
predictability. Learning about relationships during Pavlovian
conditioning requires discriminating not only temporal contiguity of paired stimuli, but also contingency between stimuli; the
learning of the associative relationship depends upon how reliably the CS predicts the occurrence of the US (Hearst 1988;
Rescorla 1988). For example, presentations of the CS in the
f. Distinction between a site of associative convergence and a
relay step in a linear cascade. In analyzing the contributions of absence of the US or presentations of the US in the absence of
the CS degrade associative learning. As yet, we know nothing
dually-regulated proteins to associative neural plasticity, it is
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Commentary /Controversies in Neuroscience III
about whether molecular integrators are sensitive to contingency. In other words, do unpaired presentations of either the
signal from the CS or the signal from the US degrade the
activation of the molecular cascade? It may be that an individual
enzyme or receptor cannot integrate across separate trials in
which stimuli are presented. The fact that a late pulse of Ca 2 +
accelerates turn-off of Aplysia neural adenylyl cyclase activated
by transmitter (or by transmitter paired with Ca 2 + ) (Abrams et
al. submitted) may cause extra unpaired CSs to reduce overall
stimulation ofcAMP synthesis; however, because cyclase activation decays in approximately 60 sec, this effect of an extra
unpaired CS could occur only within a relatively short time
window. It is also possible that other downstream proteins in the
intracellular cascade(s) that have longer "memories" than the
integrator protein itself (e.g., a protein kinase) feed back,
modulating the integrator protein; such interactions could cause
activation of the associative integrator protein by paired stimuli
to be influenced by unpaired stimuli at earlier times. As yet
there have been no serious attempts to evaluate whether the
activation of either Ca 2+ /CaM-sensitive adenylyl cyclase or the
NMDA receptor by paired stimuli is decreased by a previous
unpaired presentation of one of the two stimuli.
ACKNOWLEDGMENTS
The experimental results cited were obtained with support from a
National Institutes of Health grant NS 25788 to the author and from a
Dana Foundation grant to the University of Pennsylvania.
The determination of rhodopsin structure
may require alternative approaches
Arlene D. Albert ab and Philip L. Yeagleb
Departments of Biochemistry" and Ophthalmology, University at Buffalo
School of Medicine (SUNY), Buffalo, NY 14214.
bchphll(H)ubvms.cc.buffalo.edu
Abstract: The structure of rhodopsin is a subject of intense interest.
Solving the structure by traditional methods has proved exceedingly
challenging. It may therefore be useful to confront the problem by a
combination of alternate techniques. These include FTIR (Fourier
transform infrared spectroscopy) and AFM (atomic force microscopy) on
the intact protein. Furthermore, additional insights may be gained
through structural investigations of discrete rhodopsin domains.
[HARCRAVE] Determination of rhodopsin structure is central to
understanding the function of rhodopsin in visual transduction.
The importance of a structure of rhodopsin is emphasized by its
membership in the family of G-protein receptors. Due to the
homology among members of this family, a structure of rhodopsin will be helpful in understanding function of other G-protein
receptors. As described by HARCRAVE, while great progress has
been made in understanding the relationship between structure
and function of rhodopsin, major hurdles remain. In particular,
no true three-dimensional crystal structure has as yet been
reported for rhodopsin. Since crystallizing membrane proteins
in forms suitable for structure determination has been successful in only a very few cases so far and since the structure of
rhodopsin is so critically needed, it is apparent that alternative
methods to structure determination should be explored to
obtain as much structural information as possible.
Fourier transform infrared (FTIR) spectroscopy has recently
provided interesting information on the structure of rhodopsin.
This technique is especially appealing in that it does not require
crystallization of the protein. Consistent with earlier circular
dichroism (CD) studies, FTIR studies indicate extensive
a-helix. Furthermore, the data from these studies suggest some
P-sheet and p-turns in rhodopsin (Brown et al. 1993, Lamba et
al. 1994). Pistorius and deGrip have employed an interesting use
of FTIR to gain further structural insight. Proteolytic digestion
was used to remove exposed peptides from the cytoplasmic side
of rhodopsin, and the FTIR spectral results before and after
proteolysis were compared. These experiments provided evidence for P-sheet and a P-turn near the C terminal region
(Pistorius & deGrip 1994).
It may be useful to consider structural studies of rhodopsin
from the standpoint of protein structural domains. The structures of many proteins have been shown to consist of multiple
structural domains that are independently stabilized. Membrane proteins also exhibit structural domains. A notable example of this is glycophorin which has both extramembranous and
intramembranous domains. Circular dichroism studies have
shown that these domains retain their secondary structure after
tryptic cleavage (Schulte & Marchesi 1979).
Rhodopsin has also been shown to exhibit intramembranous
structural domains (Albert & Litman 1978, Litman et al. 1982).
After proteolysis the rhodopsin fragments remain associated as a
complex. However, after detergent solubilization and exposure
to light the fragments disassociate. Although when detergent
solubilized rhodopsin is bleached there is loss of ot-helical
structure, the protein retains at least 30% of its helical structure.
Interestingly, the secondary structure which remains after
bleaching intact rhodopsin, is not further lost when the opsin
proteolytic fragments are separated. These fragments may serve
as a useful starting point for additional structural work. If they
were isolated in a fully deuterated detergent they may be
amenable to NMR studies.
Recently, several studies have reported interesting results
using fragments of rhodopsin. For example, a peptide incorporating residues 325-338 of the carboxyl terminal of rhodopsin
was reported to interact with transducin (Phillips & Cerione
1994). A peptide incorporating the sequence of the third cytoplasmic loop of rhodopsin inhibited binding between rhodopsin
and arrestin (Krupnick et al. 1994). Finally, phosphorylated
derivatives of the sequence 337-348 of rhodopsin were found to
bind to rhodopsin kinase (Pullen et al. 1993).
The sites of palmitoylation of rhodopsin are on the carboxyl
terminal of the protein. The role of these sites in receptor
function has not been identified. Recent fluorescent labeling of
these sites with a fluorescent acyl CoA opens the possibility of
exploring the function of these sites, and of gaining additional
structural information about rhodopsin through fluorescence
techniques (Moench et al. 1994).
Atomic force microscopy (ATM) may also soon prove to be a
powerful tool for investigating rhodopsin structure. This technique has been shown to be sensitive enough to detect changes
in the structure of lysozome upon substrate binding (Radmacher
et al. 1994). This technique may then be especially useful in
investigating the changes occurring upon metarhodopsin II
formation.
Mutant forms of rhodopsin have provided extensive information regarding the importance of particular residues. Together
with the biophysical structure techniques there is reason to
anticipate progress on a molecular structure of rhodopsin in the
near future.
Finally, it is important to consider the role of the lipid bilayer
on the structure of rhodopsin. This is especially important in
considering the structure of Metarhodopsin II. A number of
studies have shown that the formation of this intermediate is
dependent upon an appropriate lipid environment. These include studies in reconstituted systems on the role of chain length
and unsaturation (Baldwin & Hubbell 1985a; 1985b) and on the
role of cholesterol (Mitchell et al. 1990; Straume et al. 1990).
The role of cholesterol is especially relevant in that it naturally
occurs in the ROS membranes and is capable of inhibiting the
cGMP cascade initiated by rhodopsin bleaching in the ROS
plasma membrane (Boesze-Battaglia & Albert 1990).
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
469
Commentary /Controversies in Neuroscience III
Mechanisms of photoreceptor degenerations
Colin J. Barnstable
Department of Ophthalmology and Visual Science, Yale University School
of Medicine, New Haven, CT 06520-8061.
colin.barnstable@quickmail.yale.edu
Abstract: The candidate gene approach has identified many causes of
photoreceptor rod cell death in retinitis pigmentosa. Some mutations
lead to increased cyclicGMP concentrations in rods. Rod photoreceptors are also particularly susceptible to some mutations in housekeeping
genes. Although many more cases of macular degeneration than retinitis
pigmentosa occur each year, there is much less known about both
genetic and sporadic forms of this disease.
[OAICER ET AL. ] The target article by DAIGER ET AL. provides an
excellent and up to date review of the cloned genes, and other
mapped loci, in which mutations result in photoreceptor degenerations. Although the title refers to retinal degenerations, the
text is focused on photoreceptor degenerations and does not
discuss other retinal diseases, such as some inherited forms of
glaucoma that lead to degeneration of retinal ganglion cells. In
addition, a number of neural degenerative diseases that can
have a strong genetic component, such as Alzheimer's disease,
lead to a significant death of retinal ganglion cells.
Table 1 in the target article lists loci defined by either linkage
or the candidate gene approach. The latter has succeeded in
increasing our understanding of photoreceptor degenerations.
In the coining years we hope genes will be assigned to the
number of anonymous loci. Several of the loci listed in Table 1 of
the target article encode housekeeping or widely distributed
proteins, suggesting that photoreceptors show a phenotypic
effect of the mutation because of some unique sensitivity.
Perhaps the best example of this is dominant gyrate atrophy,
which is caused by mutations in the ornithine aminotransferase
gene on chromosome 10. This gene is expressed in many tissues,
including liver, kidney, and brain as well as retina. Since these
other tissues show no obvious effects of the mutation it would
appear that the photoreceptors are uniquely sensitive to increased concentrations of ornithine.
The idea that photoreceptors are more sensitive to otherwise
mild variations in proteins may explain why some opsin mutations lead to degeneration. The light-sensitive outer segments
are complex membranous extensions of a cilium. To synthesize
the membrane removed each day by phagocytosis requires a
high level of protein synthesis. Small structural changes in
opsin, the major outer segment protein, may slow the movement of protein through the endoplasmic reticulum (ER) and
Golgi by just enough to disrupt outer segment biosynthesis and
thus lead to eventual cell death. Although the review suggests
that "degradation and clearance of aberrant proteins is highly
efficient" (sect. 4.2.1, para. 1) this may apply less to the metabolically hyperactive photoreceptors that to other cell types such as
fibroblasts. Support for this idea comes from studies with
transgenic mice expressing an additional normal opsin gene
(Sung et al. 1994). These mice show rod photoreceptor degeneration whose rate is roughly proportional to the level of expression of the transgene. Perhaps some of the mutations listed in
Table 1 do not cause structural changes in the opsin molecule
itself but rather mark changes in the level of expression.
One striking feature of photoreceptor degenerations is that
mutations in many components of the transduction pathway can
lead to disease. In section 3.2.4 the authors repeat some of the
current ideas about why these mutations might cause cell death.
Direct measurements of rd mutant mice, which have an inactive
rod photoreceptor cGMP-phosphodiesterase, have shown that
just before the major period of rod degeneration the levels of
cGMP are over 10-fold higher than in normal animals (Farber &
Lolley 1974; Fletcher et al. 1986). Normal levels of cGMP are
sufficient te activate only a few percent of the cGMP-gated
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
cation channels in the outer segment. A 10-fold increase in
cGMP would probably activate all the channels and cause
complete cation equilibrium, including large increases in intracellular calcium. In section 4.2.3 the authors describe mutations
that cause severe, dominant degeneration and in experimental
studies have been shown to be constitutively active. In the intact
rod photoreceptor it is not clear how the mechanisms that shut
down rhodopsin activity, namely phosphorylation and binding of
arrestin, act on the constitutively active molecules. The authors
are probably incorrect when they state that these mutations will
lead to high levels of cGMP. Activation of rhodopsin normally
leads to a reduction in cGMP levels by activation of a cGMPphosphodiesterase. If this occurs in these mutants, then it would
seem that both abnormally high and abnormally low levels of
cGMP might cause photoreceptor degeneration.
One point that is not stressed in this review is that the macular
degenerations affect many more people than the various forms
of retinitis pigmentosa. A recent estimate is that 1,700,000 people
in the United States suffer from age-related macular degeneration as compared to 100,000 people with all forms of retinitis
pigmentosa. As with many degenerative conditions that affect
the aged, it is difficult to determine what proportion of cases
have a genetic basis and what proportion are truly sporadic.
Many family studies are confounded by the death of subjects
before they show clinical symptoms of macular degeneration.
An important question in future studies of macular degeneration will be what proportion is due to defects within the retina
and what proportion to defects in the underlying tissues. The
macular region is avascular and the retina in this region is
dependent upon the underlying choroidal blood supply for
oxygen and nutrients. Anything that alters the permeability or
function of the intervening retinal pigment epithelial cells, or
alters the structure of the extracellular matrix between the
choroid and the RPE might lead to macular degeneration.
Perhaps the strongest point brought out by DAIGER ET AL.'S
target article is that studies of photoreceptor degenerations have
advanced beyond those of most other neurological degenerative
diseases. We know more mutations that can give rise to the
diseases, we have better hypotheses as to the mechanism of
action of the mutations, we have good natural and transgenic
animal models that seem to exhibit very similar phenotypes to
those seen in humans. While there is no simple drug to alleviate
the problems associated with retinitis pigmentosa or macular
degeneration, experimental use of growth factors, transplantation, and targeted gene replacement are all showing great
promise. Once again the eye may serve as a window on the brain
and knowledge gained from retinal degenerations may prove
invaluable for other conditions such as Alzheimer's, Parkinson's,
and Huntington's diseases.
Genetic and clinical heterogeneity in tapetal
retinal dystrophies
A. A. B. Bergen
The Netherlands Ophthalmic Research Institute, 1100 AC Amsterdam, The
Netherlands, bergen@amc.uva.nl
Abstract: Large scale DNA-mutation screening in patients with hereditary retinal diseases greatly enhances our knowledge about retinal
function and diseases. Scientists, clinicians, patients, and families involved with retinal disorders may directly benefit from these developments. However, certain aspects of this expanding knowledge, such as
the correlation between genotype and phenotype, may be much more
complicated than we expect at present.
[DAIGER ET AL.] DAIGER and colleagues provide another example of how molecular genotyping of a large number of patients
with hereditary tapetal retinal dystrophies has dramatically
expanded our knowledge about retinal function and disease. In
Commentary /Controversies in Neuroscience III
summary, the benefit of large-scale DNA-mutation screening of
patients with retinitis pigmentosa (RP) and allied disorders is at
least four-fold.
Specific knowledge about mutated genes in RP provides
biochemists with clues about the function of regions of the
proteins corresponding to these genes. Thus, biochemical research strategies can be adapted to address functional problems
more specifically.
Electrophysiological, pathological, and epidemiological studies
benefit directly. For the first time, the large number of clinically
different types of hereditary retinal disorders can be unambiguously grouped together on the basis of their primary DNA
defect. Thus, the results of functional, genetic, and morphological studies can be directly compared within, and between, these
specific and uniform groups of disorders (e.g., Zong-Yi et al.
1994).
The molecular characterization of RP patients is also important for future therapies. At least some possible therapies, such
as gene therapy, will most likely be mutation-specific. Thus,
patients already typed at a molecular level will benefit most
from these new developments.
Clinicians and patients also benefit directly. For a patient, a
more accurate differential diagnosis is possible if a functional
DNA mutation is found that can be implicated in his disease.
The analysis of the segregation of that functional DNA mutation
in pedigrees may also lead to improved genetic counselling.
Moreover, as DAICER and colleagues describe in their article,
more accurate prognostic information about the severity and
course of the disease may become available.
However, although the contribution of Daiger et al. is very
valuable, the data presented should be interpreted with caution. First of all, the number of patients who can be thoroughly
characterized both at the genotypic and phenotypic level, is still
limited. Unfortunately, with a few exceptions (Jacobson et al.
1994), molecular geneticists tend to summarize the ophthalmic
data in their articles, while ophthalmologists usually minimize
the amount of molecular genetic information.
Also, it is not clear in all cases whether the phenotypic
consequences of (groups of) mutations can be compared directly; for instance: How is a "mild" form of RP defined? Can
"severe" forms of RP from different studies be directly compared? Although the results of DNA studies can be compared
directly and easily, the importance of standard and internationally accepted clinical protocols must (again) be emphasized
here. These protocols are extremely useful in order to establish
a correct correlation between genotype and phenotype, and also
to obtain comparable clinical results.
Although mtrafamilial clinical variability in RP has been well
documented, little is known yet about the variability of clinical
expression within larger pedigrees in which RP is caused by
defined DNA mutations (Weleber et al. 1993). It may well be
that this clinical variability is caused by the "molecular genetic
background" in which the defective gene functions. In other
words, while a certain mutated gene may actually cause RP,
several other "modifier genes" may influence the clinical expression of the disease. These "modifier genes" may contribute
strongly to the expression of the disease (similar to the situation
in "digenic RP" (Kajiwara et al. 1994)) or only weakly, thereby
influencing only the severity, expression, or progression of the
disease. As an example, we recently described a pedigree from a
genetic isolate in The Netherlands, in which at least two different types of RP (both genetically and clinically) are segregating
within one pedigree (van Soest et al. 1994). We may have
localized a modifier gene to chromosome lq, segregating in only
part of the pedigree and causing the phenotype retinitis pigmentosa plus para-arteriolar preservation of the retinal pigment
epithelium. At present, it is our hypothesis that the location of
the gene, which actually causes RP in the entire pedigree,
remains to be elucidated.
Clearly, other genes directly implicated in RP may be candi-
date modifier genes, although other genes that are more fundamental to the cell-structure, or in DNA- or RNA- processing, or
metabolism cannot be excluded. Thus, it can be postulated that
other genetic factors besides the actual disease causing gene
may influence the clinical picture in an individual RP patient.
Finally, the phenotype of the patient may also be influenced
by nongenetic factors, such as overall physical condition, exposure to light, or smoking. Such factors clearly complicate the
phenotypic assignment of patients given their genotype. In
conclusion, in my opinion, much more extensive clinical and
molecular data of RP patients, especially in larger families, are
needed in order to establish a more definite correlation between
phenotype and genotype in inherited retinal degeneration.
Molecular insights gained from covalently
tethering cGMP to the ligand-binding sites of
retinal rod cGMP-gated channels
R. Lane Brown and Jeffrey W. Karpen
R S. Dow Neurological Sciences Institute, Portland, OR 97209 and
Department of Physiology, University of Colorado School of Medicine,
Denver, CO 80262. karpenj@essex.hsc.colorado.edu
Abstract: A photoaffinity analog of cGMP has been used to biochemically identify a new ligand-binding subunit of the retinal rod
cGMP-activated ion channel, as well as amino acids in contact with
cGMP in the original subunit. Covalent tethering of this probe to
channels in excised menbrane patches has revealed a functional
heteogeneity in the ligand-binding sites that may arise from the two
biochemically identified subunits.
[MOLDAY & HSU] In recent years we have been interested in
identifying ligand contact points within retinal rod cGMP-gated
channels, and in elucidating the mechanism by which cGMP
binding leads to channel opening. For these purposes we developed a new photoaffinity analog of cGMP (8-p-azidophenacylthiocGMP; APT-cGMP) that specifically labels the channel
from bovine rods, and irreversibly activates the channel in
excised patches from amphibian rods by covalently tethering
cGMP to the channel's binding sites. This probe has enabled us
to address, from a unique perspective, some of the issues raised
by MOLDAY & HSU regarding the subunit composition of the
channel and how each subunit contributes to overall channel
function.
In 1993, we reported that APT-[32P]-cGMP specifically labeled the 63-kDa channel subunit originally purified by Cook et
al. (1987), as well as a 240-kDa associated protein in a partiallypurified biochemical preparation (Brown et al. 1993a). We
subsequently isolated a CNBr-peptide from the 63-kDa subunit
that contained virtually all of the label. Sequence analysis
indicated that this peptide is contained within the 110 aminoacid region that shows homology with other cyclic nucleotidebinding proteins. The label was further localized to three amino
acids (val 524-ala 526) within a larger hydrophobic stretch of
residues (Brown et al. 1995). This may explain the channel's
preference for cGMP derivatives containing hydrophobic substituents at the C 8 position (Brown et al. 1993b). A model of the
cGMP-binding domain proposed by Kumar and Weber (1992),
which places these residues on a P-strand near C 8 , is consistent
with our photoaffinity labeling result.
The recent cloning of a second subunit provided explanations
for several functional discrepancies between native channels
and expressed a-subunits (Chen et al. 1993). Like most important discoveries, however, it posed a number of new questions.
First of all, the biochemical identity of the new subunit was a
mystery because no protein of the predicted molecular weight
(71 or 102 kDa, depending on the splice variant) was present in
highly-purified channel preparations. Although a 240-kDa pro-
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
471
Commentary /Controversies in Neuroscience III
tein was known to copurify with the bovine channel, it seemed
an unlikely candidate for the second subunit because of the
obvious molecular weight discrepancy. Furthermore, antibodies against this protein were shown to cross-react with
spectrin, a cytoskeletal protein from red blood cells. We were
prompted to take a closer look at the 240-kDa protein, however,
when our photoaffinity probe specifically labeled this protein in
biochemical preparations. Sequencing of a labeled 8-kDa CNBr
fragment revealed a stretch of 16 amino acids that is identical to
part of the subunit cloned by Chen et al. (1993). Furthermore,
alignment of the amino acid sequences of the two cloned
subunits based on homology indicates that the peptide labeled
in the 240-kDa protein corresponds to that labeled in the a
subunit (Brown et al. 1995). This result agrees with the conclusions of Molday and his colleagues (Chen et al. 1994) that the
cloned P subunit is contained within the 240-kDa protein
complex. Together with the molecular genetic data, these results provide compelling evidence that the 240-kDa protein
binds cCMP and is a second subunit of the native rod channel.
A second major question that arises is how the binding of
cGMP to each subunit contributes to channel activation. The
ability to covalently tether cGMP to channel binding sites in
excised membrane patches (Brown et al. 1993a) affords a unique
opportunity to investigate the allosteric mechanism of channel
activation. As the fraction of covalently-activated channels increased, the dose-response relation for the remaining closed,
but partially-liganded, channels became progressively shallower. This behavior is expected from channels that are activated
by the binding of only one or two molecules of cGMP, rather
than the full complement of Iigands. In fact, after 80% of the
channels in a patch have been covalently activated, binomial
statistics predicts that the remaining channels will be missing
only thefinalligand. Dose-response relations for this population
of channels revealed a previously unknown heterogeneity in the
channel's cGMP-binding sites. The relations were shallower
than predicted by single-site activation models; however, the
relations were fit rather well by a model assuming two populations of binding sites, present in roughly equal proportions,
which bind cGMP with widely different apparent affinities
(Brown & Karpen 1994). This heterogeneity appears to reflect
an intrinsic property of the native channel because, although
neither affinity alone was consistent with the original doseresponse relation of the patch, both affinities taken together
provided a goodfit.Although the origin of this heterogeneity is
unknown, we are testing two intriguing possibilities: it may arise
from the two subunits described above, or it may be due to
differential modification of binding sites. Gordon et al. (1992)
have reported spontaneous shifts in the channel's dose-response
relation to lower concentrations following patch excision. These
shifts were blocked by protein phosphatase inhibitors and accelerated by protein phosphatases, suggesting that phosphorylation may play a role in modulating the channel's affinity for
cGMP. Because covalent tethering of cGMP to the channel's
binding sites permits the functional isolation of channels with a
fixed number of bound Iigands, it promises to be a valuable tool
to dissect the contribution of individual subunits to the overall
function and regulation of this type of channel.
The structure of rhodopsin and mechanisms
of visual adaptation
Rosalie K. Crouch3 and D. Wesley Corson ab
Departments of "Ophthalmology and of bPathology and Laboratory
Medicine, Medical University of South Carolina, Charleston, SC 29425.
crouchro@musc.edu and corsondw@musc.edu
Abstract: Rapidly advancing studies on rhodopsin have focused on new
strategies for crystallization of this integral membrane protein for x-ray
472
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
analysis and on alternative methods for structural determination from
nuclear magnetic resonance data. Functional studies of the interactions
between the apoprotein and its chromophore have clarified the role of
the chromophore in deactivation of opsin and in photoactivation of the
pigment.
[BOWNDS & ARSHAVSKY; HARCRAVE] HARCRAVE'S target article
is a thorough and penetrating analysis of the structure and
function of rhodopsin that is current up through 1993. Little
new structural information has been added since that time.
The author summarizes three methods of obtaining atomic
level structural data for proteins: x-ray diffraction of threedimensional crystals, electron diffraction of two dimensional
crystals, and nuclear magnetic resonance analysis of rhodopsin
in solution. The author dismisses the latter technique due to the
size of rhodopsin. However, the latter point is debatable as data
are now being obtained on proteins and receptor-ligand complexes in the 35-40kD range (Clore & Gronenborn 1994).
Considerable progress has been made on obtaining both twoand three-dimensional crystals suitable for x-ray diffraction and
the majority of the paper is a discussion of suggestions for
improving this process.
The importance of the choice of detergent was emphasized.
Since HARCRAVE'S target article was written, Schafmeister et al.
(1993) have reported the design of a homogeneous peptide for
use as a detergent to solubilize integral membrane proteins. The
general plan was to have a peptide that spanned the membrane
and which would "pack around the protein in a rigid, wellordered, parallel a-helical arrangement. " This detergent was
found to solubilize both rhodopsin and bacteriorhodopsin. The
approach offers clear advantages to the more traditional detergents which are likely to cause disorder in the crystals.
As HARGRAVE has considered, the inherent photosensitivity
of rhodopsin is a major problem for crystallization. A potential
solution to this problem is to reconstitute the pigment with a
chromophore that cannot isomerize and is therefore not photosensitive. The use of ring locked retinal derivatives would seem
to be the most straightforward approach. Unfortunately, as
discussed in the target article, activation of these pigment
analogues is still observed, even in the absence of full isomerization about a double bond. An alternative approach might be to
use fragments of retinal that do not form a Schiff base with the
protein but which have been shown to induce a "rhodopsin-like"
conformation by physiological studies (Jin et al. 1993). Partial
occupation of the binding site by fragments of retinal may serve
to maintain the protein in a sufficiently rigid conformation for
crystallization without the problems arising from light activation
of extended chromophores.
A third approach which might be considered is to use peptides from transducin to stabilize the Meta II state of rhodopsin
for crystallization in a more soluble complex. Recent studies
have shown binding of peptides from transducin to the activated
form of rhodopsin (Hamm & Rarick 1994). This approach has
recently been used to crystallize the complex of alpha transducin (Noel et al. 1993). Although this latter study did not
involve integral membrane proteins, peptides binding to the
activated form of rhodopsin may impart sufficient stability to the
complex for crystallization to occur.
BOWNDS & ARSHAVSKY provide a thorough and perceptive
overview of adaptation. They cover recent work on photoreceptor adaptation up through July of 1994. A few comments on
background and bleaching adaptation are in order.
In considering bleaching adaptation, that is, the total desensitization that occurs after bleach of a substantial amount of
pigment, it is important to distinguish among the forms of
desensitization produced by (1) pigment depletion, (2) the
short-lived effects of background adaptation produced by the
bleaching light, (3) the long-lived but finite effects of accumulated bleaching photoproducts (see Lamb 1990), and (4) the
effects of accumulated opsin which persist indefinitely in cells in
Commentary
the absence of 11-cis retinal (Corson et al. 1990; Perlman et al.
1982).
BOWNDS & ARSHAVSKY emphasize recently discovered similarities between background adaptation and opsin desensitization (see Cornwall & Fain 1994). However, several significant
differences remain to be explained. First, the diffusional space
constant for opsin desensitization is considerably smaller than
that of background adaptation (Cornwall & Pan 1985; Cornwall
ct al. 1983). Second, Fain & Cornwall (1993) have recently
reported that the noise associated with opsin desensitization is
not similar to the photon shot noise produced by dim backgrounds or the shot noise that occurs transiently after small
bleaches. Third, the two mechanisms display markedly different sensitivities to IBMX (Cornwall et al. 1990). Finally, although a sufficiently bright background light can completely
suppress the light response, this is not the case with bleaching
adaptation (Cornwall et al. 1990). These differences are not
easily reconciled with a simple single mechanism that explains
both phenomena.
Recent experiments on adaptation have probed the physiological lifetime of photoactivated pigment (R*) (see Pepperberg
et al. 1994). One measure of the physiological lifetime of R* can
be derived from the intensity dependence of the response
saturation following bright flashes (Pepperberg et al. 1992). By
this measure, the physiological lifetime of R* (~2 seconds at
~20°C in amphibian rods) appears to be independent of both
background and bleaching adaptation (Corson et al. 1994b). It
can, however, be modified through manipulation of the chromophore as might be expected for this fundamental process (Corson et al. 1994a; 1994b). However, other physiological experiments by Lagnado & Baylor (1994) suggest that R* is subject to
control by the intracellular calcium concentration during a
narrow window at the onset of a flash.
Finally, other recent work is directed to the role of the
chromophore in adaptation and pigment cycling. Detachment
of the all-trans chromophore from opsin, conversion to retinol
and export from the photoreceptors have recently become
active topics of investigation (Hofmann et al. 1992; Palczewski et
al. (1994) but see Jones et al. 1989).
ACKNOWLEDGMENT
Supported by N1H grants EY04939 and EY07543 and Research to
Prevent Blindness.
The key to rhodopsin function lies in the
structure of its interface with transducin
Edward A. Dratz
Department of Chemistry and Biochemistry, Montana State University,
Bozeman, MT 59717. uched@msu.oscs.montana.edu or
dratz@chemistry.montana.edu
Abstract: Light activated rhodopsin functions by catalyzing the exchange of GTP for GDP on numerous copies of transducin. Peptide
mapping has shown that at least six regions, three on rhodopsin and
three on the transducin alpha subunit, are involved in the active
interface between the two proteins. The most informative structural
studies of rhodopsin should include focus on the transducin interaction.
[HARCRAVE] Achieving a detailed understanding of biological
mechanisms requires knowledge of the molecular structures
and dynamics of the component molecules and of the functional
complexes between components. The first step of signal amplification in visual excitation is accomplished by the interaction of
light excited rhodopsin with the CTP binding protein, transducin. Until very recently, concepts of rhodopsin structure have
of necessity relied on theoretical models. For example, the
model of rhodopsin proposed by Dratz and Hargrave (1983)
contained a bundle of seven (bent) transmembrane helices
/Controversies in Neuroscience III
(7TMH) and a detailed alignment of the amino acid sequence in
the membrane. This and related models helped to organize
thinking about the structure of rhodopsin and other G-proteincoupled receptor (GPCR) proteins. The general features of the
1983 vintage rhodopsin model survive today. For example,
proline residues, that were proposed to bend the transmembrane helices, have been found to be among the most
conserved features of the growing super-family of GPCR that
now includes perhaps 850 members. However, most features of
the model have not been tested with experimental data.
2D crystals thought to be due to rhodopsin, were grown and
studied by Corless et al. (1982) and similar crystals shown to be
due to rhodopsin by Dratz et al. (1985), were consistent with the
membrane surface are of the 7TMH model. The crystals were
stained with heavy metals to improve contrast, which restricted
resolution to ca. 20 A and structural detail was limited. Recently,
Schertler et al. (1993) were able to obtain much better resolution
data (ca. 9 A in plane) on unstained, two dimensional crystals of
rhodopsin, where some of the long-imagined transmembrane
helices could be visualized in the structure for the first time and
the approximate packing of the helices was apparent.
Transducin can be eluted from the retinal rod disk membrane
surface in soluble form and has been much more amenable to
crystallization and structural study than rhodopsin. Noel et al.
(1993) recently published a high resolution (2.2 A) x-ray crystal
structure of the a subunit of transducin. Lambright et al. (1994)
were able to further refine the transducin a structure (to 1.8 A)
and show structural changes that occur in the crystals between
the Ga complex with GDP (the "off" state of the protein) and the
Ga complex with a nonhydrolyzable GTP analog (the "on" state
of the protein). This elegant work on Ga structure did not,
however, reveal Ga interactions with the excited rhodopsin,
where Ga has an empty nucleotide binding site (the required
intermediate between GDP release and GTP uptake).
Secrets of visual excitation and the function of rhodopsin lie in
the interaction between rhodopsin and transducing. Peptide
mapping has provided information on the interacting interfaces
between rhodopsin and transducin. Hamm and coworkers
(1988) first reported that peptides which mimicked three regions of Ga (residues 1-23, 311-329, and 340-350) were able to
block formation of the complex between light excited rhodopsin
(Mil) and transducin. Soon after, Konig and coworkers (1989)
reported that peptides mimicking three regions of rhodopsin,
thought to be on cytoplasmic loops of the structure, were also
able to block the Mf]-transducin complex. Therefore, at least six
regions, three on each protein, are involved in the active
interface between Mil and Ga.
The peptide mapping experiments stimulated Dratz and
coworkers (1990; 1991) to apply NMR techniques to study the
structures of the interfacial peptides when they are bound to
their intact protein partner. Recently, Dratz and coworkers
(1993) reported the use of NMR methods to study the three
dimensional structures of Ga peptide homolog, N-acetyl 340350 (K341R), when it was bound to rhodopsin in the dark (called
the "precoupled" state). In addition, structural changes were
found in the bound peptide when rhodopsin was excited by
light. The peptide interactions with their intact protein interfaces have rather low affinity but appear to be quite specific. For
example, changes of single amino acids from Gly—»Ala or
Cys—»Ser or amidination of the C-terminal carboxyl group in Ga
340-350 each greatly reduce the activity of the peptides.
The crystal structure of Ga was not useful for comparison with
the NMR structure of Ga 340-350 bound to rhodopsin or Mil,
because most of the 340-350 region was disordered in the
crystals (Noel et al. 1993; Lambright et al. 1994). Furthermore,
binding to rhodopsin or Mil would be expected to affect the
structure. The NMR approach reported by Dratz et al. (1993)
showed feasibility, but the results were preliminary since structure refinement (such as used in the development of x-ray
structures) was not yet reported. These NMR methods are
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
473
Commentary /Controversies in Neuroscience HI
yielding more detailed information on the structure of the
rhodopsin/Mfl-transducin interface as structure refinement
and data acquisition proceed on several of the peptides on the
contract interface between the two proteins (Busse, Furstenau
& Dratz, in preparation). In addition, enhanced NMR methods,
using G protein peptides combined with constitutive rhodopsin
mutants (Cohen et al. 1993) and rhodopsin mutants which bind
but do not activate G proteins upon bleaching (Franke et al.
1990), may be expected to illuminate the molecular basis of the
activation mechanism. NMR methods also have the potential for
obtaining information on the dynamics of the structure and the
structural interfaces. However, it is not yet clear if the resolution
of the structures determined by NMR on bound peptides,
which mimic the protein-protein interfaces, will be as high as
those obtainable with x-ray diffraction on high quality 3D
crystals. Therefore, approaches to obtain better ordered 2D and
3D crystals should certainly be vigorously pursued.
Current effort to improve the 2D crystals of rhodopsin,
described by HARGRAVE in the target article, are providing new
information on the structure of rhodopsin. HARGRAVE cites a
manuscript in preparation in 1994, by Schertler and Hargrave,
where the planer (2D) projection of frog rhodopsin was obtained
with 6 A resolution. If tilt series data can be reconstructed on
such 2D crystals, a 3D structure could be obtained with a ca.
12A resolution perpendicular to the plane of the membrane.
Such a structure would be sufficient to reveal the location of
helices rather clearly, but would not resolve even the largest
amino acid side chains. Many years of work were required on
bacteriorhodopsin to obtain a 3.5 A 2d/ca. 5.5A 3D structure,
which was sufficient to resolve the positions of about 21 of the
largest amino acid side chains in the transmembrane helices and
thus reveal much of the alignment of the amino acid sequence in
the structure (Henderson et al. 1990).
The electron microscopy method uses a series of 2D images,
obtained by tilting the specimen, to mathematically reconstruct
the 3D electron density profile of the protein in the 2D array.
This process suffers from the so called "missing cone" problem
since data can be obtained up to a maximum tilt angle of about
60°. Therefore, with this method the 3D reconstruction of a
cylindrical helix yields a long, skinny "morphed" football shape
with tapered ends. The most detailed information is obtained by
tilt series image reconstruction in the center of the membrane,
whereas most of the information on the structure of the interconnecting loops on the surface is lost in the missing cone. The
regions of rhodopsin that are implicated in coupling to transduction by peptide mapping (Konig et al. 1989) are thought to be
located in or near the cytoplasmic surface loops.
Formation of transducin complexes with light-excited 2D
crystals of rhodopsin would shift the center of the electron
density to near the interface of interest between the two proteins. However, it has not been possible to pack transducin
(GotB-y = 80kD) above about 1 transducin per 4 rhodopsins in
the native membrane. The surface density of rhodopsin in the
native membrane is 2-3 times lower than in 2D crystals and so it
may not be possible to bind more than 1 transducin per 8-10
rhodopsins in 2D rhodopsin crystals, and this would presumably
provide poorly ordered transducing arrays. Therefore the
smaller 40 kDa Got subunit of transducin should be used instead
to bind light excited 2D rhodopsin crystals, since it should be
possible to obtain a much tighter packing of Got than the whole
transducin. Presumably, it would be important to attain a 1:1
complex of Mfl rhodopsin and Got, since Got stabilizes Mil
extremely well and uncomplexed Mfl would decay to later
intermediates and disorder the structure. If the whole Got
subunit cannot be made to pack in a 1:1 complex with Mfl in 2D
crystals, perhaps a large fragment of Got could be produced that
would do so.
It seems that the most desirable approach to provide the
structural information of interest is to seek 3D crystals contain-
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
ing rhodopsin. By far the most desirable type of 3D crystals
would be complexes of light excited rhodopsin and transducin
(Mil-Gap"/) or light-excited rhodopsin and the Got subunit
(Mil-Got). Hargrave presents arguments that it might be easier
to crystallize these complexes than pure rhodopsin, since crystal
packing forces are strengthened by increasing the size of the
aqueous domains. A major problem impeding progress is the
"tradition" in structural biology in the USA that investigators
generally cannot obtain grant support for growing crystals of
macromolecules. Investigators in Europe can often obtain support for seeking crystals, and so much of the leading work is
going on there. Some of the labs in the United States that work
with these proteins have worked on the crystallization problem,
but they have had to do so on a relatively small scale with little
direct support.
I urge that attacks on the crystallization and structure determination of rhodopsin/transducin be funded and pursued more
vigorously. Progress is most likely to occur if financial support is
offered to consortia of investigators who combine a wide range of
different expertise. As pointed out by HARGRAVE, many different types of expertise can be identified as being important for
this eflFort: protein purification; site specific mutagenesis; protein expression; amphiphile, detergent, and lipid synthesis;
protein characterization; crystal growth; crystal evaluation; synchrotron source and crystallography. I take the opportunity in
this commentary to urge (and ask readers to urge) the US
National Eye Institute to issue a Request for Proposals (RFP)
inviting the formation of competing consortia to attack the
crystallization and structure determination of rhodopsin and the
rhodopsin-G protein complex. If the attack were widened to
include other GPCR, than several NIH Institutes could reasonably team to cooperatively fund crystallization/structure determination consortia and perhaps more consortia could be added.
It may well also be possible to obtain industrial/pharmaceutical
partners for such an effort. Members of each consortium could
be located anywhere in the world and communicate efficiently
by Internet and video conferencing.
A detailed 3D structure of any one of the GPCR complexes
with its cognate G protein would provide an enormously increased understanding of this major signal transduction pathway
in biology. Such an effort would also be expected to develop new
methodology for structural studies of other membrane proteins.
Discovery of the detailed molecular structures of membrane
receptors would greatly facilitate structure-based design of
improved drugs to specifically modulate different signal transduction pathways.
The atomic structure of visual rhodopsin:
How and when?
R. Michael Garavito
Department of Biochemistry and Molecular Biology, The University of
Chicago, Chicago, IL 60637. garavito@biovax.uchicago.edu
Abstract: Strong arguments are presented by Hargrave suggesting that
the crystallization of visual rhodopsin for high resolution analysis by
X-ray crystallography or electron microscopy is feasible. However, the
eflFort needed to achieve this goal will most likely exceed the resources of
a single laboratory and a concerted approach to the research is
necessary.
1. Introduction: The dilemma, [HARGRAVE] Twenty years has
elapsed since Henderson and Unwin (1975)firstpresented their
low resolution structure of bacteriorhodopsin from purple membrane. Since 1975, the atomic structures of several integral
membrane proteins have been reported (for a recent overview,
see Sowadski 1994) that have significantly altered the way we
view membrane protein structure. Moreover, this emphasizes
Commentary
that effective techniques for growing 3-dimensional and 2-dimensional crystals of membrane proteins have been developed
(Garavito 1995; Garavito & Picot 1990; Kuhlbrandt 1988; 1992).
While this seems to promise a bright future, the handful of
successes pale when compared to the exponential growth in the
number of structure determinations of soluble proteins (see
Bowie et al. 1991). The bottom line is that the effort needed to
bring a membrane protein structure project to fruition remains
enormous and long-term.
The questions posed by my title I consider critical: how
should investigators approach this goal and how long might it
take? As molecular biology and computer modeling (Baldwin
1993), has improved, some have argued that we will gain more
from these techniques, over the same span of time, than from
experimental structure determination, HARCRAVE raises this
issue with regard to visual rhodopsin and discusses many of the
facets of the research problem that highlights the dilemma that
most investigators in this field face.
2. The growth and analysis of 3-dlmenslonal crystals of rhodopsin. X-ray crystallography requires well ordered, 3-dimensional crystals for the analysis to succeed. The physical, biochemical, and biological characteristics of rhodopsin satisfy
most, though not all, of the criteria (Garavito 1995) I feel are
essential before engaging in a membrane crystallization experiment: (1) the protein should be obtainable in milligram quantities; (2) the protein is stable under a reasonably wide range of
detergent conditions; (3) native (although perhaps inactivated)
protein can be isolated and purified; (4) the purified protein is
chemically homogeneous; and (5) the detergent-solubilized protein is physically monodisperse (i.e., it forms a unform population of protein-detergent aggregates).
Most preparations of rhodopsin satisfy all but the last two
criteria. Not only is rhodopsin heterogeneously glycosylated
(Hargrave, sect. 9, para. 1) but may also be contaminated by
phosphorylated opsin due to incomplete dark adaptation (Hargrave, sect. 9, para. 3) and nonstoichiometric palmitoylation
(Hargrave, sect. 9, para. 3). The possibility of chemical heterogeneity being introduced after rhodopsin is purified, particularly from stray-light induced opsin formation (Hargrave, sects.
17 and 18) and depalmitoylation (Hargrave, sect. 9, para. 3),
remain major concerns. Furthermore, the physical state of the
rhodopsin-dctergent complex, although well-studied compared
to other membrane proteins (Hargrave, sect. 11), is still not
sufficiently defined.
The idea that monovalent Fab! fragments from monoclonal
IgCs might be used to add 50 kDa of "soluble" protein mass to a
rhodopsin molecule (Hargrave, sect. 20, para. 2) is an excellent
one and has been bandied about for more than 10 years. If
specific, high avidity monoclonal IgGs are available, these
experiments should be done. Moreover, the idea of crystallizing
fusion proteins (Hargrave, sect. 20, para. 4) may also work for
smaller (<50 kDa), monomeric membrane proteins although
the linker region between the fused proteins may need to be
carefully engineered. The lectin-rhodopsin complexes (Hargrave, sect. 20, para. 3) may be less likely to succeed as lectin
binding would be expected to be less specific and more heterogeneous given the glycosylation present. For both the Fabrhodopsin and fusion protein "complexes," they will still need to
be solubilized in detergent to prevent nonspecific aggregation.
However, the increase in the polar surface area should decrease
the detergent-dependence on crystallization (Garavito & Picot
1990).
If a chemically and physically homogeneous preparation
of rhodopsin, preferably light-stable, can be produced, the
chances of finding appropriate conditions for the growth of X-ray
quality crystals are greatly improved. Poor quality 3-dimensional crystals of rhodopsin have already been obtained (Hargrave, sect. 6), so the potential is there. However, the successful
determination of a crystal structure by X-ray diffraction depends
/Controversies in Neuroscience HI
on crystal quality: they should display well-ordered Bragg diffraction to better than 3.4 A resolution if a moderately accurate
atomic model is to be built. The history of the bacteriorhodopsin
crystal structure research (Michel 1982; Michel & Oesterhelt
1980; Schertler et al. 1993b) demonstrates the effort and time
expended in searching for conditions which yield X-ray quality
crystals. However, the crystallization potential of purified,
detergent-solubilized rhodopsin warrants further, rigorous
investigation.
3. The growth and analysis of 2-dlmenslonal crystals of
rhodopsin. The advances made by high resolution electron
microscopy (Henderson et al. 1990; Kuhlbrandt et al. 1994;
Subramaniam et al. 1993) have clearly demonstrated that this
technique has come of age and promises to be quite powerful.
The consensus of the field (Robert Glaeser, University of California, personal communication) is that upcoming technical
advances and refinements promise even wider application.
Kuhlbrandt (1992) presents the straight-forward methodology
for obtaining 2-dimensional crystals, although the exact roles of
many of the components (e.g., specific lipids) in the crystallization process remain to be determined (Nussberger et al. 1993).
However, a distinguishing difference between X-ray and electron microscopic crystallographic techniques is that even poor
2-dimensional crystals can yield useful low resolution structures
of membrane proteins (Hargrave, sect. 13, para. 2; Kuhlbrandt
1992), including rhodopsin (Hargrave, sect. 13, para. 3; Schertler et al. 1993a). It may also be more feasible to devise experiments to trap light-induced structural changes in 2-dimensional
crystals (Subramaniam et al. 1993).
4. Conclusions: Where should one go? A careful reading of
the target article by HARGRAVE reveals two important aspects of
the research. First, the potential for the successful determination of the rhodopsin atomic structure is excellent, either by
X-ray diffraction or electron microscopy. Second, the work
ahead promises to be exhausting and time-consuming. It is no
coincidence that most of the successful structure determinations
of membrane proteins have required the collaboration of two or
more laboratories and many investigators. Thus, in many respects, the real issue is: can sufficient resources be brought to
bear on the problem over the long-term? Hargrave shows it is
feasible, but how long remains a question to be answered.
Does calmodulin play a functional role
in phototransduction?
Mark P. Gray-Keller and Peter B. Detwiler
Department of Physiology and Biophysics, University of Washington School
of Medicine, Seattle, WA 98195. mgkeller@u.washington.edu and
detwiler@ii.washington.edu
Abstract: Molday and Hsu review results from in vitro experiments,
which indicate that Ca-bound calmodulin reduces the cGMP sensitivity
of the cyclic nucleotide-gated channel of photoreceptor cells, and
speculate about the role they might play in the recovery of the light
response. We discuss results from in vivo experiments that argue against
the participation of Ca-calmodulin in photorecovery.
[MOLDAY & HSU] In vitro experiments are discussed by MOLDAY
& HSU that indicate the cGMP binding affinity of the cyclic
nucleotide-gated channel from retinal rods is reduced by calmodulin in a Ca-dependent manner. The authors speculate
about the possibility that Ca-calmodulin regulation of the channel plays a role in shaping the recovery of the light response
under physiological conditions. In previous studies on functionally intact rod outer segments we have found, however, that
neither the amplitude of circulating dark current nor the sensitivity and kinetics of the electrical light response were affected
by either exogenous calmodulin (Gray-Keller et al. 1993) or high
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
475
Commentary /Controversies in Neuroscience III
concentrations of calmodulin inhibitors (trifluoperazine, W-7,
calmidazolium, and mastoparan).
Our brief commentary focuses on possible explanations for
the differences between the results of in vitro and in vivo studies
of the role of calmodulin in phototransduction. One option is to
ignore the inhibitor experiments on the grounds that negative
results are notoriously problematic in their interpretation.
Other studies, however, using truncated rod outer segments
(Lagnado & Baylor 1994) and excised outer segment membrane
patches (Gordon et al. 1994) have also failed to demonstrate
effects of calmodulin inhibitors. Taken together, these experiments on more intact preparations than those used by Hsu and
Molday (1993) suggest that the negative results with calmodulin
and its antagonists deserve consideration.
The observation that exogenous calmodulin had no effect on
the light response in dialyzed rod outer segments may indicate
the presence of a saturating amount of endogenous calmodulin.
The ineffectiveness of calmodulin inhibitors suggests that, during a light response, internal free Ca (Ca,) does not fall low
enough to dissociate Ca-calmodulin from the channel complex.
Using Indo-dextran, a fluorescent Ca indicator, we have recently measured Ca( in dialyzed rod outer segments in the dark
and during subsaturating flash and background responses (GrayKeller & Detwiler 1994). In darkness steady-state Ca( was 554 ±
25 nM (n = 28) and briefly declined to a minimum of—325 nM
shortly (~600 msec) after the peak of a flash response that
transiently suppressed the circulating dark current by ~70%.
On the basis of results reported by Hsu and Molday (1993) the
calmodulin-dependent shift in the cGMP affinity of the channel
would not be expected to be triggered by a fall in Ca, from 550 to
325 nM.
Hsu and Molday found that calmodulin had its largest effect
on the channel when Cas was between 50 to 100 nM. Our
measurements suggest that Cas would reach these values only
during very bright illumination. In steady saturating light, for
example, after permanent closure of all cGMP-gated channels,
Ca, dropped to a minimum level of about 50 nM in approximately 20 seconds. Another possible explanation for the negative results with the calmodulin inhibitors is that rods contain an
endogenous factor that affects the cyclic nucleotide affinity of
the channel-like calmodulin but is not calmodulin. This suggestion is supported by recent experiments on truncated rods
(Nakatani et al. 1995) and excised patches (Gordon et al. 1995),
which show a large irreversible increase in cGMP-induced
current following exposure to low Ca (tens of nanomolar). These
results are consistent with an endogenous factor that continually
reduces the cGMP binding affinity of the channel and can be
removed when Ca levels are in the vicinity of 20 to 60 nM. In this
scenario, the endogenous inhibitory factor would only dissociate
from the channel in functionally intact cells during bright steady
light when Ca, falls to the lower end of its physiological range.
In summary, the physiological importance of the interaction
between the channel and Ca-calmodulin, or some other unidentified endogenous Ca-binding protein, has not been established. Recent studies suggest that the Ca-dependent regulation of the cGMP binding affinity of the channel may only
operate in bright steady light when Cas falls low enough to
dissociate Ca from the channel-associated binding protein.
describing how structure determines function is only just beginning.
The discovery that the affinity of the rod channel for its agonist can be
modulated indicates that the relationship between intracellular cGMP
and the channel's open probability (current) during the course of the
photoresponse may be more complex than previously thought.
Structure and physiology of photoreceptor
cGMP-gated cation channels
The presence of an S4-like region (the presumptive voltagesensor of voltage-gated channels) in these nearly (rod) or completely (cone) voltage-insensitive channels is another mystery.
As MOLDAY & HSU point out, the S4 region of the cGMP-gated
channels have lost three or more of the positively charged repeats. This might explain the loss of voltage-dependence in the
gating process, but on the other hand, parts of the gating process are strongly voltage-dependent even though there is no
net voltage-dependence. This can be seen from the voltage-
Lawrence W. Haynes
Department of Medical Physiology, University of Calgary, Calgary, Alberta,
T2N 4N1, Canada, haynes@acs.ucalgary.ca
Abstract: The primary sequence of two subunits of the rod and one
subunit of the cone cGMP-gated channel have been described, but
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
[MOLDAY & HSU] An excellent review of the cloning and expression of the vertebrate rod photoreceptor's cGMP-gated cation
channel has been provided by MOLDAY & HSU. It clearly
demonstrates the power of molecular biology and just as clearly
shows the limitations of this approach. These limitations are apparent both in what the authors say and in what they are forced
to omit. Although we have learned a great deal about these
channels, what we have yet to learn about their structure and
how structure determines function is enormous.
If the goal of those of us working on the cGMP-gated channels
is to understand how, at the molecular level, these channels
function, then cloning the proteins that comprise the channel is
a necessary first step, after which investigation of structurefunction relationships using site-directed mutagenesis can be
undertaken. These studies, in turn, rely implicitly upon knowing the biophysical properties of the native channel since this is
the standard against which the mutations must be judged. Thus,
it was clear from the gating properties and diltiazem sensitivity
of the expressed homomeric channel as cloned by Kaupp et al.
(1989) that, while they had a cGMP-gated channel, it was not the
same as the rod channel. Something was missing. This, in turn,
led to the discovery of a second subunit by Chen et al. (1993).
Several questions arise. First, are their more subunits? MOLDAY
& HSU suggest a third subunit that is tightly associated with the
P subunit in vivo, but the function of this subunit is not understood. Second, howdoesthePsubunitinteractwiththeasubunit
to alter the gating of the channel? Third, what is the stoichiometry for the subunits of the channel?
While knowing the primary sequence of the channel is important, knowing the three dimensional structure is crucial to
understanding how the protein functions. At present, our
knowledge of the structure of these channels allows us to draw
only crude cartoons about how we imagine the pieces may fit
together. This can be misleading since, as pointed out above, we
do not yet know if we have all of the pieces to this puzzle.
Nevertheless, the gaps in the picture can be illuminating. For
example, simply examining the amino acid sequence of the
clone can lead to some mistaken conclusions. Consider the
sequence of the putative pore region of the cloned rod and cone
a subunits. This sequence is identical in the two types of
channel, yet the electrophysiological evidence obtained from
native channels suggest striking differences between the rod
and cone channels. Under identical physiological conditions,
the rod channel shows strong outward rectification while the
cone channels show exponential increases in current at both
hyper-and depolarized potentials (reviewed in Yau & Baylor
1989). Clearly, the putative pore sequence cannot explain such
differences. Either there is a cone p subunit that changes the
permeation properties of the channel, or there are other regions
of the channel a subunit that also form part of the pore and
which differ in sequence between rods and cones. Site-directed
mutagenesis studies can help answer this question, but knowing
the 3D structure of the channel would help greatly by indicating
which regions of the protein to probe.
Commentary/Controversies in Neuroscience III
dependence of diltiazem "block.' Diltiazem does not, in fact,
block the pore of the channel, but is instead a gating-modifier
(Haynes 1992). One can speculate that diltiazem immobilizes a
segment of the protein that must move during the gating
process. One can further speculate that there are at least two
such segments moving in opposite directions (or the same
direction but having opposite charge) during normal gating.
This would give rise to a gating process with no net voltageindependence except when one of the two segments is immobilized by diltiazem. If this scheme is correct, which segments
are moving and how far do they travel? Since the P subunit
confers both diltiazem sensitivity and modifies the gating of the
a subunit, the binding site for diltiazem is probably located
within the P subunit. The location of this site and how it affects
the conformation of the channel need to be determined. This is
only part of a larger question, which is how the interaction of a
ligand with its binding site in one portion of the channel protein
leads to conformational changes that give rise to the opening of a
pore in another, distant, part of the channel. The description of
the gating of these channels at a level deeper than rather crude
kinetic models is a complete mystery.
The observation of Hsu and Molday (1993) that calciumcalmodulin reduced the apparent affinity of the rod cGMP-gated
channel for its ligand in vitro is very interesting. Together with
the observations of Gordon et al. (1992) that the rod channel may
undergo phosphorylation-dephosphorylation reactions, this offers a possible mechanism that can explain the high degree of
variability in the apparent affinity of these channels for their
ligand as well as a mechanism that may accelerate the recovery
of the response to light. It remains to be seen if this process
works in vivo, however. In the only experiment dealing with the
physiology of calmodulin, Gray-Keller et al. (1993) found dialysis of calmodulin into a rod from a patch pipette had little effect
on the shape of the light response. This might be expected if the
concentration of calmodulin within the rod outer segment was
enough to saturate any calmodulin binding sites. It would be
interesting, therefore, to see if the calmodulin inhibitor mastoparen prolongs the light response without affecting the rising
phase as Hsu and Molday's hypothesis predicts.
The time-course of modulation is, of course, of crucial importance here. It is unfortunate that Hsu and Molday's experiments
could not provide this information. If the affinity of calmodulin
for calcium is - 6 0 iiM (Fig. 3 of Hsu and Molday 1993), then the
mean time that it takes calcium to unbind from calmodulin is
~170 msec (assuming diffusion limited binding and no cooperativity). Clearly, this length of time is not important when
considering the long responses elicited by bright flashes or
steps, but it may be significant when considering responses to
dim flashes.
One might expect that cones, with their shorter light responses, would undergo modulation by calmodulin to a greater
degree. Contrary to this expectation, I have found that while
catfish rod channels are modulated by calmodulin, cone channels are not under identical conditions. This would represent a
major departure between rods and cones in terms of the mechanisms used for phototransduction. It may be that the unbinding
rate for calcium is too slow for the cone response and so this
mechanism is not used. If, as MOLDAY & HSU'S Figure 6 sug-
gests, the calmodulin binding site is on the P (or 7) subunit then
the cone analog of this subunit must lack the calmodulin binding
site. Testing this prediction must await the cloning of the cone
channel's other subunits.
ACKNOWLEDGMENT
The author is an Alberta Heritage Foundation for Medical Research and
Medical Research Council of Canada Scholar. This work was supported
by the MRC.
Long term potentiation and CaM-sensitive
adenylyl cyclase: Long-term prospects
Warren Heideman
Division of Pharmacology, School of Pharmacy, University of Wisconsin,
Madison, Wl 53706. heldeman@macc.wisc.ed
Abstract: The type I CaM-sensitive adenylyl cyclase is in a position to
integrate signals from multiple inputs, consistent with the requirements
for mediating long term potentiation (LTP). Biochemical and genetic
evidence supports the idea that this enzyme plays an important role in
LTP. However, more work is needed before we will be certain of the role
that CaM-sensitive adenylyl cyclases play in LTP.
[XIA ET AL.] The work of Westcott et al. (1979), published some
15 years ago, describing an adenylyl cyclase that could respond
to both GTP and to calmodulin (CaM) pointed to a potential
crossroad for two major signal transduction systems. The fact
that this enzyme could respond to both G protein regulation and
to signals regulating calcium and calmodulin pointed to the
possibility that this form of adenylyl cyclase could play a pivotal
role in integrating multiple signals coming into neuronal cells.
XIA ET AL. have collected data showing that this integration of
signals indeed exists. In particular, this integration can take the
form of a synergistic burst of cAMP production that is much
greater than that produced in response to a single input. This
synergism is relevant to long term potentiation (LTP) in that,
with LTP, combinations of signals produce responses that are
distinctly different from those produced by individual signals
alone.
Experiments manipulating cAMP, and Ca + 2 in neuronal cells
also point to a role for these systems in LTP. Thus, activation of
these two pathways correlates with induction of LTP.
The evidence from invertebrates is perhaps even more compelling (Abrams et al. 1991a). It is perhaps remarkable that
learned responses can be observed in organisms that are enormously divergent. Thus, the molecular mechanisms that underlie this process must have been conserved over hundreds of
millions of years. The fact that calmodulin-sensitive adenylyl
cyclases are associated with learning in creatures as divergent as
nudibranchs and fruit flies suggests more than a coincidence.
This is strengthened by the finding that the CaM-sensitive type
I adenylyl cyclase is restricted to only certain regions of the
mammalian brain.
With these arguments in mind, can we say that the CaMsensitive adenylyl cyclases play a role in neuroplasticity? It
seems likely that indeed they must play at least some role. More
work remains ahead before we can describe that role in exact
terms. The disruption experiments should be very revealing,
but the tantalizing early results reported by XIA ET AL. point to
the possibility that this is a redundant pathway in rodents. The
modest effects on CaM-sensitive adenylyl cyclase activity and
learning suggest that there may be another CaM-sensitive
adenylyl cyclase that is not the type I and perhaps not the type
III that can also mediate learning in the absence of the type I
enzyme. Putting this aside, the genetic experiments in both
mice and Drosophila have some limitations in interpretation. It
is possible, for example, that the CaM-sensitive adenylyl cyclase
plays a permissive role in the process rather than a regulatory
role. In such a model, the CaM-sensitive adenylyl cyclase would
be necessary for neuroplasticity by allowing some important
process to occur but would perhaps not play a direct role in
regulating the response. With either a direct or permissive role,
a loss of function mutation in the CaM-sensitive adenylyl cyclase
will block LTP.
Ultimately, we will need to set up some sort of mammalian
model along the lines of the Aplysia system in which we can
manipulate the production of cAMP and measure the effects on
neuronal activity. Also, because the mechanisms involved in
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
477
Commentary /Controversies in Neuroscience III
LTP probably can be better represented by a web of signals
rather than as a pathway, we will need a thorough understanding
of the messengers produced in response to the external stimuli,
as well as the downstream events, including the genes that are
transcribed and are set in motion by these signals.
Channel structure and divalent cation
regulation of phototransduction
Richard L. Hurwitz,3 Devesh Srivastava," and
Mary Y. Hurwitza
"Departments of Pediatrics and Cell Biology, Baylor College of Medicine,
Houston, TX 77030 and bCollege of Optometry, University of Houston,
Houston, TX 77204. rhurwltz@msmail.his.tch.tmc.edu
Abstract: The identification of additional subunits of the cGMP-gated
cation channel suggests exciting questions about their regulatory roles
and about structure/functional relationships. How do the different
subunits interact? How is the complex assembled into the plasma
membrane? Divalent cations have been implicated in the regulation of
adaptation. One often overlooked cation is magnesium. Could this ion
play a role in phototransduction?
& ARSHAVSKY; MOLDAY & HSU] An understanding of
the photoreceptor response to light requires an insight into the
structure/function of the individual members of the phototransduction cascade and an understanding of the interactions
between them and the regulatory mechanisms that modulate
their response. The importance of both calcium and cGMP in
visual signal transduction is recognized in the target articles by
[BOWNDS
MOLDAY & HSU a n d BOWNDS & ARSHAVSKY.
MOLDAY & HSU have detailed a thorough view of exciting
recent discoveries concerning the structure and function of
the cGMP-gated cation channel which regulates the lightdependent intracellular concentration of cations in the photoreceptor. As in all good investigations, these discoveries point to
many more questions that need to be answered in order to
understand the relationships of the various channel subunits (oc
and P) to each other and to the regulation of phototransduction.
Chen et al. (1993) have demonstrated the existence of the putative P subunit of the cGMP-gated channel which, when coexpressed with the a subunit in a mammalian cell line, imparts
diltiazem sensitivity to the cation flux. Molday and his colleagues have found that the deduced amino acid sequence of this
P subunit is identical to sequenced polypeptides derived from
the 240 kDa polypeptide (sect. 2.4, para. 1) that copurifies with
the a subunit. The p subunit cDNA, however, codes for a protein that is less than half the size of the 240 kDa polypeptide.
Unraveling the mystery of the 240 kDa polypeptide may provide
substantial insight into the structure of the channel complex. In
our hands, the purified, reconstituted channel was inhibited by
1 cis diltiazem and this inhibition could be eliminated by
preincubation with trypsin (Hurwitz & Holcombe 1991). Cook
et al. (1987) have also purified the channel, however their
preparation did not appear to be 1 cis diltiazem sensitive. Both
of these channel preparations contain the 240 kDa polypeptide
and therefore would be predicted to be affected by diltiazem.
Definition of the requirements necessary for effective interaction between the a and P subunits should provide insight into
the function of the channel complex in vivo.
Localization of the channel exclusively in the plasma membrane (Cook et al. 1989) raises another fascinating question: How
is the channel segregated into the plasma membrane while rhodopsin is directed to both the plasma and disk membranes? The
mechanism of this specific localization may provide insight into
membrane protein trafficking for other macromolecules as well.
The observation that the amino terminus of the channel
appears to be missing when the channel is isolated from photoreceptors but not when the channel is isolated from in vivo
478
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
expression systems using oocytes or mammalian cultured cell
lines (Molday et al. 1991) may suggest a functional role for this
apparent posttranslational modification. Molday and his colleagues provide suggestive evidence that this cleavage may
occur in vivo and is not an artifact of the channel purification.
Both the mechanism of this proteolysis and any functional
differences that may result should provide additional insight
into the functional channel complex.
BOWNDS & ARSHAVSKY provide a thorough discussion and
summary of the diverse work that addresses the topic of photoreceptor adaptation. The authors correctly point out that experiments have been performed using various rod outer segment
preparations, stimulus conditions, and, at times, nonphysiological assay conditions (sect. 3, para. 2), which make interpretation
of the literature as well as arriving at basic conclusions difficult.
The authors associate light-induced changes in intracellular
calcium concentrations with the kinetics of the adaptive process,
but they acknowledge that calcium may not be the sole regulator
of the adaptive process (sect. 9, para. 6). Another cation that may
regulate the kinetics of both excitation and recovery is magnesium.
Magnesium ions comprise 5% of the current that flows
through the cGMP-gated cation channel (Yau & Baylor 1989).
The manner by which magnesium exits the photoreceptor is not
known. Somlyo and Walz (1985) have used electron probe
analysis to demonstrate that light decreased the total concentration of magnesium in both the inner and outer segments of the
frog photoreceptor. The free concentration of intracellular magnesium ([Mg2+],) and the effect of light on [Mg2+], are unknown. In other tissues, [Mg2+]j has consistently been ~0.5
mM (Chen et al. 1992; Heinonen & Akerman 1987; Quamme &
Rabkin 1990; Raju et al. 1989).
What impact would potential changes in [Mg2+], have on
mechanisms that contribute to adaptation? Bownds and Arshavsky identified major sites of adaptation (sect. 10, para. 2).
These sites affect cGMP hydrolysis and synthesis and the
phosphorylation of rhodopsin. These reactions all require Mg2+
as a cofactor. In our laboratory we demonstrated an almost 2-fold
decrease in the Vmax of the isolated, trypsin-activated bovine rod
phosphodiesterase when [Mg2+] was decreasedfrom500 to 100
\x.M (Srivastava et al. 1994). The Km of the enzyme also decreased as [Mg2+] decreased. We further demonstrated that
Mg2+ and cGMP bind to the phosphodiesterase in a random
order prior to the hydrolysis of cGMP. The substrate for guanylate cyclase, rhodopsin kinase, and protein kinase C is a Mg-NTP
complex. A decrease in [Mg2+]( would result in a decrease in
available substrate. A decrease in available substrate would
result in a decrease in the rate of cyclic nucleotide production or
protein phosphorylation by cyclases of kinases, respectively.
Therefore, a light-induced decrease in [Mg2+], may decrease
the kinetics of excitation or recovery.
With the acceptance of the essential nature of Mg2+, its
contribution to the current through the cGMP-gated channel,
and its effect on the kinetics of numerous reactions, perhaps this
cation deserves more attention. First, future experiments will
need to determine whether there are light-dependent changes
in the free [Mg2+],. Second, these potential changes need to be
related to each step of excitation and recovery. Third, the
mechanism and regulation of the extrusion of Mg2+ to the
extracellular space needs to be identified. Once these are better
understood, perhaps our knowledge of the mechanisms of photoreceptor adaptation will be more complete.
Linking genotypes with phenotypes in
human retinal degenerations: Implications
for future research and treatment
Michael W. Kaplan
fl. S. Dow Neurological Sciences Institute, Portland, OR 97209-1595.
kaplanm@ohsu.edu
Commentary /Controversies in Neuroscience HI
Abstract: Although undoubtedly it will be incomplete by the time it is
published, the target article by DAJCER ET AL. organizes mutations in
genes that produce retinal degenerations in humans into categories of
clinically relevant phenotypes. Such classifications should help us understand the link between altered photoreceptor cell proteins and
subsequent cell death, and they may yield insight into methods for
preventing consequent blindness.
[DAICER ET AL. ] The application of genetic analysis and molecular biology to determine mutations responsible for retinitis
pigmentosa in humans has been both informative and surprising. Although variability in the inheritability, course, and severity of the disease suggest multiple etiologies, the number of
underlying genetic defects that have been discovered so far is
both daunting and bewildering. Studies of specific pedigrees
provide a rapidly expanding genetic taxonomy of the disease
that will be important for deciphering how the underlying
mutations lead to retinal cell death. The review by DAICER ET
AL. organizes the individual mutations into phenotypic families,
and provides a useful framework for building general principles
of how the consequent disease progresses. Grouping the mutations into functional and clinical phenotypes should also facilitate the development of any strategies for treatment and
remediation.
Faced with the multiplicity of genetic defects, are there
reasonable strategies that can be adopted for developing therapies for inherited retinal degenerations? The answer to this
question will depend upon whether information about specific
mutations can be translated into an understanding of how the
resulting aberrant protein produces cell death and whether
therapeutic intervention is possible. Further understanding will
be facilitated by appropriate animal models, cell cultures, and
cell-free systems. The correspondence of naturally occurring
retinal degenerations in mice (rd, rd-3, rds, nr, pcd) (Chang et
al. 1993; Mullen et al. 1976; Sidman & Green; 1965; 1970; Van
Nie et al. 1978), dogs (Aguirre et al. 1978), cats (Narfstrom 1983),
and other species to retinitis pigmentosa in humans is not exact.
The genotypes usually differ from human mutations, and there
may be species-dependent effects of altering given proteins
upon photoreceptor cell development and ultimately upon
photoreceptor cell death. The value of natural mutations of
photoreceptor-specific murine and canine genes such as the
genes for PDEp (rd) (Bowes et al. 1990; Farber et al. 1992;
Pittler & Baehr 1991; Suber et al. 1992) and rds/peripherin (rds)
(Council et al. 1991; Travis et al. 1989; 1991) has been to show
that their abnormal gene products can cause photoreceptor cell
death. They have focused the search for mutations in the
equivalent human genes. The outcome has been an extensive
catalog of mutations in human PDEB and r<is/peripherin and a
depressingly varied list of the categories of retinitis pigmentosa
that they cause.
An approach to understanding the consequences of given
mutation identified in humans has been to develop a corresponding transgenic animal model incorporating the same mutation. Transgenic mice potentially can answer many important
questions about the physiological consequences of a particular
mutation (Huang etal. 1993; Naashetal. 1993; Roof etal. 1994).
For example: How does the mutation affect the function of its
gene product? Does it affect the transport and localization to
appropriate sites within the cell? Does it affect the way that its
gene product interacts with other proteins? Why do different
mutations of the same gene sometimes have profoundly different consequences in terms of the course and severity of the
retinal degeneration? How and why does the mutation cause
photoreceptor cells to die? Why does a mutation in a rod-specific
gene ultimately cause the death of cone cells? Why do mutations
in some proteins of the visual transduction cascade cause autosomal dominant disease (opsin) while mutations in other transduction proteins (PDEB) cause autosomal recessive disease? Given
the extent of the ever expanding list of mutations that cause
retinal degeneration in humans, it is unlikely that equivalent
transgenic animal models will be developed for them all. A more
reasonable expectation is that categories of disease phenotypes,
such as those presented by Daiger et al., will be used to select
for critical analysis those mutations that represent a particular
category or functional domain or retinal degeneration.
Unsolved issues in S-modulin/recoverin
study
Satoru Kawamura
Department of Physiology, Keio University School of Medicine, Shinanomachi 35, Shinjuku-ku, Tokyo 160, Japan
Abstract: S-Modulin is a frog homolog of recoverin. The function and the
underlying mechanism of the action of these proteins are now understood in general. However, there remain some unsolved issues including; two distinct effects of S-modulin; Ca 2+ -dependent binding of
S-modulin to membranes and a possible target protein; S-modulin-like
proteins in other neurons. These issues are considered in this
commentary.
[HURLEY] S-modulin in frog and recoverin in bovine were
reported almost at the same time in 1991. Even though their
reported molecular weights and molecular characteristics were
very similar, the reported functions were different: S-modulin
was shown to inhibit phosphodiesterase activation at high
[Ca 2+ ] and recoverin to activate guanylate cyclase at low [Ca 2+ ].
There had been a dispute about the function(s) of these proteins.
However, as reviewed in HURLEY'S target article, it now appears
that recoverin is a bovine homolog of S-modulin and does not
activate the cyclase but instead regulates PDE (phosphodiesterase) activation through inhibition of rhodopsin phosphorylation.
In section 2.3 of HURLEY'S target article, studies of S-modulin
are briefly reviewed, but there remain some unsolved issues
that are not addressed in the target article. I will elaborate on
these issues to contribute to future studies of this protein family
(for details, see Kawamura 1994).
Two distinct effects of S-modulln. S-modulin seems to consist
of (at least) two components, each of which exerts a distinct
effect. This notion came from the observation that S-modulin
purified with rod outer segment (ROS) membranes only affects
the fractional activation of the cGMP phosphodiesterase (PDE)
(Kawamura 1992; Kawamura & Murakami 1991) while S-modulin purified with a phenyl Sepharose column affects not only
the fractional activation of PDE but also the inactivation of PDE
(Kawamura 1993). In both purifications, S-modulin was obtained as a single band on an SDS-PAGE gel. From these
results, there seem to be two populations of S-modulin whose
hydrophobic characteristics are different. Unfortunately, no
attempts have been made so far to isolate these components. As
reviewed in HURLEY'S target article, one of the S-modulin
effects, namely that on PDE inactivation, is attained by suppressing rhodopsin phosphorylation. The mechanism of the
other effect, the effect on the fractional activation, is not known.
However, the fractional activation also seems to be attained by
regulation of phosphorylation by S-modulin, since without ATP
or S-modulin, this effect was not seen (Kawamura 1993). It is
interesting to note that ATP has two distinct effects on PDE; one
was observed on the fractional activation of PDE with a half
effect at 50 n-M ATP and the other on the PDE inactivation with
a half effect at 2 p,M ATP (Kawamura 1983). Recent electrophysiological work has revealed that PDE fractional activation is
influenced by both Ca 2 + and a soluble Ca 2+ -binding protein;
this effect is distinct from the regulation of PDE inactivation
(Lagnado & Baylor 1994). This observation supports the possibility mentioned above.
Ca2+-dependent binding of S-modulln to membranes. S-mod-
ulin (Kawamura et al. 1992) and recoverin (Dizhoor et al. 1993;
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
479
Commentary /Controversies in Neuroscience III
Zozulya & Stryer 1992) bind to ROS membranes in a Ca 2 + dependent manner. It has been thought that this binding is
essential for S-modulin function. However, the [Ca 2+ ] required
for the half-effect of the membrane-binding of S-modulin (10
|j.M; Kawamura et al. 1992) is almost 100 times higher than
for half-inhibition of rhodopsin phosphorylation (=100 nM;
Kawamura 1993). In addition to this, it has recently been
found that N-myristoylation of recoverin is essential for the
membrane-binding (Dizhoor et al. 1993; Zozulya & Stryer 1992)
but not for the inhibition of rhodopsin phosphorylation (Kawamura et al. 1994). These two observations raised the possibility that membrane-binding at \x.M range of [Ca 2 + ], and
therefore N-myristoylation, is not essential for the function of
S-modulin.
S-modulin is washed out from a truncated ROS at 1 u.M Ca 2 +
(Kawamura & Murakami 1991). Thus, the binding affinity to the
membrane is low in intact cell in agreement with the binding experiment mentioned above. To elucidate the role ofthe membranebinding and thus N-myristoylation, further work is required.
Target molecule of S-modulin. Even though S-modulin exerts
its effect by inhibiting rhodopsin phosphorylation, the target
molecule has not been clearly identified. Rhodopsin kinase and
photolyzed rhodopsin are the potential candidates for the target
molecule (Kawamura 1993). Actually, it has been claimed that
recoverin binds to the rhodopsin kinase in a Ca 2+ -dependent
manner (Gorodovikova & Philippov 1993). However, the [Ca 2+ ]
used in this study was very high (100 u,M). At this very high
calcium concentration, physiologically less important membranebinding of recoverin takes place. Therefore the observed binding may not be physiologically relevant; we wish to see the
binding at physiological (<(j,M) [Ca 2 + ]. In the original report of
the finding of recoverin (Dizhoor et al. 1991), this protein was
isolated as a binding protein of rhodopsin. It remains to be
determined which of the molecules, rhodopsin kinase or photolyzed rhodopsin, is the real target.
At [Ca 2+ ] over 1 p.M, S-modulin/recoverin has been reported
to bind to ROS membranes or phospholipids (Dizhoor et al.
1993; Kawamura et al. 1992; Zozulya & Stryer 1992), which
raises the possibility that S-modulin/recoverin first binds to the
membrane and then interacts with rhodopsin kinase or photolyzed rhodopsin. However, the membrane-binding is not essential for the inhibition of rhodopsin phosphorylation (Kawamura
et al. 1994), and thus this possibility seems to be slight.
S-modulln homologs In other tissues. As reviewed in section 3
of HURLEY'S target article, many S-modulin/recoverin homologs
have been found in nonretinal tissue. These homologs can be
classified into three groups (Nef et al., submitted.) We picked up
S-modulin and recoverin from group 1, NCS-1 from group 2,
and Vilip 1 and hippocalcin from group 3, and examined the
effects of these proteins on rhodopsin phosphorylation. All of
the proteins inhibited rhodopsin phosphorylation at 10 u,M
Ca 2 + (Nef et al., submitted; Kawamura & Takamatsu, unpublished observation cited in Kawamura 1994). The site (or
sites) that interact with rhodopsin or its kinase must be conserved in this protein family. Even though the real functions of
these proteins may not be the same as in photoreceptors, they
could possibly regulate phosphorylation reactions in a Ca 2 + dependent manner in the host-cells.
Crucial steps in photoreceptor adaptation:
Regulation of phosphodiesterase and
guanylate cyclase activities and
Ca 2+ -buffering
Karl-Wilhelm Koch
Institut fur Biologische Informationsverarbeitung, Forschungszentrum
JQIich, Postfach 1913, 0-52425 Julich, Germany.
Inboo8ez.am00l.zam.kfa-juelich.de
480
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Abstract: This commentary discusses the balance of phosphodiesterase
and guanylate cyclase activities in vertebrate photoreceptors at moderate light intensities. The rate of cGMP hydrolysis and synthesis seem to
equal each other. Ca2+ as regulator of both enzyme activities is also
effectively buffered in photoreceptor cells by cytoplasmic buffer
components.
[BOWNDS & ARSHAVSKY; HURLEY] Since the light-triggered
cGMP cascade of visual transduction has been investigated in
more detail, a comprehensive description of its amplification
and the kinetics of its activation steps has become possible (Pugh
& Lamb 1993). Recently, interest has also been focused on other
aspects of phototransduction, for example, the inactivation steps
that restore the dark state of a photoreceptor cell and the
adaptational processes of which a significant part takes place in
the photoreceptor itself. A rich complexity of biochemical control functions has emerged in the last 3-4 years following the
initial observation from electrophysiological recordings that an
important signal of light adaptation is the change in intracellular
Ca 2 + (Matthews et al. 1988; Nakatani & Yau 1988). Bownds and
Arshavsky in this volume discuss many steps on phototransduction as putative points in the modulation of photoreceptor
recovery and light adaptation, emphasizing that the most important site of light adaptation cannot be determined at present
(sect. 4). This is certainly true; it will become necessary in the
future to work out the quantitative contribution of the different
control steps and feedback loops (involving phosphodiesterase,
guanylate cyclase, the cGMP-gated channel, and other unidentified proteins). I will comment mainly on two points related to
the target articles of BOWNDS & ARSHAVSKY and HURLEY: (1)
What can be concluded about the balance between guanylate
cyclase (GC) and phosphodiesterase (PDE) activity after light
stimulation or "is cyclase activation sufficient to contribute to
adaptation during background light?" (BOWNDS & ARSHAVSKY
para. 10). (2) What is known about the functional role of intracellular Ca2+-bufFers in photoreceptors?
1. Balance of PDE and GC activities. The discrepancy between the kcat values of activated GC (GC*) at low free Ca 2 + and
activated PDE (PDE*) after light stimulation is obvious (kcat of
GC = 10 sec" 1 ; kcat of PDE* = 2000 sec" 1 ) and would point to
an effective shutdown of PDE* activity under control of the
Ca 2+ -feedback. However, considering the cellular situation,
the rate of cGMP hydrolysis should be compared with the rate of
cGMP synthesis. According to Pugh and Lamb (1993) the rate of
cGMP hydrolysis is (equation 11 in their paper):
dcG/dt = -PDE* (t) [k^, (Km Nnv Vcyl BP "']cG
BP is the buffering power of the cytoplasm for cGMP. Nnv is
the Avogadro number and Vcyt is the cytoplasmic volume of a
rod. The free substrate concentration is cG. A similar equation
should be valid for the synthesis of cGMP by the GC. The rate of
transforming GTP to cGMP is:
-dGTP/dt = dcG/dt = GC*(t) [keal (Km Nov Vcyl )"']CTP
A half-saturating response activates about 180 rhodopsin molecules (Rh*) per sec in bovine photoreceptors (Nakatani et al.
1991). With an activation of 2500 transducin molecules (G*) per
Rh* and per sec at 30°C (Kahlert & Hofmann 1991), we have 4.5
x 105 PDE*. With the numbers for a mammalian rod provided
by Pugh and Lamb (1993):
2 kcat = 4000 sec-1; Km = 100 |xM; Vcyt
cG = 4 (xM
40fl;BP = 2;
the rate of cGMP hydrolysis by PDE* is dcG/dt = 720 (J.M
sec"1.
Assuming that at half-saturating responses the cytoplasmic Ca 2 +
drops to a value below 0.1 (iM in one second by operation of
the powerful Na:Ca,K-exchanger (Lagnado et al. 1992) all GC
molecules become activated by the Ca z+ -feedback. The GC to
Commentary/Controversies in Neuroscience III
rhodopsin ratio in a bovine photoreceptor was determined to be
1:100 yielding about 1.5 x 106 GC* (Koch 1991). Free GTP is
about 1 mM and the Km of GC with MG 2 + as cofactor is 1 mM
(Koch et al. 1990).
Abstract: Light adaptation is modulated almost exclusively by changes
in intracellular Ca 2 + concentration, and other Ca 2+ -independent mechanisms are likely to play only a minor role. Changes in Ca 2+ , may be not
only necessary for light adaptation to take place but sufficient to cause it.
the rate of cCMP synthesis by GC* is dcG/dt = 600 (xM sec-1.
In their target article BOWNDS & ARSHAVSKY say that calcium is
unlikely to be the sole regulator ofadaptation in vertebrate photoreceptors. Wewouldargue(withKoutalosetal. 1994) that adaptation can be accounted for by reactions that are modulated by
changes in intracellular Ca 2 + concentration (Ca2+j). We believe
recent results indicate that changes in Ca 2+ , are not only necessary
for light adaptation to take place but are sufficient to cause it.
It is well known that adaptation to steady light leads to graded
acceleration of the recovery of the response to a bright flash.
This acceleration can be abolished if the fall in Ca 2+ j induced by
light is prevented by superfusing the outer segment with low
Ca 2 + /0 Na + solution, which simultaneously minimizes Ca 2 +
influx and efflux (Matthews et al. 1988; Nakatani & Yau 1988).
The falling phase of the flash response is unaffected by prior
exposure to steady light if the light is presented in low Ca 2 + /0
Na + solution (Fain 1989). Together with other observations, this
result indicates that the light-induced fall in Ca 2 + , is necessary
for adaptation to take place (Fain & Matthews 1990).
When low Ca 2 + /0 Na + solution is used to hold the Ca 2 + in the
cell near its dark level and steady light is presented, the
circulating current can be completely suppressed by quite a dim
background light (Fain et al. 1989). Partial inhibition of the PDE
(phosphodiesterase) with IBMX can then be used to restore the
dark current to its normal level despite the continued presence
of the background. Under these circumstances, the response to
a dim flash exhibits dark-adapted kinetics, instead of the more
rapid kinetics normally seen in the presence of the background
when Ca 2 + , is allowed to fall to the appropriate light-adapted
level (Matthews 1992b; 1995). This observation shows that adaptational changes in the kinetics of the dim flash response require a
fall in Ca 2 + i ( which cannot be mimicked by light alone. However,
it should be noted that this approach is intended to restore the
cyclic G M P concentration to around its normal value in darkness,
and so cannot directly rule out a feedback role for cyclic GMP.
If the photoreceptor is allowed to adapt to steady light in
Ringer and then exposed to low Ca 2 + /0 Na + solution, the Ca2"1",
can be held near the value appropriate for the steady light
intensity. If the background is then extinguished, the response
to a subsequently-presented bright flash is still accelerated to
the same degree as when the steady light was present, even
though in Ringer the same period of darkness would have
allowed a substantial degree of recovery of response kinetics
(Matthews 1995). Comparable acceleration of bright flash response recovery can be seen when Ca 2 + | is artificially reduced in
darkness by removing Ca 2+ O (Matthews 1992a; 1995). These
results strongly suggest that lowered Ca 2+ j is sufficient to cause
adaptation even in the absence of light itself.
These observations place severe constraints on the possible
significance of any adaptational mechanism operating other than
through changes in Ca 2+ ,. However, Ca 2+ ( can only be reliably
controlled by supervision with low Ca 2 + /0 Na + solution for 1015 s (Fain et al. 1989). Thus these results do not directly exclude
the possibility that some other calcium-independent mechanism might operate on a longer time scale. However, as
the great majority of adaptation takes place within 10-15 s
in Ringer, the quantitative significance of any calciumindependent mechanism would seem likely to be rather limited.
Apparently, both enzyme activities almost match each other at
moderate light intensities. A decrease of free cGMP would only
affect PDE* since CC activity is independent ofcGMP(but GC is
inhibited by the other product pyrophosphate; see for example
Hakki & Sitaramayya 1990; Yang & Wensel 1992). Thus, regulation of PDE* by substrate level seems to be significant. A similar
conclusion is drawn by Miller and Korenbrot (1993) based on
their observations on cone photoreceptors of striped bass.
2. Recoverin as Ca2*-buffer. Ca 2 + as modulator of phototransduction exerts its effects mainly via Ca 2+ -binding proteins
(see recent review, Koch 1994). Another crucial aspect of Ca 2 +
and its role in recovery and light adaptation is the Ca 2 + buffering in an intact photoreceptor cell. Ca 2+ -binding proteins
are good candidates to serve this function, HURLEY discusses
properties of recoverin, a prototype of a new class of neuronal
Ca 2+ -binding proteins originally isolated from photoreceptor
preparations. There is presently good evidence that recoverin or
the amphibian homolog S-modulin prolongs the lifetime of
PDE*. I will add a few remarks on a possible function of
recoverin as a Ca2+-buffer. Lagnado et al. (1992) have demonstrated the existence of two intracellular Ca2+-buffers in tiger
salamander rods. One buffer is present in high amounts and has
a low affinity for Ca 2 + , the other buffer is present in smaller
amounts, but has a higher affinity. Particularly the high-affinity
buffer has important functions: (a) It determines the rate of
relaxation in intracellular Ca 2 + after closure of the lightsensitive channel. Thus the onset of Ca 2+ -dependent light
adaptation would be controlled by this buffer (Lagnado et al.
1992). (b) It would continuously supply Ca 2 + from its binding
sites and therefore be crucial for the operation of the Na:Ca,Kexchanger. (c) It would participate in any new steady state of
Ca 2+ -homeostasis at different background light intensities.
Lagnado et al. (1992) have derived some molecular properties
of the high-affinity buffer from their electrophysiological recordings. The buffer is a diffusible macromolecule with a buffering
capacity of 35 |i,M in aequorin-loaded rods or 241 u,M in intact
rods. The apparent Michaelis constant Kbllfrwas £ 0.7 u,M. DO
the properties of recoverin fulfill these requirements? Recoverin can be isolated in high amounts from rod outer segments at
least in a ratio of 1:100 to rhodopsin corresponding to a cellular
concentration of 30 u,M (reviewed in Koch 1994). Although KD
values for Ca 2+ -binding have not been documented, the threedimensional structure of recombinant unrnyristoylated recoverin gave first insight into its Ca 2+ -binding properties (Flaherty
et al. 1993). Recoverin has two canonical EF-hands that probably bind Ca 2 + with KD-values of 0.15 u.M and 2 u.M. Therefore,
due to its relative abundance and high-affinity binding of Ca 2 + ,
recoverin could function as a prominent Ca2+-buffer in photoreceptor cells. For a full description of Ca 2+ -homeostasis it will
be important to determine the intracellular concentrations of
photoreceptor Ca 2+ -binding proteins and their corresponding
KD-values.
Reduced cytoplasmic calcium concentration
may be both necessary and sufficient for
photoreceptor light adaptation
H. R. Matthews and G. L. Fain
Physiological Laboratory, University of Cambridge, Downing Street,
Cambridge CB2 3EG, England and Department of Physiological Science,
University of California at Los Angeles, Los Angeles, CA 90025-1527.
hrml@phx.cam.ac.uk; gordonf@physcl.lifesci.ucla.edu
Gene therapy, regulatory mechanisms, and
protein function in vision
James F. McGinnis
Department of Anatomy and Cell Biology, University of California, Los
Angeles, Los Angeles, CA 90024. ianw|fm@mvs.oac.ucla.edu
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
481
Co7nmentari//Controversies in Neuroscience III
Abstract: Hereditary retinal degeneration due to mutations in visual
genes may be amenable to therapeutic interventions that modulate,
either positively or negatively, the amount of protein product. Some of
the proteins involved in phototransduction are rapidly moved by a lightdependent mechanism between the inner segment and the outer segment in rod photoreceptor cells, and this phenomenon is important in
phototransduction.
[DAICER ET AL.; BOWNDS & ABSHAVSKY; HURLEY] T h e target
article by DAIGER ET AL. has done an excellent job of documenting, organizing, and correlating specific mutations, especially in
rhodopsin, with hereditary blindness. Although it is still not yet
possible to predict the severity of the disease based on precise
mutations in rhodopsin, the amount of knowledge accumulated
enables one to begin to envision therapeutic methods that can
be devised to prevent the onset or to stop the progression of the
degeneration of photoreceptor cells. In those cases where the
accumulation of defective rhodopsin molecules is correlated
with blindness, a logical strategy would be to inhibit the production of such molecules. This could be accomplished through the
development of "antisense" therapies using either antisense
oligonucleotides and/or vectors that express antisense molecules capable of distinguishing between the "good" mRNA and
the "bad" mRNA. Similarly, studies that identify and characterize cts-acting and trans-acting regulatory regions may provide
alternative approaches to suppress the synthesis of a specific
mRNA. In those cases of blindness due to the absence of a
specific function, the development of appropriate vectors that
can target the expression of the correct gene to the photoreceptor cells offers a real possibility to preserve vision by preventing
the death of the cells. A "good" gene product, even at half the
concentration found in the normal individual, should be sufficient to stop the progression of the disease. Cell culture techniques for growing and maintaining retina tissues and cells in
vitro will provide the opportunity to develop and test the
efficacy of such approaches. These in vitro techniques may also
enable small numbers of photoreceptor cells to be removed from
an individual, be maintained and "modified" in vitro, and then
transplanted to the same or different person to provide a source
of hormone, nutrient, growth factor, or drug for nearby cells or
as functional replacements for defective cells. The compilation
and analysis of mutations in photoreceptor cell-specific genes
such as presented in this article are necessary for progressing to
the next level of research on retinal degeneration; the preservation and/or restoration of vision in patients with retinitis pigmentosa or other hereditary eye diseases.
The target article by BOWNDS & ARSHAVSKY does not include a
discussion of the contributions and/or effects of the lightdependent movement of some of the proteins involved in
phototransduction. We have shown, at the light microscopic
level, that the intracellular localization of transducin and arrestin
in mouse photoreceptors is transient and dependent on the
lighting environment to which the retina is exposed (McGinnis et
al. 1992b; Whelan & McGinnis 1988). Similar data have been
obtained in rats and frogs by other laboratories (Brann & Cohen
1987; Mangini & Pepperberg 1988; Philip etal. 1987). In the dark
when the rods are most active, the inhibitory protein, arrestin, is
in the inner segments while the activating protein, transducin,
is in the outer segments. When the lights are turned on, or
with sunrise, the positions of these proteins rapidly and simultaneously reserve with the majority of arrestin moving to the
outer segment and transducin moving to the inner segment. This
movement has already begun at the earliest time point tested (30
seconds) and is readily reversed by changing the lighting environment (McGinnis et al. 1992b). The outer segments from lightadapted mice were shown to contain at least three times as much
arrestin as the outer segmentsfromdark adapted mice (Whelan &
McGinnis 1988), supporting the conclusion that the proteins are
actually moving between the inner and outer segments. Temporal and spatial changes in the cellular and subcellular concentrations of photoreceptor cell gene products appear to be important
482
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
features of phototransduction in rod photoreceptor cells. The
positional shifts of arrestin and transducin result in dramatic
changes in the subcellular concentrations of these photoreceptor specific proteins and, therefore, dramatic changes in the
rates of the reactions catalyzed by these proteins. We think that
these light-dependent translocations are functionally related to
the mechanism of phototransduction and photoreceptor adaptation and that they may be incorporated into any model that seeks
to explain the molecular basis of these processes.
I would like to add to the information presented in the target
article by HURLEY. Using a novel immunological approach, we
(McGinnis & Leveille 1985) identified four proteins as being
photoreceptor cell-specific. One of these, 23kDa, was subsequently cloned (McGinnis et al. 1992a) from our mouse and
bovine retina cDNA expression libraries in lambda gtll and
shown to be identical to the amino acid sequence published for
the protein cloned and named recoverin (Dizhoor et al. 1991)
and the cancer-associated retinopathy protein (Polans et al.
1991; Thirkill et al. 1992) or S-modulin (Kawamura & Murakami
1991). We subsequently mapped the mouse 23kDa (recoverin)
gene to chromosome 11 adjacent to the tumor suppressor gene
trp53 (McGinnis et al. 1993) and the human recoverin gene to
17pl3.1, adjacent to the human tumor suppressor gene p53
(McGinnis et al. 1994). Recoverin was shown to be developmentally regulated in the mouse and to be present in the retinas of a
variety of species (Stepanik et al. 1993). We cloned frog recoverin, showed it to be identical to frog S-modulin and demonstrated, using coupled in vitro transcription and translation of a
rhodopsin kinase cDNA, that rhodopsin kinase specifically
binds to mouse and frog recoverin (Subbaraya et al. 1994). This
supports our suggestion that the function of recoverin is to
inhibit the phosphorylation of rhodopsin that would prevent its
interaction with arrestin and thereby prolong the light state.
Structure of the cGMP-gated channel
Daniel D. Oprian
Graduate Department of Biochemistry and Volen Center for Complex
Systems, Brandeis University, Waltham, MA 02254
Abstract: The subunit structure of the cGMP-gated cation channel of
rod photoreceptors is rapidly being defined, and in the process the
mode of regulation by Ca 2+ -calmodulin unraveled. Intriguingly, early
results suggest that additional subunits of unknown function are associated with the channel and remain to be identified.
[MOLDAY & HSU] The target article by MOLDAY & HSU provides a
timely review of the recent history of the cGMP-gated channel
of rod photoreceptor cells. Thisfieldhas advanced by leaps and
bounds since the seminal observation by Fesenko et al. (1985)
that the cation conductance of rod cell plasma membranes was
controlled by cGMP. The identity of the channel, which formed
the foundation of a lively controversy just a few years ago, is now
resolved, at least to the extent that the a-subunit cloned by Kaupp
et al. (1989) forms the cGMP-gated pore. However, the complete formation of the channel with all of its pharmacological and
electrophysiological nuance requires in addition a (5-subunit isolated by Chen et al. (1993) and perhaps other as yet unidentified
polypeptides. It is here that Molday and Hsu bring us to the current issues in the structure of the channel.
Purification of the channelfrombovine retina by immunoaffinity
chromatography using a monoclonal antibody raised to the osubunit results in two proteins identifiable by SDS-PAGE: a63 kDa
subunit corresponding to a proteolytically processed form of the
a-subunit in which the amino-terminal 92 amino acids have been
removed, and a much larger protein migrating with an apparent
mobility of240 kDa. The identity of the 240 kDa protein remained a
mystery until very recently. Hsu and Molday (1993) showed that the
affinity of the channel for cGMP is regulated by Ca2+-calmodulin,
Commentary/Controversies in Neuroscience III
and that the channel can be purified on a calmodulin affinity
column. The channel purified in this manner contains the same
two proteins that were purified by immunoaffinity chromatography and blots of SDS gels demonstrated that calmodulin binds to
the 240 kDa protein. Very recently, Molday and Yau and coworkers (Chen et al. 1994) have shown that antibodies raised to
the P-subunit bind to the 240 kDa protein and that the amino acid
sequence of peptides from this protein match closely the sequence of the cloned human P-subunit. Furthermore, electrophysiological measurements of the channel expressed in HEK
293 cells after connection of mRNA for both the a- and p-subunits
demonstrates that the P-subunit confers on the channel sensitivity to Ca 2+ -calmodulin. Prior to these studies, the p-subunit
was known to be inactive on its own, but when coexpressed with
the ot-subunit gives the channel the flickering response and
sensitivity to l-cis diltiazem long associated with the intact channel from rod outer segment plasma membranes. The more recent
results clearly demonstrate that the P-subunit is an integral component of the 240 kDa protein and that the regulatory properties of
Ca2+-calmodulin stem from an interaction with the P-subunit.
However, major questions remain. For example, what other
polypeptides are components of the 240 kDa protein and what is
the subunit composition and structure of this complex. It is clear
that there are other polypeptides. To begin, the calculated
molecular weight of the cloned p-subunit is 102 kDa not 240
kDa. Also, the P-subunit polypeptide isolated from transfected
HEK 293 cells migrates on SDS gels with a mobility of 130-150
kDa, again distinct from 240 kDa. Finally, some of the peptides
isolated and sequenced from the 240 kDa complex are clearly
different from the cloned P-subunit. It will be very interesting
to discover what additional polypeptides are components of the
240 kDa complex and why this assembly survives dissociation on
SDS gels. Answers to these questions will provide much needed
information for resolution of the channel's structure.
Recoverin is the tumor antigen in cancerassociated retinopathy
new information is now available about the role of recoverin in
the development and progression of the CAR syndrome.
Our laboratories recently identified recoverin in the tumor of a
CAR patient diagnosed with small cell carcinoma of the lung. We
performed a Western blot analysis using biopsy material and the
patient's own serum. A prominent band at 23 kDa was reactive
along with a few nonspecific bands that also reacted with normal
human sera. Sera from other CAR patients gave the identical
staining profile. Since human recoverin migrates at 23 kDa, we
used a series of anti-peptide antibodies generated against different regions of the recoverin sequence (Polans et al. 1993), and the
same 23 kDa protein was stained with each of the antibodies. Our
experiments, therefore, demonstrate that recoverin is expressed
in the tumors of CAR patients. We have analyzed similar tumors
from individuals who do not have the associated retinopathy,
and recoverin cannot be detected in those tumors. Therefore, it appears likely that the disease originates as the result of the aberrant expression of recoverin in a subset of tumors. Recoverin is
photoreceptor-specific and normally expressed in the eye, which is
an "immunologically privileged" site. Under normal conditions,
potential antigens like recoverin are unavailable for immunological
recognition at the inductive stage of the immune response due to
sequestration within photoreceptors. Owing to the expression by
the tumor, however, the released recoverin can trigger the autoimmune response, which in the patient results in both autoantibodies
and specific T-cells. The immunological response then correlates
with the degeneration of the target cells, photoreceptors.
If the disease is autoimmune mediated then a potential
autoantigen (recoverin) should induce immunological mechanisms that lead to the degeneration of photoreceptors. Two
recent studies have demonstrated this finding (Adamus et al.
1994; Gery et al. 1994). After inoculation of Lewis rats with a
single dose of recoverin, a specific immunological response
occurred. Antibodies to recoverin could be detected seven days
after the initial immunization, and the peak of the antibody
response occurred around day 28. The response was specific for
recoverin; no antibodies could be detected using arrestin (Santigen), IRBP or rhodopsin in specific ELISAs (Fig. 1). Impor-
Arthur S. Polans and Grazyna Adamus
R S. Dow Neurological Sciences Institute, Legacy-Good Samaritan
Hospital and Medical Center, Portland, OR 97209
Abstract: Considerable progress has been made toward understanding
the involvement of recoverin in a cancer-associated retinopathy (CAR)
that results in blindness. We describe the expression of recoverin in
tumors of individuals afflicted with CAR, characterize the hnmunological response towards recoverin in these patients, and demonstrate how
the disease can be induced in rodents using recoverin as an immunogen.
[HURLEY] Recoverin was identified simultaneously in 1991 by
four laboratories. Dizhoor et al. (1991) and Lambrecht and Koch
(1991) characterized the protein in terms of its possible involvement in the calcium-dependent regulation of guanylyl cyclase.
Kawamura and Murakami (1991) isolated the frog homologue of
recoverin, S-modulin, and proposed its regulation of cyclic GMP
phosphodiesterase. In studies unrelated to phototransduction,
Polaris et al. (1991) identified recoverin as the autoantigen in a
parancoplastic disease of the retina known as cancer-associated
retinopathy (CAR). In this disorder individuals with cancer at a
site other than the nervous system develop visual abnormalities
and eventually blindness. Autoantibodies specific for recoverin
were found only in the sera of CAR patients, and the presence of
such antibodies suggested that this paraneoplastic disease may
result from an autoimmune response based on antigenic mimicry.
The occurrence of autoantibodies could be the result of selfantigen(s) released from the cancerous tissue followed by a
response of the immune system to these antigens. In CAR, the
presence of such antibodies correlates with the degeneration of
rods and cones, resulting in loss of visual function. Considerable
IT)
O
<*
Q
O
PRE-IMM
REC
RHO
IRBP
SAG
ANTIGENS
Figure 1 (Polans & Adamus). ELISA of rat antiserum to various
photoreceptor proteins. Dilutions of antiserum (1:1,000) obtained from a rat after immunization with 50 p.g recoverin were
added to plates coated with 50 ng of the following retinal
proteins: rhodopsin (RHO), interphotoreceptor retinoid binding protein (IRBP), S-antigen (S-AG), and recoverin (REC). Preimmune serum from the same animal also was tested.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
483
Commentary/Controversies in Neuroscience III
B
Figure 2 (Polans & Adamus). Histopathological changes in the retinas of a human CAR patient (A) and Lewis rat 39 days after
immunization with 50 |xg recoverin (B). There is limited inflammatory infiltrate detectable in both tissues, while the photoreceptor
layers have completely degenerated (asterisks). C, choroid; I, inner nuclea layer; G, ganglion cell layer. Paraffin embedded sections
stained with hematoxylin and eosin.
tantly, there was no secondary response to other retinal antigens
that might become available during photoreceptor degeneration. As measured by a lymphocyte proliferation assay, cellular
responses towards recoverin also were detected and reached a
peak 17 days after immunization. Furthermore, noT-cells were
activated by S-antigen, IRBP or rhodopsin in comparable cell
proliferation assays. During the same period as the cellular
responses to recoverin, inflammation of the retina also was
observed. By four weeks after the single injection of recoverin,
photoreceptor cells had degenerated while the remainder of the
inner retina and pigment epithelium appeared normal, thus
paralleling the end stage of the CAR syndrome in humans (Figs.
2A and 2B). The combined data indicate that photoreceptor
degeneration is not the result of an epiphenomenal event
involving induction by other retinal antigens. Rather, development of a specific immunological response to recoverin underlies the degenerative event. Furthermore, the autoimmune
etiology of CAR is supported by the finding of recoverin in the
tumors of CAR patients, the presence of autoantibodies and
T-cells to recoverin in those patients, and studies of animal
models.
It has yet to be determined how the immune response leads to
photoreceptor degeneration, and specifically the role of autoantibodies. In previous experiments (Adamus et al. 1994), we
demonstrated that the retinopathy can be produced in Lewis
rats by passive transfer of T-cells obtained from animals immunized with recoverin, but the pathogenicity of the anti-recoverin
antibodies still needs to be studied. In regards to other experiments, it is noteworthy that the gene encoding recoverin maps
to human chromosome 17pl3.1, which also is the site of the
tumor suppressor gene encoding p53. A low frequency mutation
in p53 might explain both the development of the tumor as well
as the aberrant expression of recoverin in a subset of tumors.
This no doubt is a simplistic model, since the fine mapping
between p53 and recoverin has not been performed, nor do we
know whether other retinal/neural gene products mapped to
the same region of chromosome 17 also are expressed by the
CAR patient's tumor. The regulation of recoverin expression in
these tumors, nonetheless, will be an important aspect of future
research.
Adenylyl cyclase, G proteins, and synaptic
plasticity
Mark M. Rasenick
Departments of Physiology, Biophysics and Psychiatry, University of
Illinois, College of Medicine, Chicago, IL 60612-7342. raz@uic.edu
484
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Abstract: It has been suggested that type I adenylyl cyclase may play a
unique role in long-term potentiation, due to both unique regulatory
properties as well as a specialized distribution within the mammalian
brain. This would allow an integration of the signals wrought by
increased intracellular calcium with those conveyed into the cellular
milieu via increased cAMP. These results are discussed in the context of
changes in cellular structure, because of changing interactions between
G proteins and cytoskeletal components, which might be expected to
accompany chronic synaptic activation.
[XIA ET AL.] The target article by XIA ET AL. makes interesting
observations that suggest a role for a specific adenylyl cyclase
subtype (type I) in the synaptic events subserving long term
potentiation. A model is presented that would integrate calcium
and cyclic AMP signals and provide for the possible long-term
elevation of cAMP, which might be needed to evoke the synaptic remodeling (physical or chemical) that likely accompanies
this process.
It is suggested that type I adenylyl cyclase might serve as a
unique neuromodulator due to its unique distribution and
localization to some brain regions often associated with long
term potentiation (LTM). It must be pointed out, however, that
mRNA hybridization was used to localize this enzyme. Most of
the localization of mRNA is confined to the cell body. While
there appears to be protein synthesis in the dendrite, it is likely
that the amount of mRNA there is sufficiently small that most
hybridization techniques would not detect this. If LTM is a
process which is largely presynaptic (this author is not attempting to join the debate), then Ca2+ sensitive adenylyl cyclase at
the axon terminals would be expected to have the type I
enzyme. The cell bodies have a diffuse representation, not all of
which appears positive for type I adenylyl cyclase. Further,
axons might project some distance from the synapse. Sustained
Ca2+ entry into synaptic terminals could also activate type I
adenylyl cyclase, but the precise localization of that enzyme
awaits the development of selective antibodies of sufficient
quality to be used in immunoelectron microscopy.
The authors state that receptors and Gs do not activate the
type I enzyme in the absence of calmodulin. These unpublished
findings appear to contradict recent work (Sutkowski et al. 1994),
which demonstrates significant (five-fold) activation of Type I
adenylyl cyclase in SF9 membranes with activated Gsa. This
does not undermine the authors' intriguing proposition that
there exists some mechanism for the receptor-independent
stimulation of adenylyl cyclase.
We have suggested (Roychowdhury & Rasenick 1994; Yan &
Rasenick 1990) that, since the net neurotransmitter (quantitatively) available to stimulate adenylyl cyclase is minuscule
next to that involved in the inhibition of that enzyme, there
must be some indirect mechanism for activating that enzyme.
Commentary/Controversies in Neuroscience III
Studies from the groups of either Cooper or Gnegy (Ahlijanian
ct al. 1987; Treisman et al. 1983) suggest that Ca 2 + may indeed
play a role in such an indirect activation of the enzyme. These
studies also suggested that calmodulin activation of adenylyl
cyclase (in brain membranes) took two forms, one of which was
independent and one of which required GTP. The GTPindependent form likely represented the type I enzyme. The
calmodulin-GTP sensitive adenylyl cyclase connection need not
have been direct. In fact, we have postulated that the role of
calmodulin (in this indirect activation of adenylyl cyclase) is to
activate CAM kinase II, subsequently phosphorylating microtubule associated protein 2 (MAP2), which occurs complexed
with CAM Kinase, MAP 2 and tubulin on the post-synaptic
membrane. MAP2 subsequently reduces its affinity for tubulin,
which binds to Gsa or Gial with a Kd of about 130 nM and
activates these G proteins due to direct transfer of GTP
(Roychowdhury et al. 1993; Wang et al. 1990), and in the case
where Gsa is activated, effects a calmodulin-dependent yet
indirect activation of adenylyl cyclase. Tubulin is capable of
transferring GTP to a recombinant Got under conditions where
that Cot is incapable of binding nucleotide from the medium
(Roychowdhury & Rasenick 1994). In fact, tubulin is capable of
bypassing a "tightly coupled" receptor to activate a G protein
(Popova et al. 1994). It is suggested that tubulin, perhaps in
response to a neurotransmitter not normally coupled to a G
protein, can be engaged to activate Gs or Gil and their attendant intracellular effectors. This could be especially important
with respect to the possibility that adenylyl cyclase can be
activated (depending on subtype) by f}"y subunits, liberated,
perhaps from a Ga coupled to a receptor nor normally associated
with the stimulation of adenylyl cyclase (Andrade 1993). Such
stimulation cannot occur without the prior activation of Gsa.
The mechanism proposed here would allow tubulin, liberated,
perhaps from the bonds of MAP2 on the post-synaptic membrane in response to an increase in intracellular Ca 2 + , to activate
Gs, Gil, orGq in a receptor-independent fashion. Thus, tubulin
may provide a conduit for the interactions among neurotransmitters.
Clearly, there is a relationship between the ordered environment of the synaptic membrane and G protein-mediated signal
transduction systems. This might be particularly true in the
nervous system, where rapid and discrete response is a hallmark
of synaptic transmission. Further, as pointed out by the authors,
changes in synaptic form may accompany changes in synaptic
efficiency. Ca 2+ -calmodulin-mediated processes may well be
involved in such shape changes. Further, it is possible that G
proteins at the synapse may be involved in synaptic shape
change through their modulation of the assembly state of synaptic microtubules (Want & Rasenick 1991). In this way, signals
through a variety of second messenger systems may interface to
orchestrate the cellular events that create memory.
Regulation of adenylyl cyclase in LTP
Erik D. Roberson and J. David Sweatt
Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030.
erikr@llgand.neusc. bcm.tmc.edu and david@ligand.neusc.
bcm.tmc.edu
Abstract: Our results on hippocampal long-term potentiation are considered in the context of Xia et al.'s hypothesis. Whereas the target
article proposes presynaptic PKC involvement in adenylyl cyclase
activation by phosphorylation of nenromodulin, we suggest an additional postsynaptic role involving RC3/nenrogranin. Finally, we examine the possibility that the adenylyl cyclase mutant mouse may display
normal learning with a selective impairment of memory.
[XIA ET AL.] The target article by XIA ET AL. reviews the
evidence supporting the hypothesis that a general mechanism of
long-term synaptic modulation is activation of adenylyl cyclase
by calcium and calmodulin, leading to altered gene expression
via activation of PKA. Our research on the molecular mechanisms of long-term potentiation (LTP) in area CA1 of the rat
hippocampus strongly supports this model. Xia et al. document
the fact that adenylyl cyclase is activated during LTP induction
(Chetkovich et al. 1991; Frey et al. 1993). This activation is
blocked by preventing calcium flux through NMDA receptors
(Chetkovich et al. 1991; Frey et al. 1993) and requires calmodulin (Chetkovich & Sweatt 1993). Although small, the LTPassociated increase in cAMP concentration is sufficient to
support activation of PKA (Roberson & Sweatt, unpublished
observations). In addition, the fact that the rise in cAMP is
transient (Chetkovich & Sweatt 1993) is consistent with the
model in which only a brief activation of PKA is required to
initiate the cascade leading to changes in gene expression.
The target article ably synthesizes the growing body of evidence supporting a role for calmodulin-stimulated adenylyl
cyclases in LTP. We would like to open two additional lines of
discussion:
1. Is the Interaction with PKCpre- or postsynaptic^ An issue of
great debate in LTP is whether the modifications that support
potentiation occur pre- or postsynaptically. Addressing the role
of PKC in activating adenylyl cyclase in section 7.2, Xia et al.
have focused on regulation of calmodulin buffering by neuromodulin, which is localized presynaptically in axon terminals
(Goslin et al. 1988). However, in light of data suggesting that
some changes in gene expression during LTP occur postsynaptically (Mackler et al. 1992), we feel that the hypothesis should
be extended to include a role for the postsynaptic calmodulinbinding protein RC3/neurogranin (Baudier et al. 1989; Watson
et al. 1990). RC3 is a 17 kD protein homologous to neuromodulin in its PKC phosphorylation and calmodulin-binding
domains that, like neuromodulin, releases calmodulin when
phosphorylated by PKC. However, unlike neuromodulin, RC3
is localized postsynaptically and enriched in dendritic spines
(Represa et al. 1990; Watson et al. 1992).
We have demonstrated an increase in PKC activity and in the
post hoc phosphorylation of a 17 kD PKC substrate during LTP
(Klann et al. 1991; 1992; 1993). This 17 kD protein is biochemically and immunologically indistinguishable from RC3
and its phosphorylation in situ increases during LTP as demonstrated by back phosphorylation (Chen, Klann & Sweatt, unpublished observations). Thus, RC3 may serve to release calmodulin postsynaptically during LTP and contribute to the
activation of adenylyl cyclase and other postsynaptic effectors,
such as the retrograde messenger generator.
2. A selective memory knockout? In section 8.4, XIA ET AL.
describe the deficits of the adenylyl cyclase knockout mouse in
the Morris water maze task. They stress the defect in the
transfer test, attributing the mutant's normal latency of escape
onto the hidden platform to the low sensitivity of that index.
This is a valid interpretation of the data, but there is an alternative explanation.
The hidden platform task is by its nature a relatively immediate test of learning and short-term memory, especially when
trials are massed; on the other hand, the transfer test is usually
administered after days of training on the hidden platform task
and thus is a measure of longer-term memory. It is possible that
the mutation in adenylyl cyclase selectively interferes with longterm memory, leaving learning and short-term memory, and
thus hidden platform task performance, intact. This would be
consistent with evidence suggesting that in the hippocampus,
cAMP antagonists selectively block the late phase of LTP,
having no effect on earlier stages (Frey et al. 1993). Interestingly, transgenic mice with mutations in a-calcium/
calmodulin-dependent kinase II, which lack early stages of LTP,
do exhibit deficiencies on the hidden platform task (Silva et al.
1992a; 1992b).
Our hypothesis predicts that protein synthesis inhibitors,
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
485
Commentary /Controversies in Neuroscience III
that, like cAMP antagonists, selectively block late LTP (Frey et
al. 1988), should cause deficits in the transfer test, but not in the
hidden platform task. This would be consistent with data from
many other systems, where inhibiting protein synthesis spares
initial learning, but selectively interferes with retention of the
learned behavior (Agranoff et al. 1965; Tully et al. 1994). We are
not aware of any published accounts of the effects of protein
synthesis inhibitors on hidden platform task performance, but
rats injected with these inhibitors do have deficits in the transfer
test (Itoh et al. 1992).
XIA ET AL. have examined a strong, testable hypothesis for the
mechanism of long-lasting modulation of synaptic function, with
supporting evidence in systems as diverse as Aplysia, Drosophila, and the mammalian hippocampus. Such a hypothesis provides a solid conceptual framework around which to construct a
future experimental edifice.
Modulation of the cGMP-gated channel by
calcium
Mandeep S. Sagoo and Leon Lagnado
Medical Research Council, Laboratory of Molecular Biology, Hills Road,
Cambridge CB2 2QH, England. Il1@mrc-lmb.cam.ac.uk and ms1@mrclmb.cam.ac.uk.
Abstract: Calcium acting through calmodulin has been shown to regulate the affinity of cyclic nucleotide-gated channels expressed in cell
lines. But is calmodulin the Ca-sensor that normally regulates these
channels?
[MOLDAY & HSU] In the first five sections of their target article,
MOLDAY & HSU provide a concise overview of the biochemical
and functional properties of the cGMP-gated channel in photoreceptors. In the next two sections they concentrate on the
evidence that calcium acts through calmodulin to regulate the
affinity of cGMP-gated channel for its ligand. Recent work from
their laboratories has demonstrated that this modulatory action
cannot be measured in the homo-ologomeric channel formed by
the human alpha subunit expressed in a cell line, but it can be
measured when the alpha subunit is coexpressed with the
longer of the two splice forms of the beta subunit. The modulatory action of calmodulin on the rod cGMP-gated channel is
therefore mediated through the beta subunit. In contrast, the
effect of calmodulin on the cyclic nucleotide-gated channel from
rat olfactory receptors is mediated through the alpha subunit. A
second important difference between the two types of receptor
is that calmodulin has a stronger action on the olfactory channel:
calcium-calmodulin increases the KU2 of the expressed olfactory
channel over 10-fold, whereas the K 1/2 of the rodchannel is
increased 1.5-to-2-fold.
When a rod responds to light, calcium levels fall inside the
outer segment, and MOLDAY & HSU suggest that this will relieve
the effect of calmodulin on the channel, increasing its affinity for
cGMP and therefore promoting the re-opening of channels
during the process of light adaptation. Unfortunately, we have
relatively little information about this proposed action. GrayKeller and Detwiler (1994) have reported that calmodulin or calmodulin inhibitors introduced into a transducing rod through a
patch pipette have no effect on phototransduction. We have
made similar observations using an alternative preparation, the
truncated salamander rod outer segment. This preparation
leaves the outer segment relatively intact and many properties
of the phototransduction mechanism are maintained (Lagnado
& Baylor 1994), but with the advantage that it is possible to
manipulate internal levels of calcium and cGMP. We find that
calcium over the range 200nM to lOnM regulates the channels
affinity for cGMP. At low cGMP concentrations the current is
486
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
increased about 2-fold when Ca is removed. A diffusible mediator is involved, since the ability to modulate the channel's
affinity for cGMP by changing calcium gradually washes out.
However, there does not seem to be any effect of adding
calmodulin or calmodulin inhibitors such as mastoparan. All
these observations are unpublished. A similar observation is
found in the original study of Kramer and Siegelbaum (1992) on
olfactory receptors, where the ability of calcium to modulate the
cAMP-dependent current did not seem to involve calmodulin.
Although we are still investigating a possible action of calmodulin, the obvious implication of the results we have so far is
that some other calcium-binding protein modulates the channel. Recently, three new calcium-binding proteins have been
discovered in the rod outer segment: recoverin, GCAP and p24.
All of these are "calmodulin-like," in the sense that they are
soluble, possess multiple E-F hands, and have molecular
weights of 20-24 kD. It does not seem that recoverin binds to
the channel but it will be interesting to see if GCAP or p24 can.
The idea that there may be a second calcium-sensitive modulator of the channel has also been suggested by Gordon and
Zimmerman (1994), who found evidence for an endogenous
modulator of the cGMP-gated channel which was lost at low
calcium.
ACKNOWLEDGMENT
Our work is supported by the Medical Research Council and the Human
Frontiers in Science Program.
Unique lipids and unique properties of
retinal proteins
Kamon Sanada and Yoshitaka Fukada
Department of Pure and Applied Sciences, College of Arts and Sciences,
University of Tokyo, Komaba 3-8-1, Meguro-hu, Tokyo 153, Japan
Abstract: Amino-terminal heteroacylation has been identified in retinal
proteins including recoverin and a subunit of G-protein, transducin.
The tissue-specific modification seems to mediate not only a proteinmembrane interaction but also a specific protein-protein interaction.
The mechanism generating the heterogeneity and its physiological role
are still unclear, but an interesting idea for the latter postulates a
fine regulation of the signal transduction pathway by distinct N-acyl
groups.
[HURLEY] AS reviewed in HURLEY'S target article, an interesting
aspect of recoverin has been heightened by the finding of its
unique N-terminal modification by myristate or its related fatty
acids. Protein N-myristoylation has been identified in a variety
of proteins and the modification is required for membrane
targeting of the proteins or protein-protein interactions (reviewed in Gordon et al. 1991).
In 1992, two research groups found that retinal G-protein
transducin a-subunit (Tot; Kokame et al. 1992; Neubert et al.
1992) and recoverin (Dizhoor et al. 1992) were modified by
one of four types of fatty acyl groups, 14:0, 14:1 (5-cis), 14:2
(5-cis, 8-cis) and 12:0 (Fig. 1). After that, similar modification has
been identified in other retinal proteins such as guanylyl
cyclase-activating protein (GCAP; Palczewski et al. 1994) and
a catalytic subunit (C-subunit) of cAMP-dependent protein
kinase (Johnson et al. 1994). It is interesting to note that only
myristate was detected in the C-subunits purified from bovine
heart and brain. A growing body of evidence suggests that the
heteroacylation is not a protein-specific but a tissue-specific
phenomenon.
A simple explanation for the tissue-specific heterogeneity is
that the mammalian photoreceptor may have a unique composition of each acyl-CoA pool available to the myristoyl-CoA:
protein N-myristoyltransferase (NMT), an enzyme probably
Commentary /Controversies in Neuroscience III
responsible for this modification. Then the relative amounts of
the four acyl groups linked to the retinal proteins are expected to
be similar. As shown in Figure 1, however, this is not the case. In
contrast to the relative abundance of N-myristoylated forms of
recoverin (43%) and the C-subunit (57%), only small percentages (5—7%) of Tot and CCAP are N-myristoylated. The significant difference of the relative amounts of four isoforms among
proteins (Fig. 1) raises a question about the mechanism yielding
the heterogeneity. Mammalian photoreceptors might have isozymes of protein-specific or N-terminal sequence-specific NMT
with different accessibility to each acyl-CoA. Alternatively, the
selectivity of a common NMT for acyl-CoAs and proteins might
be regulated by unknown factor(s) in photoreceptor cells. Characterization of photoreceptor NMT is required for elucidation of
the mechanism.
Although the vertebrate retina is unusual tissue enriched
with unsaturated fatty acids, C14:2(5-cw, 8-cis) has not been
found in the fatty acid composition analysis. The uncommon
fatty acid, C14:2, seems to be formed specifically for the acylation of retinal proteins. Enrichment of C14:2 in retinal proteins
(Fig. 1) raises the more important question of whether the
heteroacylation has a specific physiological function. As described by Hurley, the N-terminal fatty acid of recoverin serves
as a Ca 2+ -dependent membrane anchor, but this does not fully
account for the significance of the heteroacylation. It seems
reasonable to speculate that the acyl groups play another role in
protein function. Although no direct evidence has been presented for the functional difference caused by distinct iV-acyl
groups, a hint was given in the case of Tot by Kokame et al.
(1992). They synthesized two kinds of acylated peptide (C14:0and C12:0-peptide) corresponding to the N-terminal part of Ta
and observed that the C14:0-peptide was a more potent compet-
itor for the Ta-TfJ-y interaction than the C12:0 peptide. Extend
ing this, they synthesized two additional peptides, C14:l- and
C14:2-peptide and found the following order of magnitude for
the inhibitory effect; C14:0 > C14:l > C14:2 = CI2:0(Shono et
al., in preparation). These observations suggest that the N-acyl
group provides a specific protein-protein interaction and that
the affinity between Ta and T|}"y is altered by the heterogeneous
acylation. A weaker ot-(i"y interaction, a unique property of
retinal G-protein, due to a small percentage (5%) of N-myristoylated Ta could lead to rapid association-dissociation cycles of
transducin subunits and contribute to the rapid and highly
amplified photon-signal transduction in photoreceptor cells.
HURLEY'S observation of the complex formation between recoverin and rhodopsin kinase has opened the way to explore a
possible fine regulation of rhodopsin phosphorylation by the
heteroacylation of recoverin. In fact, the magnitude of the
inhibition of rhodopsin phosphorylation is different among recoverin isoforms with distinct fatty acyl groups (Sanada et al.
1995).
Physiological role of the heterogeneous acylation of retinal
proteins discussed here is likely to contribute to the unique
properties of high speed, high gain, and low noise signal transduction in photoreceptor cells.
ACKNOWLEDGMENTS
The work is supported in part by Grants-in-Aid from the Japanese
Ministry of Education, Science and Culture, and by grants from Toray
Science Foundation, SUNBOR, and Mochida Memorial Foundation.
K. Sanada is supported by fellowships of the Japan Society for the
Promotion of Science for Young Scientists.
a
N-terminal Structures
Ta
be
Recoverin
c
GCAP
C-subunit
43.0
C14:0
H
Gly-
28.1
C14:1 (5-c/s)
GlyC14:2 (5-c/s, 8-cis) H
O
C12:0
28.9
H
Figure 1 (Sanada & Fukada). Relative quantities of N-fatty acyl groups linked to bovine retinal proteins
"Data from Kokame et al. (1992)
'•Data from Sanada et al. (1995)
"Data from Sanada et al. (1994)
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
487
Commentary/Controversies in Neuroscience III
Na-Ca + K exchanger and Ca 2 +
homeostasis in retinal rod outer segments:
Inactivation of the Ca 2 + efflux mode and
possible involvement of intracellular Ca 2 +
stores in Ca 2 + homeostasis
Paul P. M. Schnetkamp
Department of Medical Biochemistry, University of Calgary, Calgary,
Alberta, T2N 4N1, Canada. schnetka@asb.acs.ucalgary.ca
Abstract: Inactivation of the Ca 2 + extrusion mode of the retinal rod NaCa + K exchanger is suggested to be the mechanism that prevents
lowering of cytosolic free Ca 2 + to < 1 nM when rod cells are saturated for
a prolonged time under bright light conditions. Under these conditions,
Ca 2 + fluxes across disk membranes can contribute significantly to Ca 2+
homeostasis in rods.
[BOWNDS & ARSHAVSKY] The contribution by BOWNDS & AR-
SHAVSKY summarizes the biochemical and physiological evidence that calcium acts as a messenger for light adaptation in the
outer segments of retinal rods (ROS). As this contribution does
not touch much upon Ca 2 + homeostasis in ROS, I would like to
review some of our recent findings on this subject. Dynamic
Ca 2 + fluxes occur across the plasma membrane (ROS).
In darkness, a large sustained Ca 2 + influx via the cGMP-gated
channels is balanced by Ca 2 + efflux via Na-Ca + K exchange,
while no evidence has been reported to involve a plasma membrane Ca2+-ATPase. The ROS Na-Ca exchanger differs from
the ones in other tissues: it couples Ca 2 + extrusion to both inward
Na + and outward K + gradients and operates with a maximal
capacity to change total intracellular Ca 2 + in bovine ROS by > 1
mM/s (for recent reviews on Na-Ca + K exchange, see Lagnado
& McNaughton 1990; Schnetkamp 1989). The high-capacity
Ca 2 + efflux mode was typically observed in ROS containing
unphysiologically high Ca 2 + loads. Ca 2 + influx and Ca 2 + efflux
mediated by the Na-Ca + K exchanger could cause large (|JUM)
and rapid (seconds) changes in cytosolic free Ca 2 + when operating at only 1% of its maximal capacity (Schnetkamp et al. 1991).
The cytosolic Ca 2 + buffer capacity was found to be 200 p,M total
Ca 2+ /jiM free Ca 2 + (Schnetkamp & Szerencsei 1993). NaCa(+K) exchangers were generally believed to operate in a continuous and reversible fashion with little regulation of the flux
rate. In view of this and in view of the exchanger's high capacity
to regulate cytosolic free Ca 2 + , intracellular Ca 2 + sequestration and release has received little attention and is thought to
be of little importance for regulation of cytosolic Ca 2 + in ROS.
ROS may have a 4Na:(lCa + IK) exchanger (as opposed to
3Na:lCa exchange in other tissues) as it is unlikely to reverse
under any physiological condition (e.g., depolarized rod cell
with elevated cytosolic Na + ) and carry Ca 2 + into the cell.
However, during bright daylight, rod cells are saturated and
Ca 2 + influx is suspended for hours, during which normal cytosolic Na + is expected to be restored and the 4Na-lCa + IK
exchanger is expected to lower cytosolic Ca 2 + to 0.1-0.2 nM.
The exchanger is kinetically quite capable of handling low free
Ca 2 + concentrations as judged from Ca 2 + influx data (via the
reverse mode of the exchanger): Ca 2 + influx could raise intracellular Ca 2 + to 1 u,M at an external free Ca 2+ concentration of
only 20 nM, when Na + -loaded and Ca 2+ -depleted ROS were
incubated in a KC1 medium with 7.5 mM NaCl (to mimic
intracellular milieu) (Fig. 7 in Schnetkamp et al. 1991). Thus,
the problem is: how does the exchanger avoid lowering cytosolic
Ca 2 + to undesirable low values?
Na-Ca exchangers have an intracellular regulatory site for
Ca 2 + (e.g., the cardiac Na-Ca exchanger, Matsuokaetal. 1993):
Ca 2 + influx via reverse Na-Ca exchange requires intracellular
Ca 2 + . However, a similar regulation by intracellular Ca 2+ was
not observed in ROS (Schnetkamp 1989). A different type of
regulation was suggested by the observation that the Ca 2 + efflux
mode of the Na-Ca + K exchanger failed to lower cytosolic free
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Ca 2 + below 50-100 nM when bovine ROS were exposed to
external Na + in the presence of EDTA. Instead, addition of Na +
caused a rapid efflux phase that was halted rather abruptly at
relatively high cytosolic free Ca 2 + levels. Inactivation of the
Ca 2 + efflux mode of Na-Ca + K exchange after a brief period of
high-velocity activity was shown to be the underlying molecular
mechanism (Schnetkamp & Szerencsei 1993). Cytosolic free
Ca 2 + levels of 50-100 nM could be maintained for prolonged
periods of time (1 hr) when the efflux mode of the Na-Ca + K
exchanger was reduced to < 0.03% of its maximal capacity.
Under these conditions, Ca 2 + sequestration and release from
ROS disks could contribute significantly to Ca 2 + homeostasis in
ROS. By combining total Ca 2 + flux measurements (with 45Ca)
and free Ca 2 + measurements (with fluo-3) we found that a
significant portion of Ca 2 + that enters ROS became sequestered
within disks. Ca 2 + fluxes across ROS disk membranes were
about 100-fold slower when compared with Ca 2 + fluxes mediated by the high velocity mode of Na-Ca + K exchange.
Nevertheless, Ca 2 + fluxes across disk membranes became significant when the exchanger was inactivated and contributed
equally to regulation of cytosolic free Ca 2 + .
To summarize, inactivation of the Ca 2 + efflux mode of the NaCa + K exchanger is a useful attribute to prevent lowering of
cytosolic free Ca 2 + to undesirably low values of < 1 nM when
Ca 2 + influx via the cGMP-gated channels is interrupted for a
prolonged period of time in bright illumination. The significance and molecular mechanism of Ca 2 + fluxes across disk
membranes remains to be explored fully, but observations
suggests that the Ca 2 + filling state of disks dictates the Ca 2 +
concentration at which the exchanger inactivates (Schnetkamp
& Szerencsei 1993).
Nuclear magnetic resonance studies on the
structure and function of rhodopsin
Steven O. Smith
Department of Molecular Biophysics and Biochemistry, Yale University,
Box 208114, New Haven, CT 06520. smlth@lokl.csb.yale.edu
Abstract: Magic angle spinning (MAS) NMR methods provide a means
of obtaining high resolution structural data on rhodopsin and its photointermediates. Current work has focused on the structure of the retinal
chromophore and its interactions with surrounding protein charges.
The recent development of MAS NMR methods for measuring internuclear distances with a resolution of ~0.2 will complement diffraction
methods for addressing key mechanistic questions.
[HARGRAVE] The major conclusion drawn by HARCKAVE on the
future directions of structure/function studies on rhodopsin is
that diffraction techniques, particularly electron diffraction of
two-dimensional crystals, are the sole methods for obtaining a
high-resolution structure of rhodopsin and its photointermediates. Hargrave rules out high-resolution solution NMR methods
for structural studies because rhodopsin is a high molecular
weight membrane protein. Although solution NMR methods
are in fact poorly suited for membrane proteins, I would like to
suggest that magic angle spinning NMR methods are promising
for addressing some relevant structural questions concerning
the reaction mechanism of rhodopsin. The key structural questions focus on (1) the structure and protonation state of the
retinal chromophore during the rhodopsin photoreaction, (2)
the relative position of Glull3 and the retinal Schiffbase, and (3)
the structure of the retinal binding site. The key mechanistic
questions involve (1) how the protein raises the pKa of the
retinal Schiffbase to effectively shut down protein activity in the
dark, (2) how absorbed light energy is stored in bathorhodopsin
and channeled into the protein, and (3) how structural changes
between the retinal and C l u l l 3 trigger Schiffbase deprotonation and G-protein binding.
Commentary/Controversies in Neuroscience III
Magic angle spinning (MAS) is a solid-state NMR method for
obtaining high-resolution spectra of macromolecules that are
rigid or have slow rotational correction times (Smith & Peersen
1992). MAS NMR has been used increasingly in the past six
years for structural studies on membrane proteins, in particular
on rhodopsin and bacteriorhodopsin (Creuzet et al. 1991; Han &
Smith 1995; Han et al. 1993; Harbison et al. 1984; Lakshmi et al.
1993; Smith et al. 1991; Thompson et al. 1992). The focus in
these studies has been on the structure and protein environment of the retinal chromophore (i.e., the conformation of the
retinal and how the retinal interacts with surrounding protein
charges). Analysis of the NMR chemical shifts has been used to
establish the relative positions of Glull3 and the retinal chromophore (Han et al. 1993). MAS NMR methods have also been
used to determine the configuration and protonation state of the
retinal-lysine C = N bond, and the conformation of the retinal
C 6 —C 7 bond. These measurements directly address how the
protein modulates the visible absorption of the retinal and the
pKa of the Schiff base. In the case of metarhodopsin II, NMR
measurements resolved a controversy as to whether the chromophore was attached as an unprotonated Schiff base or as a
tetrahedral carbinolamine (Smith et al. 1992).
Based on NMR constraints, we have recently been able to
position the retinal chromophore in a structural model of
rhodopsin developed by J. Baldwin (Baldwin 1993; Han & Smith
1995). We are now in a position to address questions involving
the reaction mechanism of the protein by characterizing how the
structure of the retinal and its interactions within the protein
change in each reaction intermediate. As mentioned by HARGRAVE, structural studies on reaction intermediates are possible
using low temperature trapping methods. One advantage of
NMR over absorption or diffraction methods, is that the contributions of different intermediates trapped in a low temperature
experiment can be distinguished on the basis of different NMR
chemical shifts. Using low temperature methods, we have
characterized the structural changes occurring in the formation
of a bathorhodopsin where rapid photoisomerization of the
retinal generates a conformationally distorted ll-trans chromophore (Han & Smith 1995; Smith et al. 1991). Steric interactions
between the retinal and the protein are responsible for most, if
not all, of the 33 kcal/mole of energy stored in this intermediate.
Finally, the future appears bright for MAS NMR and high
resolution distance measurements in membrane proteins. Over
the past five years, a number of methods have been developed
for measuring 13 C.... I3 C and I 3 C ... 1 5 N distances out to ~ 7 A
with a resolution on the order of 0.1 - 0.2 A (Bennett et al. 1992;
Gullion & Schaefer 1989; Raleigh et al. 1988; Tycko & Smith
1993). With the ability to incorporate specific isotopic labels into
selected sites in membrane proteins, there is the prospect that
high resolution distance measurements can be made to address
the local secondary structure and tertiary interactions in both
rhodopsin and its photointermediates. These measurements
will greatly extend in the structural and mechanistic studies
outlined above.
A novel protein family of neuronal
modulators
Ken Takamatsu
Department of Physiology, Toho University School of Medicine, 5-21-16,
Ohmori-nishi, Ohta-ku, Tokyo 143, Japan. physiken@med. toho-u.ac.jp
Abstract: A number of proteins homologous to recoverin have been
identified in the brains of the several vertebrate species. The brainderived members originally contain four EF-haml domains, but NH2terminal domain is aberrant. Many of these proteins inhibited lightinduced rhodopsin phosphorylation at high [Ca2+], suggesting that the
brain-derived members may act as a Ca2+-sensitive modulator of receptor phosphorylation, as recoverin does.
[HURLEY] Recoverin was initially discovered in bovine photoreceptors as an activating factor of guanylyl cyclase (GC) to
mediate the Ca 2+ -dependent stimulation of cGMP resynthesis
during the recovery of the electrical light response. Recent
studies indicate that recoverin does not regulate GC. Instead, it
may regulate the rate of deactivation of cGMP phosphodiesterase (PDE) following photoexcitation. Rhodopsin phosphorylation and in consequence cGMP hydrolysis in bovine rod
outer segments (ROS) are Ca 2+ -dependent in the presence of
ATP. The level of rhodopsin phosphorylation decreases and the
lifetime of active PDE increases when free [Ca 2+ ] is raised from
1 nM to about 1 |J.M; in both cases the half maximal effect was
observed at 140-170 nM of free [Ca 2 + ]. The Ca 2 + effects
observed are mediated by recoverin which inhibits rhodopsin
kinase at a high [Ca 2 + ]. Although the protein structure of this
protein family was first reported in visinin (Yamagata et al. 1990)
and the actual role of recoverin is that of S-modulin, the term of
recoverin seems to be suitable for this protein family.
During a search for endogenous protein kinase C (PKC) inhibitors, the protein 21-kDa CaBP has been isolated from bovine
brain and characterized as a member of the calmodulin superfamily (Walsh et al. 1984). 21-kDa CaBP was reported to have
isoforms of 21 kDa and 23 kDa depending on the degree of glycosylation. Since 21-kDa CaBP did not inhibit PKC activity, no
further analysis has been done. After the discovery of recoverin, a
number of proteins homologous to recoverin were identified in
the brain of the several vertebrate species (Hidaka & Okazaki
1993; Kajimoto et al. 1993; Kobayashi et al. 1992; Nef et al., in
press; Takamatsu & Uyemura 1992), indicating that multiple
isoproteins exist in the brain and form the brain-derived recoverin family. These proteins have been given several different
names, such as neurocalcin a.P.'Y.S, hippocalcin and hippocalcinIike protein 1, visinin-like protein (VILIP) 1,2,3, and neural
calcium sensor (NCS) 1. These, including recoverin and Smodulin, are classified into three subfamilies according to their
sequence homology (Nef et al., in press). All of these proteins
originally contained four EF-hand domains. One of the four EFhand domains does not seem to satisfy the structural criteria of a
high affinity calcium-binding site; therefore, these proteins may
bind less than three moles of Ca 2 + per mole of protein. The NH 2 terminal sequence of these proteins meet the criteria for NH 2 terminal myristoylation: a glycine residue at the NH 2 -terminus,
and a serine or threonine residue at position 5. Many of these
proteins are known to have a blocked NH 2 -terminus, indicating
that these proteins might be NH2-myristoylated. Hippocalcin
was confirmed to incorporate myristic acid into its NH 2 -terminus
(Kobayashi et al. 1993). NH 2 -terminal myristoylation of hippocalcin was essential for its Ca 2+ -dependent membrane-binding
properties as recoverin does. The maximal membrane-binding of
hippocalcin was observed at about 30 n,M [Ca 2 + ], and halfmaximal binding at about 5 JJLM [Ca 2 + ]. Contrarily, NH 2 myristoylation was not essential for the inhibition of rhodopsin
phosphorylation by these proteins (Kawamura 1994). The physiological roles of NH2-myristoylation are yet to be determined.
The physiological function of the brain-derived members of
the recoverin family from all three subfamilies were examined
using ROS membrane preparation; S-modulin and recoverin
from subfamily I, NCS from subfamily II, VILIP and hippocalcin from subfamily III (see Kawamura 1994 for a review). All
the proteins tested in this study inhibited rhodopsin phosphorylation at high (10 |xM) [Ca 2 + ], suggesting that all members of
the recoverin family act in a similar way as does S-modulin in
ROS. At least five G protein-coupled receptor kinases (GRKs)
exist in the brain (Inglese et al. 1993). Phosphorylation of
receptor molecules by GRKs may be involved in agonistdependent desensitization, however, the mechanism behind
the modulation of phosphorylation in the neurons shows diversity and is totally different from the mechanism in the vertebrate
photoreceptors. The effect of the brain-derived members on
receptor phosphorylation by GRKs are yet to be determined.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
489
Commentary/Controversies in Neuroscience III
It has been known that Ca 2 + affected both the rate of rise
(PDE) activity) and the time course of the recovery (PDE deactivation) of a photoresponse in the vertebrate photoreceptors. It
is noteworthy that S-modulin purified using ROS membranes
affected the PDE activity but not the PDE deactivation (Kawamura & Murakami 1991); however, S-modulin purified using
phenylsepharose affected both the PDE activity and deactivation (Kawamura 1993). Recently, Lagnado and Baylor (1994)
observed the Ca 2 + effect on the PDE activity using internally
dialyzed ROS preparation and found that the Ca 2 + effect seems
to be mediated by a soluble Ca 2+ -binding protein. Although the
isoforms of S-modulin have not been characterized, thus two
Ca 2 + effects seem to be mediated by two distinct forms of
S-modulin, one isoform is responsible for the PDE deactivation
and the other isoform for the PDE activity. Two distinct Ca 2 +
effects on PDE activation are observed only in the presence of
both ATP and S-modulin. The mechanism of the Ca 2 + effect on
the PDE activity is not known, but is possibly regulated by
rhodopsin phosphorylation.
Frequenin, a member of the recoverin family identified in
Drosophila, modulates neurotransmitter release at the neuromuscular junction (Pongs et al. 1993) and also modulates
K + -channel in muscle cells (Poulain et al. 1994) in a Ca 2 + dependent manner. The molecular mechanism of the frequenin
actions is not known. The brain-derived members are distributed heterogeneously in the brain. Many of these proteins are
distributed in neurons. In some cases, the same neuron expresses at least two different members of this family (Saitoh et al.
1993; 1994).
It is not clear whether each protein plays a different role or
shares the same role in its host cells, however, the common
action on rhodopsin phosphorylation lead us to postulate that all
the members of this protein family regulate the signal transduction systems by modulating phosphorylation reaction in their
host cells.
Glutamate accumulation in the
photoreceptor-presumed final common
path of photoreceptor cell death
Makoto Tamai
Department of Ophthalmology, Tohoku University School of Medicine, 1-1
Seiryomachi, Aoba-ku Sendai, 980-77 Japan
Abstract: Genetic abnormalities of three factors related to the photoreceptor mechanism have been reported in both animal models and
humans. Apoptotic mechanism has also been suggested as a final
common pathway of photoreceptor cell death. Our findings of increased
level of glutamate in photoreceptor cells in rds mice suggest that amino
acid might mediate between these two pathological mechanisms.
[DAICER ET AL.] An excellent summary of genetic abnormalities
of rhodopsin, peripherin/RDS, and phosphodiesterase b-subunit and phenotypic variations in human is provided by DAICER
ET AL. They also describe almost all possible mechanisms of
photoreceptor cell death caused by these mutations and proteins. I agree with their descriptions about molecular abnormality and their explanation of the variety of phenotypes, but
the abnormality of proteins related to signal transduction does
not seem to be enough to explain the biological mechanisms
underlying retinitis pigmentosa and the heterogeneity of phenotypes of this disease entity. From our observations, other
mechanisms may exist between genetic mutations and trigger of
cell death.
We have reported increased level of glutamate-like immunoreactivity in the interior of segments of photoreceptors at 2, 3,
and 9 weeks postnatally of rds/rds mice comparing to the control
BALB/c mice (ARVO, abstract #501,1994). It is very interesting
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
that the accumulation was observed to start earlier than the
beginning of photoreceptor degeneration. The accumulation of
this amino acid has also been reported in the chick retina by
Ulshafer et al. (1990). They reported that the uptake of glutamate in photoreceptors occurred normally but that the release of
this neurotransmitter did not because glutamate was accumulated and hence the retinal degeneration took place.
At present, we have no data about why glutamate is accumulated in rds/rds photoreceptors. We also cannot explain why the
accumulation of this amino acid was related to the apoptosis of
photoreceptors. (Chang et al. 1993). Kure et al. (1991) have
reported that the chromosomal DNA of cultured neurons was
degraded into nucleosomal-sized DNA fragments by the addition of glutamate prior to the glutamate-induced neuronal
death. Oxidative stress induced by light exposure or other
mechanisms might also result in apoptosis-type cell death by the
depletion of glutathione (Ratan et al. 1994). Ratan et al. have
shown that glutamate could induce glutathione depletion in
immature embryonic cortical neurons. Intracellular mechanisms of accumulation and the mode of action of glutamate in
photoreceptors need to be studied.
In conclusion, as pointed out by DAICER ET AL., retinal
dystrophy has genetic and allelic as well as clinical heterogeneity. Such heterogeneity may be affected by environmental
or systemic conditions, which might affect retinal circulation
and might also be affected by daily lighting conditions or
nutrition via the level of vitamin A or trace elements such as
M g + + or Z n + + (Wu et al. 1993).
The genetic kaleidoscope of vision
Douglas Wahlsten
Department of Psychology, University of Alberta, Edmonton, Alberta,
Canada T6G 2E9. wahtsten@psych.ualberta.ca
Abstract: Site-specific phenotypic effects of the 73 known alleles in the
rhodopsin gene that cause retinal degeneration are difficult to interpret
because most alleles are documented in only one case or one family,
which means variation in effects could actually arise from interactions
with other loci. However, sample sizes necessary to detect epistatic
interaction may place an answer to this question beyond our grasp.
[DAICER ET AL.] The comprehensive review of DAICER ET AL.
documents genetic heterogeneity, allelic heterogeneity, and
clinical heterogeneity in a diverse collection of mutations causing degeneration of the human retina. Listed here are 42
mapped loci, one of which (rhodopsin) has at least 73 alleles
causing a wide array of phenotypes in terms of the quality,
severity, and consistency of effects. While proposing a classification scheme to bring some order to a bewildering complexity in
a single gene, the authors point out that many vital aspects of the
gene's role in development remain unexplored. A great deal has
been accomplished in the biochemical genetics of retinal degeneration, but continuing study suggests a limitless diversity and
provokes questions about the goals of this research and the
likelihood of achieving them.
1. The chemically complete retina. Understanding a system of
42 genes is challenging enough, yet there must be thousands of
genes expressed in the retina, many of which are fixed in the
population and do not produce disease. It makes good sense to
concentrate on genes associated with disorders of vision if one
hopes to someday prevent blindness. At the same time, the
connectedness of the metabolic system in the retina dictates that
the effects of a mutation at one locus will not be well understood
until its associated protein can be placed in a biochemical
context that includes the protein products of fixed loci. Thus, it
would be helpful to know how many genes are expressed in the
retina and how many are unique to the retina. Is it important for
science to identify and characterize all of these, and will this
Commentary /Controversies in Neuroscience III
knowledge benefit those who suffer from retinal dystrophy? The
target article has set forth much that is already known but has
given little more than a hint at the goals of this enterprise. In
particular, will the knowledge of the DNA sequence of various
alleles causing some form of retinal degeneration be used
primarily to screen for genetic defects and possibly abort embryos destined to suffer reduced vision in later life, or is there
hope for ameliorating these hereditary conditions when the
specific allele is known? A simple catalog of alleles and phenotypes is sufficient for eugenic purposes, whereas practical
intervention to prevent retinal degeneration will necessitate a
much more comprehensive understanding of the relations between rhodopsin and its environment.
2. Site-specific phenotyplc effects? The answers to these
questions will depend critically on the general properties of
biochemical gene action. In particular, should we expect that
changing a specific amino acid in a protein should result in a
specific array of phenotypic effects at the level of retinal function
in anyone possessing that allele? If yes, then there is no need to
understand much about associated proteins derived from fixed
loci or polymorphic loci not strongly tied to disease. The familiar
catalog of loci related to specific disease syndromes can be
expanded into a catalog of alleles or groupings of alleles, each
with a characteristic syndrome. This seems to be the answer
provided by the authors when they state "the clinical phenotype
is a consequence of where and when the mutation affects the
function of rhodopsin" (sect. 4.2, para. 1).
There is no doubt that an amino acid substitution in a critical
protein can alter neural function. Rigorously controlled studies
of oisogenic mice differing at a single amino acid in only one
protein find that the mutants deviate phenotypically from their
normal siblings, at least when group averages are compared.
However, it does not follow that the average phenotypic difference is attributable solely to the amino acid difference.
Coisogenic littermates share thousands of other genes and a
multifaceted laboratory environment in common, and the genetic difference occurs in a context that may be critically
important for the phenotypic outcome. For example, epistatic
interaction with the genetic background can markedly alter the
consequences of mutations such as diabetes and reeler in mice
(Caviness et al. 1972; Coleman 1981) and interaction with the
rearing environment can sometimes be quite dramatic (e.g.,
Lee & Bressler 1981).
The appropriate research designs for assessing epistasis and
gene-environment interaction always require individuals with a
specific genotype at a designated locus to be combined with
different genetic backgrounds or reared in different circumstances. To achieve this, the investigator must have access to a
fairly large number of individuals carrying the identical mutation. In this regard, the yellow flag for caution begins to wave
when the authors inform us in section 4.1.1 that "The majority of
mutations in Table 2 are unique, occurring in one patient or one
family only." Is it not possible or even likely that certain
mutations with apparently variable effects are working in concert with other retinal genes that, while not causing disease on
their own account, are interactants that strongly influence the
consequences of a rare rhodopsin allele? And might not some of
the apparently consistent effects of other mutations simply
reflect the lack of genetic variation in interacting loci within
certain families? The great phenotypic diversity attributed in
this article to allelic heterogeneity may perhaps result from
epistatic interaction or even gene-environment interaction, as
DAICEK ET AL. acknowledge briefly in section 3.2.2.
Of course, the argument can work both ways. When a mutation in a gene results in substantially different disease in different people, epistasis might be invoked to explain this, but the
effect could just as well arise from allelic diversity. In this
respect, reliable knowledge about the DNA sequence in the
rhodopsin gene should help to discriminate between these two
kinds of causes. We begin with the observations that there are in
fact many alleles of the rhodopsin gene and there are also many
phenotypes. But are the two variations closely associated and, if
so, why?
3. Interaction, the night blindness of statistical analysis. It is
instructive to explore the kinds of data that will be needed to
address two important questions about allelic heterogeneity.
Both of these involve the question of sample size required to
render a statistical procedure sufficiently sensitive to more
subtle genetic effects.
One question is simply whether two alleles do indeed differ
significantly in the frequency of associated phenotypes. Suppose most cases can be dichotomized into early and later onset of
retinal degeneration, and let the probability of the early form be
p, and p 2 f° r two alleles. The null hypothesis is that p, = p 2 = p,
whereas the alternative hypothesis is that p , = p + c and p2 = P
— c, where c is the deviation from an average of the two alleles. If
one plans to test the significance of a difference; in sample
proportions of the early onset form using a Z test with a Type I
error probability of a, two-tailed, and wants the test to have
Type II error probability P, equivalent to statistical power of 1 —
P, then the appropriate sample size is given by a formula based
on a normal approximation to the binomial distribution.
N
= •
Suppose p = .5. To achieve power of 90% when a = .05, the
necessary sample sizes per group are indicated in Table 1. It
could be quite difficult to find enough humans with each of the
rare alleles. In this regard, it appears the authors are on the right
track by seeking commonalities in terms of domain of action of
various alleles, where samples could reasonably be pooled
across similar alleles.
In order to test the classification scheme more rigorously, it
would help not only to work with adequate samples but also to
obtain continuous measures of the phenotypes associated with
retinal degeneration, such as age at onset of symptoms, rate of
degeneration, and percent of the retina impaired. These dependent variables could then be assessed with multivariate statistics
to determine the significance and strength of relationships with
region occupied by a mutation (as in sects. 4.2.2 to 4.2.5). This
would allow for exceptions, such as the 1289dell7 and 1312del24
mutations, to weaken an interesting relationship without negating it altogether.
The other important question about phenotypic consequences of allelic heterogeneity concerns the possible role of
epistasis. Two alleles might have quite different effects on one
genetic background but appear to differ less radically on another
background. Consider the example in Table 2 where hypothetical group means are given in arbitrary units. The allelic difference is twice as large on background B compared to A. A simple
formula that provides a normal approximation to the noncentral
t distribution is convenient for estimating the necessary sample
sizes (Wahlsten 1991). Suppose the standard deviation within a
group is o" = 7.5 units and a planned contrast is used to test the
difference between the effects of the two alleles without regard
to genetic background. To achieve power of 80% when a two-
Table 1 (Wahlsten). Sample size to yield power of 90%
c
Pi
P2
N
.05
.10
.15
.2
.55
.6
.65
.7
.45
.4
.35
.3
128
32
14
8
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
491
Commentary /Controversies in Neuroscience III
Table 2 (Wahlsten). Model of epistatic interaction
Background A
Background B
Allele 1
Allele 2
10
11
15
21
tailed test with a = .05 is used, the investigators must study
about 10 patients in each of the four groups. However, to test the
hypothesis of interaction between allele and genetic background
with the same level of power, the sample size must be 73
patients per group! As a general rule, the necessary sample size
is inversely proportional to the square of the size of the effect.
Thus, the sample sizes appropriate for studying many kinds of
interactions in a serious way are substantially larger than those
that investigators commonly employ when looking at the average effects of an allelic difference [see Wahlsten "Insensitivity of
the Analysis of Variance to Heredity-Environment Interaction"
BBS 13(1) 1990].
The tyranny of numbers appears to render unanswerable the
question of epistatic interaction when there are so few families
with rare alleles. If we cannot address this question in a serious
way, I believe investigators should inform readers that they
cannot argue strongly for the reductionistic thesis that each
allele or molecular domain codes for a specific clinical syndrome.
Given the statistical difficulties of human genetic research,
perhaps it might be helpful to assess allelic interactions in
animal models of retinal degeneration using transgenic methods
to create animals with different rare alleles. Once accomplished,
large numbers of retinal degenerate mice could be produced.
More answers about cGMP-gated channels
pose more questions
Theodore G. Wensel and Joseph K. Angleson
Verna and Mans McLean Department of Biochemistry, Baylor College of
Medicine, Houston, TX 77030. twensel@bcm.tmc.edu
Abstract: Our understanding of the molecular properties and cellular
role of cGMP-gated channels in outer segments of vertebrate photoreceptors has come from over a decade of studies which have continuously altered and refined ideas about these channels. Further examination of this current view may lead to future surprises and further refine
the understanding of cGMP-gated channels.
[MOLDAY & HSU] In the decade or so since the first reports of
cGMP-gated ion flux in rod outer segments (Carretta & Cavaggioni 1983, Fesenkoetal. 1985), the standard dogma concerning
the channels responsible for it has gone through numerous
revisions. A comparison of earlier notions with current views
summarized by MOLDAY & HSU highlights both the frequent
modification of simplified models that have been necessary, and
the clues to new breakthroughs that have been revealed, however dimly at the time, by pesky details that failed to fit neatly
into these models. Close examination of remaining loose ends of
the otherwise tightly wrapped package presented in the target
article may provide hints of future surprises about the structure
and function of cGMP-gated channels.
Examples of the seeds of new discoveries in the imperfect
understanding of old ones abound in this story. For example,
early reports of cGMP-gated .cation channels in ROS membranes (Caretta & Cavaggioni 1983; Cavaggioni & Sorbi 1981)
met with some skepticism, because several features were hard
to fit into ideas then-extant of how visual transduction should
work. Channels located in disk membranes (Caretta et al. 1979;
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Koch & Kaupp 1985) and releasing Ca 2 + from intradiskal pools
in response to increased cGMP were hard to reconcile with
light-induced hydrolysis of cGMP (Bitensky et al. 1981) accompanied by Ca 2 + release from outer segments (Gold & Korenbrot
1980; Yoshikami et al. 1980) generally assumed to be due to
increased cytoplasmic [Ca 2 + ]. These puzzling features actually
reflected important information about the channels and about
the organization of photoreceptor membranes. They arose from
the facts (appreciated only later) that the plasma membrane
channel is highly permeable to Ca 2 + , a property that figures
prominently in shaping the light response, and that disk membranes are so strongly attached to plasma membranes that
standard preparations of "disks" contained many plasmamembrane-derived vesicles (Molday & Molday 1987).
Following the identification of a 63 kDa protein with the
channel by protein purification (Cook et al. 1987) and functional
expression of cDNA (Kaupp et al. 1989), and the ruling out of
rhodopsin or proteins of similar size as likely candidates (Cook et
al. 1989; Molday et al. 1990), a simple picture emerged. The
simplest model was that the 63 kDa channel formed a multimeric channel with a central pore, similar to the heteropentameric complex formed by other ligand-gated channels, with
cGMP-binding sites on each subunit. The channel was exclusively (or nearly so) located in plasma membranes (Cook et al.
1989). Nonetheless, nagging questions remained: Why was the
electrophoretic mobility of the purified channel protein so
different from that of the heterologously expressed protein?
Why was the purified channel almost always accompanied by a
240 kDa spectrin-like protein? Why did the detailed properties
(such as sensitivity to 1-cw-diltiazem) of the purified or expressed
channel differ from those of the channel in photoreceptor
membranes? As detailed in the target article, answers (or at least
partial answers) to these questions not only cleared up some
discrepancies, but provided important new information about
the channels; for example, photoreceptor-specific proteolytic
processing and the existence of an additional channel subunit.
Additional discrepancies have persisted, and the unfolding of
new explanations for them has continued to deepen our understanding of how these channels work.
Here then, are some remaining questions whose answers may
provide new insight into channel structure and function; the
reader may well be able to think of many more:
What is the native channel subunit stoichiometry and how it is
governed? Is there only one such stoichiometry, or might there
be functional channels with different ratios of a to B subunits?
What is responsible for the apparent heterogeneity of cGMP
binding sites revealed in excised patches from outer segments
(Brown & Karpen 1994)? Is there a function for alternative
splicing of the B subunit? Might there be additional regulatory
subunits (e.g., Ca 2+ -binding proteins) lost in standard purification schemes?
Why are some preparations of the channel apparently devoid
of the 240 kDa band corresponding to the B subunit, whereas
other preparations following the same procedure yield apparently different ratios of a subunit to the 240 kDa band (Cook et
al. 1987)? What is the function of the peptide in the 240 kDa
band that is not the B subunit?
What is the functional role of proteolytic cleavage of the a
subunit amino-terminal peptide? Where and when does this
processing occur? Why is the purified channel sensitive to 1-ctsdiltiazem when purified on a cGMP affinity column (Hurwitz &
Holcombe 1991) but not when ion exchange and/or dye affinity
chromatography is used, even though all these preparations
presumably include the B subunit? MOLDAY & HSU cite evidence
indicating that proteolytic processing is not the deciding factor,
but could it be that cGMP binding enhances subunit-subunit
interactions or stabilizes a diltiazem-sensitive domain?
How is the channel localized so precisely? Does subunit
composition or processing affect the targeting of channels to
outer versus inner segments? How do subunit compositions of
Commentary /Controversies in Neuroscience III
rod and cone channels compare, and what is the molecular
nature of cCMP-gated channels recently reported in cone inner
segments and synaptic processes (Rieke & Schwartz 1994).?
Is there an in vivo role for modulation by calcium binding
proteins or phosphorylation? Is the rather small effect of calmodulin on cGMP sensitivity the predominant effect, and if so,
why do calmodulin inhibitors appear to have little effect on rod
responses (Cray-Keller & Detwiler 1994)? Are there additional
calcium binding proteins that can modulate channel function, as
has been suggested (Gordon & Zimmerman 1994). What, if any,
role does phosphorylation play? Why do phosphatase inhibitors
show significant effects in excised patch experiments (Gordon et
al. 1992), but not in dialyzed rods (Gray-Keller & Detwiler
1994)?
Answers to these questions may well result in more surprises
which may lead to yet more questions about cGMP-gated
channels.
Cyclic nucleotides as regulators of lightadaptation in photoreceptors
Barry M. Willardson, Tatsuro Yoshida, and
Mark W. Bitensky
Biophysics Group, Physics Division, Los Alamos National Laboratory,
University of California, Los Alamos, NM 87545. tatsuro@beta.lanl.gov
Abstract: Cyclic nucleotides can regulate the sensitivity of retinal rods
to light through phosducin. The phosphorylation state of phosducin
determines the amount of G, available for activation by Rho*. Phosducin
phosphorylation is regulated by cyclic nucleotides through their activation of cAMP-dependent protein kinase. The regulation of phosphodiusteru.se activity by the noncatalytic cGMP binding sites as well as
Ca 2+ /calmodulin dependent regulation of cGMP binding to the cation
channel are also discussed.
[BOWNDS & ARSHAVSKY; HURLEY; MOLDAY & HSu] T h e target
articles by HURLEY, BOWNDS & ARSHAVSKY, and MOLDAY & HSU
address various aspects of light adaptation in photoreceptors.
They reflect a time-honored tradition in vision research to
understand the electrophysiological responses that underlie rod
signal transduction in terms of the interactions of their highly
specialized gene products. The research summarized in these
articles continues to elucidate a light adaptation scheme that is
orchestrated by the second messengers, cGMP and Ca 2 + , and
appears to be regulated at practically every locus of the light
activation cascade.
f. Phosducin. A fundamental mechanism of signal down regulation mentioned only in passing by BOWNDS & ARSHAVSKY is
the regulation of transducin activation by phosducin. Phosducin
can compete with G,a for binding to C,P"y (Lee et al. 1992;
Yoshida et al 1994). The ability of phosducin to compete with
C,a for binding sites on Gfiy depends directly on the phosphorylation state of phosducin (Yoshida et al. 1994). When phosducin was phosphorylated with PKA, its ability to compete with
G,a for a binding site on G,P"y was dramatically reduced. This is
an important point because, in rods, phosducin is found in its
phosphorylated state in the dark and becomes dephosphorylated upon illumination (Lee etal. 1984). Moreover, the endogenous kinase appears to be PKA (Lee et al. 1990), and rod PKA
may be activated by p-M concentrations of cGMP. From these
data, a mechanism can be postulated in which phosducin regulates the concentration of G,a(3-y in a light dependent manner: in
unilluminatcd rods, cyclic nucleotide levels are elevated, PKA
is active, and phosducin remains phosphorylated. Under these
conditions, phospho-phosducin would not compete with G,a for
G,p7 binding, allowing G t a and Gfiy to be maximally associated in their activatable heterotrimeric state. After illumination,
cyclic nucleotide levels fall, PKA is inactivated, and phosducin
becomes dephosphorylated by unopposed rod phosphatases.
Dephospho-phosducin avidly binds to G,P"y and prevents G t a
from associating with G,P7. The rate of activation of G,a by
Rho* is linearly dependent upon free [C,p-y]. Thus when
dephospho-phosducin sequesters Gfiy, only a fraction of the
G t a normally activated during the lifetime of a Rho* will be
activated in the light-adapted, phosducin-activated state. In this
manner, phosducin sequestration of Gfiy could contribute to
the smaller response amplitude and shorter response times that
are widely observed in light-adapted photoreceptors. Following
a light response, cyclic nucleotides eventually return to dark
levels and PKA reactivates. Phosducin is subsequently phosphorylated even while complexed to Gfiy (Willardson et al.,
unpublished observations). Under these conditions, G t a can
displace phospho-phosducin from GtP"y, restoring the rod's full
compliment of G t aP7, and the system is returned to its high
sensitivity state.
A similar mechanism of phosducin regulation is also observed
in other signaling pathways. Bauer and others (Bauer etal. 1992)
have shown that phosducin isolated from bovine brain also
inhibits p-adrenergic receptor activation of adenylyl cyclase in
A431 cells, and phosphorylation of phosducin with PKA reverses this inhibition. Moreover, they demonstrated that phosducin inhibits the GTPase activity of G s , G,, and Go in a
phosphorylation dependent manner.
Phosducin may also regulate direct activation of effector
enzymes by GP"y subunits. Stimulation of P-adrenergic receptor
kinase activity by Gp-y is inhibited by phosducin in a phosphorylation dependent manner (Hekman et al. 1994). All of the
findings mentioned above suggest that phosducin plays an
important role in downregulating signaling in retinal rods as well
as in other G-protein mediated signaling pathways.
2. Noncatalytic cGMP binding sites on PDE. Since their discovery over a decade ago, the noncatalytic binding sites on PDE
have been an enigma, BOWNDS & ARSHAVSKY have now de-
scribed an attractive mechanism for limiting phosphodiesterase
(PDE) activity in amphibian rods by an actual sensing of [cGMP]
by the noncatalytic cGMP binding sites on PDE. In assessing
the function of the noncatalytic cGMP binding sites on PDE
regulation, the rate of release of cGMP from the noncatalytic
sites is of critical importance. Depending on this rate, the
noncatalytic sites may participate in light adaptation, as suggested by Bownds & Arshavsky, and/or in turning off PDE
activity within a single light activation cycle. Recently, Cote and
others (Cote et al. 1994) demonstrated that the activation of
PDE by G,orGTP"yS accelerates the dissociation of cGMP from
the non-catalytic sites. One component of the dissociation
process was rapid, with a kll2 of 25 sec, whereas the other
component was as slow as that from inactivated PDE with a kl/2
of 4.4 min. The faster rate is comparable with light-adaptation of
rods under background illumination, as suggested by Bownds &
Arshavsky. Although this rate is too slow for the noncatalytic
sites to play an active role in PDE turn-off within a single light
response, there are two reasons to believe that the dissociation
rate of cGMP from the noncatalytic sites during activation would
be more rapid in vivo. First, in preparing disrupted rod outer
segments, a significant portion of G, is lost. Moreover, when G,ct
is activated by light and GTP-yS, much of the G,ct GTP-yS is
released to the supernatant. As a result, only a subset of PDE
may be activated. In fact, the fraction of PDE with the slower
k1/2 may simply be PDE that was not activated by G^GTP'yS.
Second, the method of exchanging prebound [ 3 H]cGMP with
lmM cold cGMP to obtain the dissociation rate assures that both
noncatalytic cGMP binding sits remain occupied; that is, when
[3H]cGMP dissociates from one noncatalytic site, the site is
immediately rebound by a cold cGMP. Thus, the two kU2 values
are valid only if the two sites are independent. However, if they
have cooperative interactions, the ko!r for the second site could
be masked by this method. In the physiological situation, both
noncatalytic sites can release cGMP as its concentration drops
during PDE activation. Once the first site is emptied, release of
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
493
Commentary /Controversies in Neuroscience III
cGMP from the second site could be accelerated. Thus, the data
of Bownds & Arshavsky do not yet rule out the possibility that
the in vivo release of cGMP from the noncatalytic sites on PDE
is sufficiently rapid to participate in the turn-off of PDE within a
single light response, in the ~ 5 second time scale.
BOWNDS & ARSHAVSKY point out that bovine PDE binds
cGMP much more tightly than does amphibian PDE, suggesting that the same mechanism of cGMP release from noncatalytic
sites may not regulate the Gtor GTPase activity and the lifetime
of active PDE in mammals. Indeed, the mechanism of acceleration of G,a GTPase by PDE in bovine rods appears to be very
different. The minimal subunit ensemble required for G t a
GTPase acceleration is PDEaP, and no G,a-GTPase acceleration was observed in the presence of PDE-y (Antonny et al. 1993;
Pages et al. 1993). Moreover, GTPase acceleration was not
effected by adding cGMP, presumably because cGMP remains
tightly bound to bovine PDE. It appears that the cGMP regulation of light adaptation in bovine rods may not be mediated by
the PDE noncatalytic sites but is mediated through cyclic
nucleotide effects on phosducin phosphorylation and perhaps
other yet to be discovered mechanisms.
3. Ca2 * /calmodulin regulation of the cGMP-gated channel. In
the section by MOLDAY & HSU, the most compelling issue (aside
from the ultimate definition of subunit composition and topology) is the significance of calcium regulation. At first blush, the
significance might appear questionable since the divergence of
the plus and minus calcium/calmodulin curves is most apparent
at concentrations above 10 u.M. Although there is a fragile consensus that the average free concentrations of cGMP may never
exceed 5 u-M; in fact, the actual concentrations of cGMP in the
vicinity of the channels are not yet precisely known. It is
noteworthy that the effects of calcium and calmodulin on the
affinity of cGMP in rod cation channels has persisted in a highly
conserved manner over eons of evolutionary time. In view of the
ruthlessness of mutational drift in protein domains without
function, it appears most prudent to assume that the calcium/calmodulin regulation of the cGMP-gated rod channel has
physiological meaning until proven otherwise.
Is the lifetime of light-stimulated cGMP
phosphodiesterase regulated by recoverin
through its regulation of rhodopsin
phosphorylation?
Akio Yamazaki
Kresge Eye Institute, Department of Ophthalmology and Pharmacology,
Wayne State University School of Medicine, Detroit, Ml 48201
Abstract: In the current model of visual transduction, the lifetime of
active cGMP phosphodiesterase depends upon the period of its interaction with GTP-bound transducin. If recoverin regulates the lifetime of
light-activated cGMP phosphodiesterase through inhibition of rhodopsin phosphorylation, rhodopsin should directly interact with cGMP
phosphodiesterase and/or GTP-bound transducin complexed with
cGMP phosphodiesterase. Is this true?
[HURLEY] In vertebrate rod photoreceptors, illuminated
rhodopsin stimulates GTP/GDP exchange on Ta (a subunit of
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
transducin) followed by dissociation of G T P T a from T(J-y (p-y
subunit of transducin). GTP T a activates PDE (cGMP phosphodiesterase) through release or displacement of Fy (inhibitory
subunit of PDE) from P a P (catalytic subunit of PDE), resulting
in a fall in cytoplasmic cGMP concentration, closure of cGMPregulated channels, and hyperpolarization of photoreceptor
plasma membranes. Therefore, illumination of rhodopsin is a
trigger for the PDE activation through stimulation of GTP/GDP
exchange on T a ; however, illuminated rhodopsin is not directly
involved for the PDE activation (Fung et al. 1981).
As reviewed concisely in HURLEY'S article, Kawamura and
coworkers (Kawamura 1993; Kawamura & Murakami 1991; Kawamura et al. 1993) proposed that recoverin regulates the
lifetime of active PDE through its regulation of rhodopsin
phosphorylation. They claimed that recoverin (S-modulin)
lengthens the lifetime of active PDE at high Ca 2 + concentration
(half-maximum effects at 200-400 nM), and that recoverin (Smodulin) inhibits (partially) rhodopsin phosphorylation (half
maximum effect at 100 nM Ca 2 + ). Their claim appears to be
supported by recent data showing that recoverin forms a complex with rhodopsin kinase (Chen & Hurley 1994; Subbaraya et
al. 1994). However, their proposal comprises several questions:
(1) how the inhibitory effect of recoverin on rhodopsin phosphorylation is connected to the longer lifetime of active PDE, and
(2) how much the partial inhibition of rhodopsin phosphorylation can regulate the PDE activity under low bleaching of
rhodopsin.
In the current model of visual transduction, inhibition of
rhodopsin phosphorylation lengthens the lifetime of active rhodopsin (meta II), resulting in the increase of number of G T P T a .
The increase in the number of G T P T a is related to amplification
of light signal through increase in the total number of active
PDE. In the process of PDE activation, GTP Ta interacts with
P 7 to release the inhibitory effect of P 7 from P a P and the
molecular ratio between G T P T a and Fy in the interaction is 1:1
(Fung & Griswold-Prenner 1989; Yamazaki et al. 1990). The
lifetime of active PDE ( P a 3 or P a P 7 ), therefore, is dependent
upon the period in which G T P T a interacts with P , but not
upon the number of GTP T a if the concentration of GTP T a is
enough for its interaction with PDE subunits. Therefore, without solid data, it is difficult for me to accept that the lifetime of
active rhodopsin is related to the lifetime of active PDE. It
should be emphasized that the lifetime of active rhodopsin is not
related to the lifetime of GTP TQ since GTP T a is believed to be
released from active rhodopsin after GTP/GDP exchange on T a
(Kiihn 1980). Moreover, the regulation of lifetime of GTP T a by
GTP hydrolysis may not be important for the turnoff of active
PDE (Erickson et al. 1991; Tsuboi et al. 1994).
If recoverin is functional for prolongation of the lifetime of
active PDE, recoverin should regulate the interaction between
G T P T a and P r In future, we may find a new regulatory
mechanism in which recoverin regulates the lifetime of active
PDE through its regulation of the interaction between P^ and
G T P T a . In such a case, change in Ca concentration may also be
crucial for the PDE regulation. I strongly feel that the role of
recoverin in visual signal transduction is still unclear.
ACKNOWLEDGMENT
The author is supported by Jules and Doris Stein Professorship from
Research to Prevent Blindness.
Response/Controversies in Neuroscience III
Authors' Responses
Distribution of commentators responded to in Authors' Responses
HARCRAVE:
BOWNDS & ARSHAVSKY:
HURLEY:
Future directions for rhodopsin
structure and function studies
How many light adaptation
mechanisms are there?
Recoverin, a calcium-binding
protein in photoreceptors
Albert & Yeagle
Crouch & Corson
Dratz
Garavito
Smith
Crouch & Corson
Hurwitz, Srivastava & Hurwitz
Koch
Matthews & Fain
McCinnis
Schnetkamp
Willardson, Yoshida & Bitensky
Kawamura
Koch
McGinnis
Folans & Adamus
Sanada & Fukada
Takamatsu
Yamazaki
XIA & STORM:
MOLDAY & HSU:
DAICER, SULLIVAN & RODRIGUEZ:
Evidence that the type I
adenylyl cyclase may be important for neuroplasticity
Further insight into the structural
and regulatory properties of the
cGMP-gated channel
Genetic and functional complexity
of inherited retinal degeneration
Abrams
Heideman
Rasenick
Roberson & Sweatt
Brown & Karpen
Gray-Keller & Detwiler
Haynes
Hurwitz, Srivastava & Hurwitz
Sagoo & Lagnado
Oprian
Wensel & Angelson
Barnstable
Bergen
Kaplan
McGinnis
Tamai
Wahlsten
Authors' Responses
Future directions for rhodopsin structure and
function studies
Paul A. Hargrave
Departments of Ophthalmology, Biochemistry and Molecular Biology,
University of Florida, Gainesville, FL 32610. hargrave@eyel.eye.ufl.edu
Abstract: NMR (nuclear magnetic resonance) may be useful for
determining the structure of retinal and its environment in
rhodopsin, but not for determining the complete protein structure. Aggregation and low yield of fragments of rhodopsin may
make them difficult to study by NMR. A long-term multidisciplinary attack on rhodopsin structure is required.
A variety of structural methods, in combination, is leading to a progressively clearer picture of the threedimensional structure of visual rhodopsin. The most informative to date has been cryoelectron microscopy of
two-dimensional crystals of frog rhodopsin (Schertler &
Hargrave 1995). This has resulted in a projection map of
frog rhodopsin to 6A resolution. More recently, using
tilted images with the same crystal form, a projection map
has been obtained that resolves each of rhodopsin's seven
helices (Unger et al. 1995). This represents the first
physical evidence for the reality of the seven transmembrane helices of rhodopsin that have been reasonably proposed to exist for over a decade.
In the target article I essentially dismissed nuclear
magnetic resonance (NMR) as a currently applicable
approach to providing us with complete structural infor-
mation for rhodopsin. Smith points out, quite correctly,
that solid state magic angle spinning NMR is an important
technique for providing selected structural information
about rhodopsin, such as the structure and protein environment of the retinal chromophore. It is this kind of
information that is most sought in order to understand
functional properties of the visual pigments. NMR
methods may well provide us with high resolution structural information for the retinal environment in a more
timely fashion than will other methods.
Crouch & Corson suggest that multi-dimensional het^
eronuclear NMR may in fact have the potential for investigating the structure of proteins as large as the 40 kDa
rhodopsin. Although the potential may be there, the
reality at present is that only proteins in the 15-25 kDa
range can be examined in solution (Clore & Gronenborn
1994). Although 40 kDa is a foreseeable frontier, only an
exceptional protein may meet the necessary criteria. The
protein would have to be highly soluble and not aggregate
at concentrations of 40 mg/ml, would have to be stable to
temperatures of over 30° for several weeks, must have
little conformational heterogeneity, and must be available
in quantity uniformly labelled with 15N and l3C (Clore &
Gronenborn 1994). Whether all of these criteria will
be met by rhodopsin, and whether advances in NMR
methodologies will allow the structures of detergentsolubilized 40 kDa proteins to be determined, should be
known during the coming decade.
Albert & Yeagle suggest that large proteolytic fragments of rhodopsin that are within the size range of
current NMR capabilities to examine might be suitable
objects of study to learn about the structure of rhodopsin.
This would certainly be the case if the isolated fragments
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
495
Response/Controversies in Neuroscience III
retained the structure that they have in the seven-helix
bundle of rhodopsin, and if they could be prepared in
quantity in concentrated solution in nonaggregated form.
Since the first two-fragment complex of thermolysindigested rhodopsin was prepared and separated on an
analytical scale (Pober & Stryer 1975), attempts have been
made to use similar methods to prepare significant
amounts of nondenatured fragments for spectral analysis.
Similar attempts in our laboratory led to aggregated
fragments in yields of less than 10% (Hargrave & Fong
1977), and we are not aware of other reports in the
literature. However, rhodopsin fragments have been coexpressed in E. coli recently (Ridge et al. 1995). It may
eventually be possible to find conditions in which some
fragments of rhodopsin may be expressed, purified, and
concentrated in "native" monomeric form, suitable for
structural examination by NMR.
Finally, both Dratz and Garavito stress that the difficulty of the task of crystallization of rhodopsin makes it a
commitment that requires long-term support. Additionally, it may require the resources and expertise of
several research groups (i.e., one lab that knows how to
purify and handle the protein, one lab that makes antibodies against it or synthesizes specialized retinal derivatives, and one lab that specializes in X-ray analysis).
Progress will probably be incremental over a period of
years, as it has been for bacteriorhodopsin. The results
will be important not only to those of us who are intrinsically interested in rhodopsin itself, but will be closely
followed by the large community who study G-proteinlinked receptors.
How many light adaptation mechanisms are
there?
M. Deric Bownds and Vadim Y. Arshavsky1
Laboratory of Molecular Biology, University of Wisconsin, Madison Wl
53706. mdbownds@facstaff.wisc.edu and vadlm@macc.wisc.edu.
Abstract: The generally positive response to our target article
indicates that most of the commentators accept our contention
that light adaptation consists of multiple and possibly redundant
mechanisms. The commentaries fall into three general categories. Thefirstdeals with putative mechanisms that we chose not
to emphasize. The second is a more extended discussion of the
role of calcium in adaptation. Finally, additional aspects of
cGMP involvement in adaptation are considered. We discuss
each of these points in turn.
The commentators made a number of interesting points,
and we are gratified that in general the main points in our
target article seem to have passed muster. We are grateful
to Drs. R. Cote, A. Dizhoor, N. Mangini, and D. Pepperberg for discussing with us different points raised by the
commentators. Our Response can be organized under the
headings shown below:
R1. Mechanisms that we did not emphasize. Crouch &
Corson give a more thorough and useful description of
bleaching adaptation than we provided in the target
article. We suggested similarities between bleaching and
background adaptation but did not want to imply that the
issue was resolved. We favor the recent suggestion of
Leibrock et al. (1994) that "a bleach induces two closely
related phenomena in rods: a process indistinguishable
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
from real light, and another process which desensitizes
and accelerates the response and causes steady current
suppression but which does not cause photon-like events.
These results can be explained ifbleached forms of rhodopsin lead both the generation of R* at a low rate and to the
direct activation of G-protein with low probability." This
model does not appear to be in conflict with most of the
observations cited by Crouch & Corson, which, as they
point out, are not easy to explain by a simple single
mechanism. Both bleaching and background illumination
might derive from a discrete collection of mechanisms. The
relative contribution of each of these mechanisms during
bleaching and background adaptation may be different.
Hurwitz et al. appropriately point out that not only
calcium but also possibly magnesium concentration inside
the photoreceptor cell changes upon illumination. The
issue is whether magnesium levels ever actually decrease
sufficiently to limit the amount of this ion available as an
enzyme cofactor. It would be most interesting to obtain
direct measurements of light-induced changes in free
magnesium concentration. If approximately 5% of the
inward dark current is carried by magnesium, this would
correspond to an inward flux on the order of 1-2 u,M/sec
compared with dark-free levels of several hundred u,M. A
cessation of the inward flux, with no adjustment of magnesium efflux, would be expected to cause a relatively slow
change in intracellular magnesium, on a time scale of
minutes rather than seconds. These kinetics are substantially slower than the decrease in intracellular calcium
known to regulate rapid aspects of light adaptation. Free
calcium concentration in the dark is only ~550 nM (GrayKeller & Detwiler 1994) and the dark influx blocked by
channel closing is ~3-fold greater than the influx of magnesium (Yau & Baylor 1989). This would appear to restrict
magnesium's role to slower aspects of light adaptation.
McGinnis mentions light-induced migration of arrestin
from inner to outer segments and a reverse movement of
transducin. Thus there might be an increase in arrestin
concentration in the light adapted outer segment, leading
to more rapid quenching of excited rhodopsin. We think it
more likely that the concentration of arrestin decreases on
illumination and that the movement of arrestin noted by
McGinnis reflects an increase in the amount of bound
rather than free arrestin in the outer segment. Mangini et
al. (1994) have convincingly shown that arrestin's movement can be explained without an active mechanism, but
rather could be the simple consequence of the appearance of arrestin binding sites in the outer segment as
rhodopsin is bleached and phosphorylated. The movement is abolished if hydroxylamine is used to remove the
arrestin binding site.
R2. Is Ca sufficient to account for adaptation? Matthews
& Fain seem to be alone among the reviewers in their
determination to exclude internal messengers other than
calcium from playing a role in light adaptation. By pushing such a restrictive agenda, they set themselves a
difficult task. While the emphasis on "necessary" and
"sufficient" reflects a proper biophysical reductionist
stance, we would suggest that biological cells care very
little about what is sufficient and often equip themselves
with multiple and/or redundant mechanisms. We should
emphasize that we completely agree that calcium changes
are a necessary and possibly sufficient explanation for
Response/Controversies in Neuroscience III
background adaptation at dim light intensities. However,
Matthews & Fain do not contest the references in section
9, last paragraph, listing dis-correlations between light
adaptation and calcium changes. One more argument can
be offered for the existence of calcium-independent
mechanisms, based on recent direct measurements of the
decrease in cytoplasmic calcium that follows saturating
illumination (Gray-Keller & Detwiler 1994; McCarthy et
al. 1994). If a continuous saturating light is applied to frog
or gecko rod photoreceptors the calcium decrease is
substantially complete after 10 seconds. However, saturating lights having durations longer than this cause
increasing shortening of recovery kinetics for periods up
to 50 seconds (cf. Cervetto et al. 1984; Coles & Yamane
1975). This longer period corresponds more closely to the
dissociation time of cGMP from the noncatalytic binding
sites of PDE, a dissociation that we suggested (sect. 7,
para. 7) might be responsible for accelerating response
kinetics. It is hard for us to imagine that a mechanism with
such detailed correspondence to response kinetics, which
operates over the physiological range of cGMP concentration changes, is a fortuitous artifact. Our idea is that
cGMP feedback on the lifetime of PDE might be particularly important at bright light levels, in receptors that
can operate over a broad range of background light intensity changes. Direct measurements of bound cGMP
levels and conductance at high background illumination
would be required to prove the point.
Both Schnetkamp and Koch make further points that
are relevant to calcium homeostasis. Schnetkamp interprets some of his data to suggest that inactivation of the
calcium efflux mechanism occurs when the level of free
calcium is reduced to ~50 nM. This agrees with recent
data from Gray-Keller and Detwiler (1994), who have
shown that free calcium in gecko photoreceptors does not
fall below ~50 nM even at light levels saturating the
photoresponse. Koch's calculations yield the same basic
conclusion as ours (see sect. 10, para. 4) namely, that at
moderate levels of illumination guanylate cyclase activation may underlie light adaptation. We find ourselves in
disagreement, however, with details of Koch's numbers.
In section 8 we discuss reasons for setting the dark cGMP
flux at approximately 4 (xM/sec, representing a turnover
of the entire free cGMP pool every second. All of the
available electrophysiological (Cornwall & Fain 1994) and
biochemical data (Dawis et al. 1988; Koch & Stryer 1988)
argue that saturating light causes no more than a 10-fold
increase in cyclase activity. Thus, a cGMP flux level of
—40 jjiM/sec represents a reasonable estimate of the
amount of PDE activity that could be countered by
cyclase. Koch's estimate of guanylate cyclase activity as
600 n-M/sec at intermediate illumination mystifies us. We
think that he has erred by overestimating the amount of
cyclase in the cell.
R3. Further comments on the possible involvement of
cGMP in adaptation. Willardson et al. make some very
thoughtful comments. They propose that regulation of
transducin activation by phosducin may be a mechanism
of signal down regulation. In detail, the light-dependent
binding of transducin (3,7 to the unphosphorylated form
of phosducin is meant to remove it from circulation. This
decreases the rate of activation of transducin's a subunit
because that activation requires the participation of the
(3,7 subunits. The suggestion is that this would both cause a
smaller response amplitude and shorter response times.
While we agree that a smaller response amplitude may
result from such a mechanism, response times are not a
function of the amount of available transducin, but rather of
the lifetimes of the activated components. We see two
major problems with depletion argument: (1) Fung (1983)
has shown in a reconstituted system that the maximum rate
of transduction a subunit activation requires only about
10% of the total (3,7 subunit pool. Current estimates of
phosducin levels in the cell suggest the presence of less
phosducin than transducin. Therefore, thereshouldalways
be an excess of (3,7 to support recycling of the transducin a
subunit. What we need to know is how much (3,7 is
required in a real cell. (2) The recent report of Kutuzov and
Pfister (1994) that the GDP-loaded a-subunit of transducin
can activate PDE raises another suggestion. The removal
of (3,7 might actually increase background PDE activity.
How does this fit into the down-regulation model?
A second major point of Willardson et al. is that the
kinetics of cGMP dissociation from PDE noncatalytic
sites may be influenced by the technique used to measure
them. Namely, the displacement of radioactive bound
cGMP by nonradioactive cGMP may affect cGMP dissociation from the second site if dissociation from this site is
dependent upon occupancy of the first. Recently we have
developed procedures for measuring cGMP dissociation
without cGMP re-binding, and find that the kinetics are
essentially the same. We agree with the argument that the
biphasic behavior described by Cote et al. (1994) may be a
consequence of incomplete activation of the PDE in the
preparation used. Our more recent measurements indicate that under conditions where the whole PDE pool is
activated the dissociation is better described by a single
exponential process that is at least four times more rapid
than dissociation from inactive PDE.
A last point raised by Willardson et al. considers the
difference between amphibian and mammalian mechanisms regulating transducin GTPase activity. We believe
that frog and bovine systems differ in the affinity of PDE
noncatalytic sites for cGMP, but not in the fundamental
mechanism of transducin GTPase acceleration. The 7
subunit of PDE catalyzes the acceleration in both cases
(Angleson & Wensel 1994; Arshavsky et al. 1994). However, in both cases it requires an additional membraneassociated factor for its manifestation. This is why Antonny et al. (1993) did not note the acceleration in a
reconstituted system lacking membranes. Arshavsky et
al. (1994) provide a detailed critique that outlines why
Pages et al. (1993) as well as Yamazaki et al. (1993) failed to
observe the acceleration.
NOTE
1. Arshavsky's present address is Howe Laboratories, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114.
Recoverin, a calcium-binding protein in
photoreceptors
James B. Hurley
Department of Biochemistry and Howard Hughes Medical Institute, SL-1S
University of Washington, Seattle, WA 98195. jbhhh@11.washlngton.edu
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
497
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Abstract: Recoverin is a Ca2+-binding protein found primarily
in vertebrate photoreceptors. The proposed physiological function of recoverin is based on the finding that recoverin inhibits
light-stimulated phosphorylation of rhodopsin. Recoverin interacts with rod outer segment membranes in a Ca2+-dependent
manner. This interaction requires N-terminal acylation of recoverin. Four types of fatty acids have been detected on the Nterminus of recoverin, but the functional significance of this
heterogeneous acylation is not yet clear.
The commentaries call attention to several important
points I did not stress in my target article on recoverin.
The alternative perspectives in the commentaries provide
a balanced view of our current understanding of recoverin
and its homologues. Since the functions of recoverin in
photoreceptors are not yet completely understood, it is
very important that the reader consider these different
perspectives because only further experimentation and
testing of alternative hypotheses can provide a more
complete understanding of this protein and its roles in
photoreceptor cells.
Kawamura, who was the first to correctly identify an
important function of recoverin, reviewed the evidence
that recoverin/S-modulin regulates stimulation of phosphodiesterase and the rate of rhodopsin phosphorylation.
A very important point raised by Kawamura that has not
yet been thoroughly investigated is that recoverin appears to have two separate activities, one on the extent of
phosphodiesterase activation, the other on the lifetime of
activated phosphodiesterase. Kawamura's framework for
thinking about these separate activities should inspire
clearer strategies for investigations of the mechanisms of
recoverin function.
The Ca 2+ -dependence of recoverin's effects on phosphorylation and on membrane binding remain controversial since several laboratories have recently found values
for the effect of recoverin on phosphodiesterase activity
and rhodopsin phosphorylation that are in the micromolar
range (e.g., Ames et al. 1995).
Takamatsu's commentary focuses attention on a family
of recoverin-related proteins and the possibility that they
play important roles in signal transduction in other types
of neurons besides photoreceptors. It is clear now that
recoverin homologues are capable of regulating rhodopsin kinase, but it is not yet clear whether other receptor
kinases are regulated by recoverin or its homologues. For
example, my laboratory has found that while recoverin
inhibits rhodopsin kinase, it fails to have any effect on the
activity of the beta-adrenergic receptor kinase. Further
studies are required to determine whether other receptor
kinases are regulated by recoverin homologues. Alternatively, recoverin homologues may regulate other types
of effectors in the same way that G-proteins regulate a
diversity of effectors.
The role of heterogeneous acylation is addressed in the
commentary by Sanada & Fukada. Fukada and his colleagues were co-discoverers of heterogeneous acylation of
transducin and it is clear that they have thought seriously
about the function of this unusual protein modification.
The differences in proportions of different types of fatty
acids on different retinal proteins is interesting, but there
is an important variable, cell type, that should be kept in
mind. For example, the C subunit of cAMP-dependent
protein kinase contains a higher proportion of myristoyl
residue than T-alpha or GCAP, but it is important to
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
remember that this protein is also present in nonphotoreceptor cells in the retina. T-alpha and GCAP are
confined to photoreceptors. (Recoverin, however, is primarily expressed in photoreceptors so the cell-type expression does not explain the high proportion of myristoyl
residues in recoverin.) It would also be interesting to
compare the distribution of N-terminal fatty acids in rods
versus cones within the same retina, but this may not be
technically feasible. Concerning differences in the ability
of nonacylated recoverin and myristoylated recoverin to
inhibit rhodopsin kinase, recent results from my laboratory show that both inhibit rhodopsin kinase, but myristoylated recoverin inhibits at ~ 5 times lower concentration than nonacylated recoverin. The myristoyl residue
enhances rhodopsin kinase inhibition, but it is not required.
Koch raises the possibility that recoverin may function
as a Ca2+-buffer within the photoreceptor cell. The concentration of recoverin appears to be in the appropriate
range for such an activity although the unresolved controversy concerning the affinity of recoverin for Ca 2 + makes
this interesting hypothesis difficult to evaluate.
I certainly agree with Yamazaki's suggestions that we
should not mistakenly confine investigations of recoverin
function to regulation of rhodopsin phosphorylation.
However, my laboratory has done one type of experiment
that suggests regulation of rhodopsin phosphorylation is
important. When hydroxylamine is used, instead of ATP,
to inactivate rhodopsin following a light flash, the rate of
phosphodiesterase deactivation in the presence of recoverin is independent of Ca 2 + . Nevertheless, this result
certainly does not preclude other regulatory interactions
of recoverin and the study of such interactions is very
important.
Finally, Polans & Adamus describe another important
process associated with recoverin, its occasional expression in tumors. When recoverin is expressed in tumors it
may cause blindness that can precede the detection of the
tumor. The diagnostic implication of this observation is
very important in itself. The finding, discussed by both
Polans & Adamus and McGinness, that recoverin maps
near to a tumor suppressor gene, suggests a possible
mechanism for expression of recoverin in certain tumors.
It will be very important to investigate the molecular
mechanisms responsible for oncogenesis in tumors expressing recoverin.
Evidence that the type I adenylyl cyclase
may be important for neuroplasticity: Mutant
mice deficient in the gene for type I adenylyl
cyclase show altered behavior and LTP
Zhengui Xia and Daniel R. Storm
Department of Pharmacology, University of Washington, Seattle, WA 98195.
dstorm@u.washlngton.edu
Abstract: The regulatory properties of the neurospecific, type I
adenylyl cyclase and its distribution within brain have suggested
that this enzyme may be important for neuroplasticity. To
address this issue, the murine, Ca2+-stimulated adenylyl cyclase (type I), was inactivated by targeted mutagenesis. Ca2+stimulated adenylyl cyclase activity was reduced 40% to 60% in
the hippocampus, neocortex, and cerebellum. Long term potentiation in the CA1 region of the hippocampus from mutants
Response/Controversies in Neuroscience III
was perturbed relative to controls. Both the initial slope and
maxim um extent of changes in synaptic response were reduced.
Although mutant mice learned to find a hidden platform normally in the Morris water task, they did not display a preference
for the region where the platform had been when it was removed. The behavioral phenotype of these mice is very similar
to that exhibited by mice which have been surgically lesioned in
the hippocampus. These results indicate that disruption of the
gene for the type I adenylyl cyclase produces changes in spatial
memory and indicate that the cAMP signal transduction pathway may play an important role for synaptic plasticity.
R1. Rasenlck. Detailed information concerning the subcellular localization of the type I adenylyl cyclase and
other adenylyl cyclases in neurons is not available because
appropriate antibodies have not been isolated. However,
we have examined the intracellular sorting of the type I
adenylyl cyclase in primary cultured neurons (Choi et al.
1992b). We have transfected primary hippocampal and
cerebellar neurons with constructs encoding epitope
tagged type I adenylyl cyclase, stained with antibodies
against the epitope, and discovered that the enzyme is
transported to neurite extremities and is not limited to
cell bodies. Whether the enzyme is postsynaptic, presynaptic, or both has not been determined.
We were also surprised to discover that the type I
adenylyl cyclase is not stimulated by Gs coupled receptors in vivo since the purified enzyme is stimulated by
addition of purified Gs-alpha (Tang etal. 1991). Treatment
of recombinant I-AC with combinations of Ca 2+ /CaM and
activated Gs-alpha in vitro produced additive stimulations that were not significantly synergistic (Tang et al
1991). We examined the sensitivity of the type I adenylyl
cyclase expressed in HEK-293 cells to beta-adrenergic
agonists or glucagon when intracellular Ca 2+ was elevated
by Ca 2+ ionophore or carbachol (Wayman et al. 1994).
Although previous studies have shown that this enzyme
can be directly stimulated by activated Gs in vitro, we
demonstrated that it is not stimulated by Gs coupled
receptors in vivo. We have also shown that the type I
adenylyl cyclase is not stimulated by beta-adrenergic
agonists in cultured neurons. However, the enzyme was
stimulated by Gs coupled receptors in vivo when it was
activated by intracellular Ca 2+ . For example, the Ca 2+
ionophore A23187 stimulated the enzyme 3 ± 0.5 fold (n
= 9), isoproterenol alone did not stimulate the enzyme,
but the combination of the two stimulated type I adenylyl
cyclase 13 ± 2 fold (n = 9) in vivo. Similarly, 500 nM
glucagon alone did not stimulate the enzyme but the
combination of A23187 and glucagon activated the enzyme 90 ± 8 fold (n = 4). Synergistic stimulation of type I
adenylyl cyclase activity was also obtained with combinations of carbachol and isoproterenol or glucagon. This
phenomenon was not observed with a mutant enzyme
that is insensitive to Ca 2+ and CaM suggesting that
conformational changes caused by binding of CaM to the
type I adenylyl cyclase enhance binding or coupling to
activated Gs. These data illustrated that this adenylyl
cyclase can couple Ca 2+ and neurotransmitter signals to
generate optimal cAMP levels, a property of the enzyme
that may be important for its role in learning and memory
in mammals.
The studies by Tang et al. (1991) clearly established that
the type I adenylyl cyclase has a domain for interaction
with Gs but it is important to note that relatively high
concentrations of recombinant Gs were used to demonstrate Gs activation of type I adenylyl cyclase in vitro.
Furthermore, these studies generally used Gs-alpha
complexed to nonhydrolizable analogous of GTP. Our
data emphasizes the importance of determining the regulatory properties of each adenylyl cyclase in vivo.
An analysis of the Ca 2+ sensitivity of adenylyl cyclase
activity in brains membranes is complicated by the
existence of several different Ca 2+ sensitive adenylyl cyclases in the same regions of brain. For example,
the type I and VIII adenylyl cyclases are both stimulated
by Ca 2+ in vivo, but the type I enzyme shows greater
Ca 2+ sensitivity and the type VIII enzyme is not synergistically stimulated by Ca 2+ and Gs coupled receptors in
vivo.
R2. Roberson & Sweatt. As emphasized in the target
article, we believe that data from Sweatt's lab showing
that LTP and NMDA activation both stimulate cAMP
levels in the hippocampus are key observations supporting the role of cAMP in synaptic plasticity.
Is the interaction with PKC pre-synaptic or postsynaptic? It has not been established whether type I adenylyl
cyclase is pre-synaptic or postsynaptic. Neuromodulin is
enriched in axons but we do not feel the protein is
exclusively presynaptic. We agree that neurogranin may
also play a role in controlling the levels of free CaM
postsynaptically. This idea is strengthened by unpublished data from Sweatt's laboratory showing that neurogranin is phosphorylated in response to LTP.
The type 1 adenylyl cyclase mutant mice. Our data with
the type I deficient mice provided the first evidence that a
specific adenylyl cyclase contributes to full development
of LTP and may be important for spatial memory in
vertebrates (Wu et al. 1995). Our data did not distinguish
between effects on long-term and short-term learning. It
is notable that the mutant mice did express normal L-LTP
but showed a dampened response in the early stages of
CA1 LTP. Since the mutant mice still expressed significant levels of Ca 2+ stimulated adenylyl cyclase activity in
the hippocampus that may be attributable to type VIII
adenylyl cyclase, mice deficient in Type VIII adenylyl
cyclase and double mutants will be required to access the
role of Ca 2+ stimulated adenylyl cyclase activity for synaptic plasticity.
R3. Abrams. As emphasized by Abrams and the target
article, the Aplasia system has been a very important tool
for demonstrating that adenylyl cyclase activity may play
an important role in associative learning. It is also important to note that there are important regulatory differences between the type I adenylyl cyclase and the Aplasia
enzyme. Furthermore, the Aplasia adenylyl cyclases
have not been cloned and their relationship to the mammalian adenylyl cyclases awaits further purification and
cloning studies.
R4. Heideman. The type I adenylyl cyclase mutant mice
show a 50% reduction in Ca 2+ sensitive adenylyl cyclase
activity in the cerebellum and hippocampus. The residual
adenylyl cyclase activity in the mutant mice is also less
sensitive to Ca 2+ than the wild type mice. It seems likely
that the type VIII adenylyl cyclase contributes to the
residual Ca 2+ sensitive adenylyl cyclase activity in mutant
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
499
Response/Controversies in Neuroscience III
mice. The type I and VIII adenylyl cyclase are, however,
not redundant activities and their regulatory properties
are quite distinct. Mice deficient in type I adenylyl cyclase
show a major deficiency in spatial memory compared to the
wild type mice, indicating that this specific adenylyl
cyclase may be important for some forms of memory.
Further insight into the structural and
regulatory properties of the cGMP-gated
channel
Robert S. Molday and Yi-Te Hsu
Department of Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, B.C., Canada V6T 1Z3. molday@unixq.ubc.ca
Abstract: Recent studies from several different laboratories have
provided further insight into structure-function relationships of
cyclic nucleotide-gated channel and in particular the cCMPgated channel of rod photoreceptors. Site-directed mutagenesis
and rod-olfactory chimeria constructs have defined important
amino acids and peptide segments of the channel that are
important in ion blockage, ligand specificity, and gating properties. Molecular cloning studies have indicated that cyclic
nucleotide-gated channels consist of two subunits that are required to reproduce the properties of the native channels.
Biochemical analysis of the cGMP-gated channel of rodcells
have indicated that the 240 kDa protein that co-purifies with the
63 kDa channel subunit contains both the previously cloned
second subunit of the channel and a glutamic acid-rich protein.
The regulatory properties of the cGMP-gated channel from rod
cells has also been studied in more detail. Studies indicate that
the beta subunit of the cGMP-gated channel of rod cells contains
the binding site for calmodulin. Interaction of calmodulin with
the channel alters the apparent affinity of the channel for cGMP
in all in vitro systems that have been studied. The significance of
these recent studies are discussed in relation to the commentaries on the target article.
R1. Structural features of the rod cGMP-gated channel.
The commentaries of Oprian, Haynes, Hurwitz et al. and
Wenzel & Angleson correctly point to the fact that although significant progress has been made on analysis of
the structural features and molecular properties of the
cGMP-gated channel of photoreceptor cells, many unresolved issues remain. Indeed, since the target article was
written, a number of important new studies have been
carried out in several different laboratories that deal with
some of these unresolved issues. Site-directed mutants
have been used to identify the glutamic acid residue
within the putative pore segment as the divalent cation
binding site (Eismann et al. 1994; Root & MacKinnon
1993) and rod-olfactory chimeras have been constructed
that begin to define regions of the channel that are
important in ligand specificity and gating mechanisms
(Goulding et al. 1994). These studies help to confirm the
general structural features of the cyclic nucleotide-gated
channels as outlined in the target article and in a recent
review article by Eismann et al. (1993). As indicated in the
commentary of Haynes, this type of approach, although
extremely valuable, will not in itself provide a refined
structure of the channel for a rigorous correlation with
functional properties. Ultimately, a high resolution threedimensional structure of the channel, such as can be
obtained by X-ray crystallographic methods, will be re500
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
quired. However, in the absence of such structural analysis, molecular biology techniques coupled with biochemical and biophysical studies should continue to provide important new insight into segments of the channel
that mediate functional and regulatory properties of these
channels.
Recent cloning studies have also revealed the presence
of cyclic nucleotide-gated cation channels in other tissues. Cyclic nucleotide-gated channels related to the
photoreceptor and olfactory channels have been reported
to be expressed in testis, kidney, and heart (Biel et al.
1994; Weyand et al. 1994). A cyclic nucleotide-gated
channel has also been cloned from Drosophila (Baumann
et al. 1994). The physiological role and properties of these
channels, however, remain to be investigated in detail.
Recent biochemical studies have also led to more insight into the subunit structure of the native rod photoreceptor cGMP-gated channel and have addressed some
of the questions raised in the commentaries. As outlined
in the target article, biochemical studies have indicated
that the purified cGMP-gated channel from rod outer
segments contains a 240 kDa polypeptide, as well as the
63 kDa a-subunit. These channel preparations, however,
do not contain polypeptides of ~70 kDa or 102 kDa, the
predicted size of the P-subunit (Chen et al. 1993). The 240
kDa protein has now been isolated and subjected to
peptide sequence analysis. These studies and immunochemical analysis indicate that the 240 kDa polypeptide contains both the P-subunit (or subunit 2) of the
channel (Chen et al. 1994) and the bovine glutamic acid
rich protein (GARP) as first cloned by Sugimoto et al.
(1991). In their commentary, Brown & Karpen have
indicated that they have also obtained a peptide sequence
corresponding to P-subunit of the channel in photoaffinity
labeling of the 240 kDa polypeptide. The primary structure of cGMP binding domains for the a-subunit and
P-subunit are similar, but not identical. Sequence differences may account for the heterogeneity in cGMP binding affinity observed in their reported studies.
The existence of a glutamic acid rich protein as a
component of the 240 kDa protein has been determined
both by peptide sequence analysis and by expression
cloning using two monoclonal antibodies that react with
the 240 kDa protein (Ming et al. 1994). What is not clear
from these studies, however, is how this glutamic acidrich protein component is linked to the P-subunit to form
the 240 kDa polypeptide. One suggestion is that they are
synthesized as individual proteins and that these proteins
are subsequently covalently linked together via a posttranslational reaction. This possibility is based on previously reported studies indicating that the glutamic acid
rich protein (GARP) is expressed as a protein of apparent
molecular mass of 65 kDa from a single 2.4 kB mRNA
transcript (Sugimoto et al. 1991) and that the human
P-subunit is expressed from one or two transcripts as a
protein of 70 or 102 kDa (Chen et al. 1993). This has led to
the suggestion that the GARP protein may be a third
subunit of the channel (Ming et al. 1994). Alternatively,
the 240 kDa protein, may be translated from a single
mRNA containing coding regions for both the P-subunit
and the glutamic acid rich protein. If this is the case, then
some of the earlier studies on the GARP protein and the
human P-subunit need to be reinvestigated. Direct experimental information on how the glutamic acid rich
Response/Controversies in Neuroscience III
protein and the p-subunit component combine to form
the 240 kDa protein remains an important area of
investigation.
On the basis of these recent studies, the earlier view
that the photoreceptor cGMP-gated channels, as well as
the olfactory channels, is composed of identical subunits
has now given way to the conclusion that these cyclic
nucleotide-gated channels consist of at least two principal
subunits (a and P subunit), both of which are needed to
reproduce the electrophysiological properties of the native channels (Bradley et al. 1994; Chen 1993; 1994;
Liman & Buck 1994). The role of the GARP protein found
in the bovine rod 240 kDa channel polypeptide is not yet
understood. Clearly, a lot still remains to be learned about
the structure-function relationships of the photoreceptor
of cGMP-gated channels and their targeting to outer segment plasma membranes as pointed out in the commentaries.
R2. Calmodulin regulation of the cGMP-gated channel.
Since the initial observation that calmodulin binds to and
modulates the affinity of the rod channel for cGMP in rod
outer segment membranes, several laboratories have confirmed this observation in different channel preparations
including patches from frog rod outer segments (Gordon
et al. 1994), purified and reconstituted channel preparations (Hsu & Molday 1994) and mammalian cells coexpressing both the a and (J-subunits of the channel (Chen
et al. 1994). Thus, the effect of calmodulin on channel
activity, although relatively small, is consistently observed, and at least in ROS membrane vesicle preparations is inhibited by mastoparan (Hsu & Molday 1994).
Calmodulin has also been observed to modulate the
activity of the olfactory channel (Chen & Yau 1994). In this
case the calmodulin mediated effect is much larger and
the binding site has been localized to a region close to the
N-terminus of the a-subunit (Liu et al. 1994). The calmodulin binding site on the rod cGMP-gated channel has
been localized to the P-subunit (Chen et al. 1994) within
the 240 kDa protein. The precise binding site has not yet
been reported, but examination of the sequence of the
P-subunit of the rod channel indicates that an amphipathic helix predicted to serve as a calmodulin binding
motif is present on the C-terminal side of the cGMPbinding domain. Preliminary studies in our laboratories
have confirmed that fusion peptides in this region do bind
calmodulin in a calcium-dependent manner (unpublished
results).
The physiological role of calmodulin modulation of the
channel in phototransduction, however, remains to be
determined. As outlined in the target article, we have
proposed that calmodulin modulation of the affinity of the
channel for cGMP may play a role in the recovery of the
photoreceptor cell to its dark state following bleaching. As
indicated in the commentary of Gray-Keller & Detwiler,
such an effect would only come into play if the physiological concentration of calcium drops to a value in the range
of the Kd for the interaction of Ca-calmodulin with the
channel. Their measurements indicate that prolonged
bleaching conditions are required to reduce Ca levels to
this range. This would suggest that calmodulin is bound to
the channel in the dark and remains bound during brief
flashes of light under conditions in which only a minimal
drop in calcium is realized. The suggestion of Gray-Keller
and Detwiler that calmodulin modulation of the channel
may occur under prolonged bleaching conditions is plausible and warrants further investigation.
Studies reported in the commentary of Sagoo & Lagnado and also by Koutalos et al. (1994) using truncated rod
outer segments show an effect of Ca on the cGMP induced
current which is consistent with a diffusable mediator.
Their studies and those of Gordon et al. (1994) lead to the
possibility that other calcium binding proteins may also
modulate the activity of the channel at least in amphibian
rod photoreceptors. It would be of interest to isolate and
characterize this calcium-binding protein or mediator for
comparison with the action of calmodulin on the channel.
We are currently looking into the possibility that other
calcium-binding proteins may also regulate the channel in
mammalian rod outer segment preparations. Another
possibility, however, that needs to be considered is that
calmodulin may regulate the channel in mammalian photoreceptors, but another calcium binding protein that is
not inhibited by mastoparan regulates the channel in
amphibian photoreceptors. This may explain the observations of Gray-Keller & Detwiler, Sagoo & Lagnado, and
Gordon et al. (1994).
As in other systems, it is likely that regulation of the
cGMP-gated channel in photoreceptors is not a simple
mechanism, but may involve multiple regulators and
modes of regulation that have yet to be discovered. A
comprehensive study of the regulatory properties of the
channel at both the biochemical and physiological level
needs to be carried out to resolve these controversial
issues and to extend our understanding of the role of the
cGMP-gated channel in phototransduction and adaptation.
Genetic and functional complexity of
inherited retinal degeneration
Stephen P. Daiger, Lori S. Sullivan, and
Joseph A. Rodriguez
Human Genetics Center, School of Public Health, The University of Texas
Health Science Center, Houston, TX 77030. daiger@gsbs21.uth.tmc.edu
Abstract: Recent findings emphasize the complexity, both genetic and functional, of the manifold genes and mutations
causing inherited retinal degeneration in humans. Knowledge
of the genetic bases of these diseases can contribute to design of
rational therapy, as well as elucidating the function of each gene
product in normal visual processes.
Our target article has two central themes. The first is that
many different photoreceptor genes (or genes expressed
in other retinal cells), and many different mutations
within these genes, can cause inherited retinal degeneration in humans. Recent developments continue to support this view. As examples, a locus for type 3 Usher
syndrome (USH3) has been mapped to chromosome
3q21-q24 (Sankila et al. 1995), one form of recessive
retinitis pigmentosa (RP14) has been mapped to 6p
(Knowles et al. 1994), and an X-linked dominant form of
cone-rod dystrophy has been mapped to Xp21.1-p21.3
(McGuire et al. 1995). In addition, a gene causing dominant Sorsby fundus dystrophy (SFD; chromosome 22ql3qter) has been cloned (Weber et al. 1994), as has a gene
causing recessive type IB Usher syndrome (USH1B;
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
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Re/erences/Controversies in Neuroscience III
chromosome Ilql3.5) (Well et al. 1995). The two cloned
genes are tissue inhibitor of metalloproteinases-3
(TIMP3) and myosin VIIA, respectively. Mutations in the
myosin VIIA gene also produce the mouse recessive
shaker-1 (shl) phenotype, with hearing loss and vestibular dysfunction but without retinal degeneration (Gibson
et al. 1995).
The second theme of our target article is that diseasecausing mutations in photoreceptor genes, such as
rhodopsin can provide valuable insights into the functional roles of particular amino acid sites and protein
domains. Thus the clinical consequences of specific mutations contribute to understanding normal biochemistry
and physiology and, in turn, biochemistry and physiology
contribute to understanding - and predicting - clinical
consequences. Our specific conclusion, regarding rhodopsin and other proteins with complex functional roles, is
that simplistic categorization of mutations, say, into
"transmembrane," or "cytoplasmic" or "intradiscal," is not
sufficient to explain the rich variety of clinical consequences. Rather, what is required is a detailed, site-bysite analysis of function. In the target article we propose a
tentative classification of functional sites in human rhodopsin, based on the current, limited number of known,
disease-causing mutations.
The commentaries provide a valuable additional prospective on these themes. Bergen suggests that the correlation between genotype and phenotype may be more
complicated than we expect at present. Moreover, he
notes that there is a need for standardized protocols for
reporting clinical findings in patients and families. We
emphatically agree with both points. The simplistic classification system used in our analysis is intended as a
starting point for more sophisticated analyses. We were
limited in our analysis by the very sketchy clinical details
provided in most mutation reports (with a few outstanding
exceptions). Thus standard reporting protocols are not
only desirable, but also essential for further progress in
understanding genotype-phenotype correlations.
Kaplan discusses the value of animal models but warns
that human physiology may differ significantly. Animal
models have been of most value in cloning disease genes
later implicated in human retinal degeneration such as
peripherin/RDS, phosphodiesterase (i and myosin VIIA.
However, the utility of transgenic animals in elucidating
the physiologic consequences of specific human mutations remains to be seen.
Tamai describes an increase in photoreceptor glutamate levels in mice homozygous for the rds insertion
mutation and suggests that increased glutamate might
trigger apoptosis. Clearly this will be a useful model for
recessive retinal degeneration.
McCinnis stresses the great promise of molecular treatments, such as gene therapy or antisense therapy, in
treating inherited retinal diseases. These treatments, of
course, require knowledge of the specific, underlying
genetic defect in each patient. This emphasizes the urgency of continued gene mapping, cloning and mutation
screening.
Barnstable notes that several forms of retinal degeneration are caused by mutations in genes expressed in many
tissues in addition to the retina. He also expresses concern that several of these diseases were not discussed in
our target article. There are over 200 distinct forms of
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
inherited retinal degeneration known in humans (McKusick
1994); the currently reported forms are likely to represent
only a small subset of all possible disease-causing genes.
We intentionally limited the discussion to photoreceptorspecific genes because these "simple" cases may best
illuminate the various mechanisms that lead to cellular
degeneration. In fact, the "housekeeping" genes implicated in retinal degeneration, such as ornithine aminotransferase in gyrate atrophy and geranylgeranyl transferase A in choroideremia, affect the entire retinal
structure, not just photoreceptors. In spite of these minor
caveats, though, we strongly agree with the points made
by Dr. Barnstable: one common element in several of
these diseases is the effect on cGMP levels, the retina
appears to be especially prone to degeneration as a result
of metabolic perturbations, and inherited retinal degeneration serves as an excellent model for other neural
degenerative conditions such as Alzheimer disease.
Finally, Wahlsten raises an intriguing philosophic
question: Is it possible that different mutations produce
different clinical phenotypes simply because of individual
variation in genetic background and/or environment?
This concern arises for two reasons. First, even within
families with the same mutation, there is substantial
clinical variation; and second, many of the reported mutations are found in only one or very few individuals; hence,
the range of expression is unknown. It is clear that genetic
and environmental factors must play a role in modulating
the clinical consequences of specific mutations. Wahlsten
correctly demonstrates the technical difficulties in identifying such factors. (Fortunately, some mutations such as
the RhoPro23His mutation, are common enough to make
statistically sound studies possible.) We disagree, though,
on the relative impact of the primary mutation versus
secondary factors. Even though there is variation within
families, this is much less than the variation often observed between individuals with different mutations. In
fact, for many specific mutations, the sample size is large
enough to argue against major consequences of modifying
factors. In addition, we intentionally chose a broad classification scheme, that is, "mild" versus "severe," to*emphasize the extremes. However, it may be that subtle
differences in clinical phenotype are strongly influenced
by genetic background or environment. We hope this is
so, since identifying these factors may suggest useful
therapies.
We look forward to rapid progress in identifying genes
and mutations causing inherited retinal degeneration in
humans. We expect that thisfloodof new knowledge will
eventually lead to effective therapies and to important
biological insights.
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521
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Printed in the United States of America
The sociobiology of sociopathy: An
integrated evolutionary model
Linda Mealey
Department of Psychology, College of St. Benedict, St. Joseph, MN
56374.'
Electronic mail: lmealey@psy.ug.edu.au
Abstract: Sociopaths are "outstanding" members of society in two senses: politically, they draw our attention because of the inordinate
amount of crime they commit, and psychologically, they hold our fascination because most ofus cannot fathom the cold, detached way
they repeatedly harm and manipulate others. Proximate explanations from behavior genetics, child development, personality theory,
learning theory, and social psychology describe a complex interaction of genetic and physiological risk factors with demographic and
micro environmental variables that predispose a portion of the population to chronic antisocial behavior. More recent, evolutionary
and game theoretic models have tried to present an ultimate explanation of sociopathy as the expression of a frequency-dependent life
strategy which is selected, in dynamic equilibrium, in response to certain varying environmental circumstances. This paper tries to
integrate the proximate, developmental models with the ultimate, evolutionary ones, suggesting that two developmentally different
etiologies of sociopathy emerge from two different evolutionary mechanisms. Social strategies for minimizing the incidence of
sociopathic behavior in modern society should consider the two different etiologies and the factors that contribute to them.
Keywords: antisocial personality; criminal behavior; emotion; evolution; facultative strategies; game theory; moral development;
psychopathy; sociobiology; sociopathy
Sociopaths, who comprise only 3%-4% of the male population and less than 1% of the female population (Davison
& Neale 1994; Robins et al. 1991; Strauss & Lahey 1984),
are thought to account for approximately 20% of the
United States prison population (Hare 1993) and between
33% and 80% of the population of chronic criminal offenders (Hare 1980; Harpending & Sobus 1987; Mednick
et al. 1977). Furthermore, whereas the "typical" U.S.
burglar is estimated to have committed a median five
crimes per year before being apprehended, chronic offenders - those most likely to be sociopaths - report
committing upward of 50 crimes per year and sometimes
as many as two or three hundred (Blumstein & Cohen
1987). Collectively, these individuals are thought to account for over 50% of all crimes in the U.S. (Hare 1993;
Loeber 1982; Mednick et al. 1987).
Whether criminal or not, sociopaths typically exhibit
what is generally considered to be irresponsible and
unreliable behavior; their attributes include egocentrism,
an inability to form lasting personal commitments and a
marked degree of impulsivity. Underlying a superficial
veneer of sociability and charm, sociopaths are characterized by a deficit of the social emotions (love, shame,
guilt, empathy, and remorse). On the other hand, they are
not intellectually handicapped, and are often able to
deceive and manipulate others through elaborate scams
and ruses, or by committing crimes that rely on the trust
and cooperation of others, such as fraud, bigamy, and
embezzlement. The sociopath is "aware of the discrepancy between his behavior and societal expectations, but
he seems to be neither guided by the possibility of such a
discrepancy, nor disturbed by its occurrence" (Widom
© 1995 Cambridge University Press
OUO-525X/95$9.OO+.1O
1976a, p. 614). This cold-hearted and selfish approach to
human interaction at one time garnered for sociopathy
the moniker "moral insanity" (Davison & Neale 1994;
McCord 1983).
Sociopaths are also sometimes known as psychopaths or
antisocial personalities. Unfortunately, the literature reflects varied uses of these three terms (Eysenck 1987;
Feldman 1977; Hare 1970; McCord 1983; Wolf 1987).
Some authors use one term or another as a categorical
label, as in psychiatric diagnosis or in defining distinct
personality "types"; an example is the "antisocial personality" disorder described in the Diagnostic and Statistical
Manual of the American Psychiatric Association (1987).
Other authors use the terms to refer to individuals who
exhibit, to a large degree, a set of behaviors or personality
attributes that are found in a continuous, normal distribution among the population at large; an example of such
usage is "sociopathy" as defined by high scores on all three
scales of the Eysenck Personality Questionnaire: extraversion, neuroticism, and psychoticism (Eysenck 1977;
1987).
Other authors make a distinction between "simple" and
"hostile" (Allen et al. 1971), or "primary" and "secondary"
psychopaths or sociopaths (Fagan & Lira 1980). These
authors reserve the term "simple" or "primary" for those
individuals characterized by a complete lack of the social
emotions; individuals who exhibit antisocial behavior in
the absence of this emotional deficit are called "hostile" or
"secondary" psychopaths or sociopaths, or even "pseudopsychopaths" (McCord 1983). Other authors also make
a typological distinction, using the term "psychopath" to
refer to antisocial individuals who are of relatively high
523
Mealey: The sociobiology of sociopathy
intelligence and middle to upper socioeconomic status
and who express their aberrant behavior in impressive
and sometimes socially skilled behavior which may or
may not be criminal, such as insider trading on the stock
market (Bartol 1984). These authors reserve the term
"sociopath" for those antisocial persons who have relatively low intelligence and social skills or who come from
the lower socioeconomic stratum and express their antisocial nature in the repeated commission of violent crimes
or crimes of property.
I will begin by using the single term "sociopath" inclusively. However, by the end of the paper I hope to
convince the reader that the distinction between primary
and secondary sociopaths is an important one because
there are two different etiological paths to sociopathy,
with differing implications for prevention and treatment.
My basic premise is that sociopaths are designed for
the successful execution of social deception and that they
are the product of evolutionary pressures which, through
a complex interaction of environmental and genetic factors, lead some individuals to pursue a life strategy of
manipulative and predatory social interactions. On the
basis of game theoretic models this strategy is to be
expected in the population at relatively low frequencies in
a demographic pattern consistent with what we see in
contemporary societies. This strategy is also expected to
appear preferentially under certain social, environmental, and developmental circumstances which I hope to
delineate.
In an effort to present an integrated model, I will use a
variety of arguments and data from the literature in
sociobiology, game theory, behavior genetics, child psychology, personality theory, learning theory, and social
psychology. I will argue that: (1) there is a genetic predisposition underlying sociopathy, which is normally distributed in the population; (2) as the result of selection to fill a
small, frequency-dependent, evolutionary niche, a small,
fixed percentage of individuals - those at the extreme of
this continuum - will be deemed "morally insane" in any
culture; (3) a variable percentage of individuals who are
less extreme on the continuum will sometimes, in response to environmental conditions during their early
development, pursue a life strategy that is similar to that
of their "morally insane" colleagues; and (4) a subclinical
manifestation of this underlying genetic continuum is
evident in many of us, becoming apparent only at those
times when immediate environmental circumstances
make an antisocial strategy more profitable than a prosocial one.
1. The model
1.1. The evolutionary role of emotion
As the presenting, almost defining characteristic of sociopaths is their apparent lack of sincere social emotions in
the absence of any other deficit such as mental retardation
or autism (Hare 1980), it seems appropriate to begin with
an examination of some current models of emotion.
Plutchik (1980) put forth an evolutionary model of
emotion in which he posits eight basic or "primary"
emotions (such as fear, anger, and disgust) which predate
human evolution and are clearly related to survival.2
524
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
According to the model, everyone (including sociopaths)
experiences these primary emotions, which are crosscultural and instinctively programmed. "Secondary" and
"tertiary" emotions, on the other hand, are more complex, specifically human, cognitive interpretations of
varying combinations and intensities of the primary emotions.3 Because they are partly dependent upon learning
and socialization, secondary emotions, unlike primary
emotions, can vary across individuals and cultures. Thus,
the social emotions (such as shame, guilt, sympathy, and
love), which are secondary emotions, can be expected to
exhibit greater variability.
Griffiths (1990) points out that most of the important
features of emotion argue for an evolutionary design:
emotions are generally involuntary, and are often "intrusive" (p. 176); they cause rapid, coordinated changes in
the skeletal/muscular system, facial expression, vocalization, and the autonomic nervous system; they are to a
large extent innate, or at least "prepared" (see Seligman
1970); and they do not seem as responsive to new information about the environment as do beliefs. Griffiths argues
that emotional responses to stimuli (he calls them "affectprograms" after Ekman 1971) are informationally encapsulated, complex, organized reflexes, which are "adaptive
responses to events that have a particular ecological significance for the organism" (p. 183). That is, they are likely
to be highly specialized reflexive responses elicited spontaneously by the presence of certain critical stimuli,
regardless of the presence of possible mediating contextual cues or cognitive assessments.
Nesse (1990) likewise posits an evolutionary model in
which emotions are "specialized modes of operation,
shaped by natural selection, to adjust the physiological,
psychological, and behavioral parameters of the organism
in ways that increase its capacity and tendency to respond
to the threats and opportunities characteristic of specific
kinds of situations" (p. 268). He attributes a particular role
to the social emotions, a role he couches in the language of
reciprocity and game theory. Presenting a classic Prisoner's Dilemma matrix, he notes which emotions would
be likely to be associated with the outcomes of each of the
four cells: when both players cooperate, they experience
friendship, love, obligation, or pride; when both cheat or
defect, they feel rejection and hatred; when one player
cooperates and the other defects, the cooperator feels
anger and the defector feels anxiety and guilt.
Given that these emotions are experienced after a
behavioral choice is made, how could they possibly be
adaptive? Nesses explanation is based on the models of
Frank (1988) and Hirshleifer (1987), which posit that ex
post facto feelings lead to behavioral expressions that are
read by others and can be used to judge a person's likely
future behavior. To the extent that the phenomenological
experience of emotion serves to direct a person's future
behavior (positive emotions reinforce the preceding behavior whereas negative emotions punish and, therefore,
discourage repetition of the behavior), the outward expression of emotion will serve as a reliable indicator to
others of how a person is likely to behave in the future.
Indeed, that there exist reliable, uncontrollable outward
expressions of these inner experiences at all suggests that
the expressions must be serving a communicative function (Dimberg 1988).
Mealey: The sociobiology of sociopathy
If, however, as in the case of the Prisoner's Dilemma,
the most rational strategy is to be selfish and defect, why
should the positive (reinforcing) emotions follow mutual
cooperation rather than the seemingly more adaptive
behavior of defection? Here lies the role of reputation. If a
player is known, through direct experience or social
reputation, always to play the "rational" move and defect,
then in a group where repeated interactions occur and
individuals are free to form their own associations, no
player will choose to play with the known defector, who
will thus no longer be provided the opportunity for any
kind of gain - cooperative or exploitative. To avoid this
social "shunning" based on reputation4 and hence, to be
able to profit at all from long-term social interaction,
players must be able to build a reputation for cooperation.
To do so, most of them must in fact, reliably cooperate,
despite the fact that cooperation is not the "rational"
choice for the short-term. Frank (1988) and Hirshleifer
(1987) suggest that the social emotions have thus evolved
as "commitment devices" (Frank) or "guarantors of threats
and promises" (Hirshleifer): they cause positive or negative feelings that act as reinforcers or punishers, molding
our behavior in a way that is not economically rational
for the short term but profitable and adaptive in situations where encounters are frequent and reputation is
important.
Frank (1988) presents data from a variety of studies
suggesting that people do often behave irrationally (emotionally) in many dyadic and triadic interactions - sometimes even when it is clear that there will be no future
opportunity to interact again with the same partner.
These studies support the suggestion that in social situations, one's emotional response will often prevail over
logic, the reason being that such behavior is, in the long
run, adaptive under conditions when one's reputation can
follow or precede one. (See also Alexander 1987; Anawalt
1986; Axelrod 1986; Caldwell 1986; Dugatkin 1992; Farrington 1982; Frank et al. 1993; and Irons 1991 for more
on the role of reputation.)
According to these models, emotion serves both as a
motivator of adaptive behavior and as a type of communication: the phenomenological and physiological experience of emotion rewards, punishes, and motivates the
individual toward or away from certain types of stimuli
and social interactions, whereas the outward manifestations of emotion communicate probable intentions to
others.
Once such reliable communicative mechanisms have
evolved, however, when communication of intent precedes interaction, or when one's reputation precedes one,
the conditions of interaction become vulnerable to deception through false signaling or advance deployment of
enhanced reputation (e.g., Caldwell 1986). Those who
use a deceptive strategy and defect after signaling cooperation are usually referred to as "cheaters," and, as many
authors have pointed out (e.g., Alexander 1987; Dennett
1988; Quiatt 1988; Trivers 1971), the presence of cheaters
can lead to a coevolutionary "arms race" in which potential
cooperators evolve fine-tuned sensitivities to likely evidence or cues of deception, while potential cheaters
evolve equally fine-tuned abilities to hide those cues. 5
As long as evolutionary pressures for emotions as reliable communication and commitment devices leading to
long-term, cooperative strategies coexist with counterpressures for cheating, deception, and "rational" shortterm selfishness, a mixture of phenotypes will result, such
that some sort of statistical equilibrium will be approached. Cheating should thus be expected to be maintained as a low-level, frequency-dependent strategy, in
dynamic equilibrium with changes in the environment
which exist as counter-pressures against its success. This
type of dynamic process has been modelled extensively
by evolutionary biologists who use game theory: the topic
I turn to next.
1.2. Game theory and evolutionarily stable strategies
Game theory was first introduced into the literature of
evolutionary biology by Richard Lewontin (1961), who
applied it to the analysis of speciation and extinction
events. It was later taken up in earnest by John Maynard
Smith and colleagues (e.g. Maynard Smith 1978; 1974;
Maynard Smith & Price 1973 [See also Maynard Smith:
"Game Theory and the Evolution of Behaviour" BBS 7(1)
1984.]) who used it to model contests between individuals. Maynard Smith showed that the "evolutionarily
stable strategies" (ESSs) that could emerge in such contests included individuals' use of mixed, as well as fixed,
strategies. Alexander (1986) writes: "it would be the worst
of all strategies to enter the competition and cooperativeness of social life, in which others are prepared to alter
their responses, with only preprogrammed behaviors"
(p. 171).
The maintenance of mixed ESSs in a population can
theoretically be accomplished in at least four ways (after
Buss 1991): (1) through genetically based, individual
differences in the use of single strategies (such that each
individual, in direct relation to genotype, consistently
uses the same strategy in every situation); (2) through
statistical use by all individuals of a species-wide, genetically fixed, optimum mix of strategies (whereby every
individual uses the same statistical mix of strategies, but
does so randomly and unpredictably in relation to the
situation); (3) through species-wide use by all individuals
of a mix of environmentally contingent strategies (such
that every individual uses every strategy, but predictably
uses each according to circumstances); (4) through the
developmentally contingent use of single strategies by
individuals (such that each individual has an initial potential to use every type of strategy but, after exposure to
certain environmental stimuli in the course of development, is phenotypically canalized from that point on to
use only a fraction of the possible strategies). To Buss's
fourth mechanism can be added a differential effect of
genotype on developmental response to the environment, thus adding another mechanism: (5) genetically
based individual differences in response to the environment, resulting in differential use by individuals of
environmentally-contingent strategies (such that individuals of differing genotypes respond differently to environmental stimuli in the course of development and are thus
canalized to produce a different set of limited strategies
given the same, later conditions).
Following the leads of Cohen & Machalek (1988); Harpending & Sobus (1987); Kenrick et al. (1983); Kofoed &
MacMillan (1986); and MacMillan & Kofoed (1984), I
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Mealey: The sociobiology of sociopathy
would like to suggest an evolutionary model in which
sociopaths are a type of cheater-defector in our society of
mixed-strategy interactionists. I will be arguing that
sociopathy appears in two forms, according to mechanisms 1 and 5 (above): one version that is the outcome of
frequency-dependent, genetically based individual differences in use of a single (antisocial) strategy (which I will
refer to as "primary sociopathy") and another that is the
outcome of individual differences in developmental response to the environment, resulting in the differential
use of cooperative or deceptive social strategies (which
I will refer to as "secondary sociopathy"). To support
this model, I will provide evidence that there are predictable differences in the use of cheating strategies
across individuals, across environments, and within individuals across environments; this evidence will integrate
findings from the fields of behavior genetics, child psychology, personality theory, learning theory, and social
psychology.
2. The evidence
2.1. Behavior genetics
For decades, evidence has been accumulating that both
criminality and sociopathy have a substantial heritable
component, and that this heritable component is to a
large extent overlapping; that is, the heritable attributes
that contribute to criminal behavior seem to be the same
as those which contribute to sociopathy. Although there is
no one-to-one correspondence between those individuals
identified as criminals and those identified as sociopaths,
(indeed, the definitions of both vary from study to study),
it is clear that these two sets of individuals share a variety
of characteristics and that a subset of individuals share
both labels (Ellis 1990b; Gottesman & Goldsmith 1993;
Moffitt 1987; Robins et al. 1991).
2.1.1. Studies of criminal behavior. The behavior-genetic
literature on criminal behavior suggests a substantial
effect of heredity across several cultures.6 Christiansen
(1977a; 1977b), Cloninger & Gottesman (1987), Eysenck
& Gudjonsson (1989), Raine (1993) and Wilson & Herrnstein (1985) review studies of twins which, taken as a
whole, suggest a heritability of approximately 0.60 for
repeated commission of crimes of property. (Heritability
is a measure of the proportion of variance of a trait, within
a population, that can be explained by genetic variability
within that population; it thus ranges theoretically from
0.00 to 1.00, with the remaining population variance
explained by variance in individuals' environment.)
Adoption studies (reviewed in Cloninger & Gottesman
1987; Eysenck & Gudjonsson 1989; Hutchings & Mednick 1977; Mednick & Finello 1983; Mednick et al. 1987;
Raine 1993; and Wilson & Herrnstein 1985) arrive at a
similar conclusion.7
Several adoption studies were also able to demonstrate
significant interactive effects not discriminable using the
twin methodology. Cadoret et al. (1983); Crowe (1972;
1974); Mednick & Finello (1983); and Mednick et al.
(1984) report significant gene-environment interactions,
such that adoptive children with both a genetic risk
(criminal biological parent) and an environmental risk
(criminality, psychiatric illness, or other severe behav-
526
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
ioral disturbance in an adoptive parent), have a far greater
risk of expressing criminal behavior than do adoptees with
no such risk or only one risk factor, and that increased risk
is more than simply an additive effect of both risk factors.
In addition, Baker et al. (1989) report an interaction based
on sex, in which females are more likely to transmit a
genetic risk to their offspring than are males.
2.1.2. Studies of sociopathy. The literature on sociopathy
suggests a pattern similar to that on criminality: Cadoret
(1978); Cadoret & Cain (1980); Cadoret & Stewart (1991);
Cadoret et al. (1987); Crowe (1974); and Schulsinger
(1972) demonstrate a substantial heritability to sociopathy; Cadoret et al. (1990) found a gene-environment
interaction similar to the one found for criminal behavior;
and Cadoret & Cain (1980; 1981) found an interaction
involving sex, such that male adoptees were more sensitive to the influence of environmental risk factors than
were female adoptees.
The similarity of the patterns described in these two
domains is to some extent due to the fact that the diagnosis of sociopathy is often based in part upon the existence of criminal activity in a subject's life history. On the
other hand, consider the following facts: (1) criminal
behavior and other aspects of sociopathy are correlated
(Cadoret et al. 1983; Cloninger & Gottesman 1987; Eysenck 1977; Morrison & Stewart 1971; Patterson et al.
1989; Wolf 1987); (2) criminal activity is found with increased frequency among the adopted-away children of
sociopaths (Moffitt 1987); (3) sociopathy is found with
increased frequency among the adopted-away children of
criminals (Cadoret & Stewart 1991; Cadoret et al. 1990).
This all suggests that criminality and sociopathy may
share some common heritable factors. For this reason,
early researchers and clinicians (e.g. Cadoret 1978; Schulsinger 1972) suggested using the term "antisocial spectrum" to incorporate a variety of phenotypes considered
likely to be manifestations of closely related genotypes.8
The existence of this spectrum suggests a multifactorial,
probably polygenic, basis for sociopathy and its related
phenotypes. Using an analogy to "g," [See Jensen: "The
Nature of the Black-White Difference on Various Psychometric Tests" BBS 8(2) 1985.] which is often used to refer
to the common factor underlying the positive correlations between various aptitude measures, Rowe (1986)
and Rowe & Rodgers (1989) use "d" to refer to the
common factor underlying the various expressions of
social deviance.
2.1.3. Sex differences and the "two threshold" model.
Cloninger put forth a "two threshold" polygenic model to
account for both the sex difference in sociopathy and its
spectral nature (Cloninger etal. 1975; 1978). According to
the model, sociopaths are individuals on the extreme end
of a normal distribution whose genetic component is (1)
polygenic and (2) to a large degree, sex limited. (Sexlimited genes, not to be confused with sex-linked genes,
are those which are located on the autosomes of both sexes
but are triggered into expression only within the chemical/hormonal microenvironment of one sex or the other.
Common examples include beard and mustache growth
in men, and breast and hip development in women.) If a
large number of the many genes underlying sociopathy
are triggered by testosterone or some other androgen,
Mealey: The sociobiology of sociopathy
many more men than women will pass the threshold of
the required number of active genes necessary for its
outward expression.
According to the two-threshold model, those females
who do express the trait must have a greater overall "dose"
or "genetic load" (i.e., they are further out in the extreme
of the normal distribution of genotypes) than most of the
males who express the trait. This proposition has been
supported by data showing that, in addition to the greater
overall risk for males as opposed to females, there is also a
greater risk for the offspring (and other relatives) of female
sociopaths as compared to the offspring (and other relatives) of male sociopaths. This phenomenon cannot be
accounted for either by sex linkage or by the differential
experiences of the sexes.
Besides providing a proximate explanation for the
greater incidence of male sociopathy and crime, the twothreshold model also explains on a proximate level the
finding that males are more susceptible to environmental influences than females. Somewhat paradoxically, although a male will express sociopathy at a lower "genetic
dose" than is required for expression in a female, the
heritability of the trait is greater for females, meaning that
the environmental component of the variance is greater
for males.9
The two-threshold model thus explains in a proximate
sense what sociobiologists would predict from a more
ultimate perspective. The fact that males are more susceptible than females to the environmental conditions of
their early years fits well with sociobiological theory in
that the greater variance in male reproductive capacity
makes their "choice" of life strategy somewhat more risky
and therefore more subject to selective pressures (Buss
1988; Mealey & Segal 1993; Symons 1979). Sociobiological reasoning thus leads to the postulate that males should
be more sensitive to environmental cues that (1) trigger
environmentally contingent or developmentally canalized life history strategies or (2) are stimuli for which
genetically based individual differences in response
thresholds have evolved. (Recall mechanisms 3, 4, and
5 for the maintenance of mixed strategy ESSs in a
population).
If the evolutionary models apply, then when, specifically, would sociopathy be the best available strategy?
What would be the environmental cues that, especially
for boys, would trigger its development? To answer these
questions, I turn to the child psychology literature, with a
special focus on studies of life history strategies, delinquency, and moral development.
2.2. Child psychology
2.2.1. Life history strategies. Beginning with Draper and
Harpending's now-classic 1982 paper on the relationship
between father absence and reproductive strategy in
adolescents, there has been an increasing effort to view
development as the unfolding of a particular life strategy
in response to evolutionarily relevant environmental cues
(Belsky et al. 1991; Crawford & Anderson 1989; Draper &
Belsky 1990; Draper & Harpending 1982; Gangestad &
Simpson 1990; MacDonald 1988; Mealey 1990; Mealey
6 Segal 1993; Moffitt et al. 1992; Surbey 1987). These
models are based either implicitly or explicitly on the
assumption that there are multiple evolutionarily adap-
tive strategies and that the optimal strategy for particular
individuals will depend both upon their genotype and
their local environment. To date, most developmental life
history models address variance in reproductive strategies (for example: age at menarche or first sexual activity,
number of mating partners, and amount of parental investment), but this type of modeling can also be applied to
the adoption of social strategies such as cheating versus
cooperation.
Perhaps the most oft-mentioned factor suggested as
being relevant to the development of a cheating strategy,
especially in males, is being competitively disadvantaged
with respect to the ability to obtain resources and mating
opportunities. Theoretically, those individuals who are
the least likely to outcompete other males in a status
hierarchy, or to acquire mates through female choice, are
the ones most likely to adopt a cheating strategy (see,
e.g., Daly & Wilson 1983; Gould & Gould 1989; Thornhill
& Alcock 1983, regarding nonhuman animals, and Cohen
& Machalek 1988; Kenrick et al. 1983; Kofoed & MacMillan 1986; MacMillan & Kofoed 1984; Symons 1979;
Thornhill & Thornhill 1992; Tooke & Camire 1991, regarding humans). In humans, competitive disadvantage
could be related to a variety of factors, including age,
health, physical attractiveness, intelligence, socioeconomic status, and social skills.
Criminal behavior, one kind of cheating strategy, is
clearly related to these factors. Of the seven cross-cultural
correlates of crime reported by Ellis (1988), three seem
directly related to resource competition - large number
of siblings, low socio economic status, and urban residency. The four others - youth, maleness, being of black
racial heritage, and coming from a single-parent (or otherwise disrupted) family background - can be plausibly
argued to be related to competition as well (see, e.g.,
Cohen & Machalek 1988; Ellis 1988; Kenrick et al. 1983;
Wilson & Herrnstein 1985). Empirical data suggest that
deficits in competitive ability due to psychosis (Hodgins
1992), intellectual handicap (Hodgins 1992; Moffitt &
Silva 1988; Quay 1990a; Stattin & Magnusson 1991), or
poor social skills (Dishion et al. 1991; Garmezy 1991;
Hogan & Jones 1983; Simonian et al. 1991) are also
associated with criminal behavior. Likewise, the competitive advantages conferred by high intelligence (Hirschi &
Hindenlang 1977; Kandel et al. 1988; Silverton 1988;
White et al. 1989; Wilson & Herrnstein 1985) or consistent external support (Garmezy 1991), can mitigate the
development of criminal or delinquent behavior in those
who are otherwise at high risk.
Rape and spouse abuse, other forms of cheating strategy, appear to be related to the same life-history factors as
crime (Ellis 1989; 1991a; Malamuth et al. 1991; Thornhill
& Thornhill 1992). In fact, Huesmann et al. (1984), Rowe
and Rodgers (1989), and Rowe et al. (1989) present evidence that there is a common genetic component to the
expression of sexual and nonsexual antisocial behavior.
Given the overlaps between rape, battering, and criminality in terms of life history circumstances, genetics, and
apparent inability to empathize with one's victim, it
would be parsimonious to postulate that they might be
expressions of a single sociopathy spectrum. As such,
these antisocial behaviors could be considered to be
genetically influenced, developmentally and environmentally contingent cheating strategies, utilized when a
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
527
Mealey: The sociobiology of sociopathy
male finds himself at a competitive disadvantage (see also
Figueredo & McCloskey, unpublished manuscript).
Along these lines, MacMillan and Kofoed (1984) presented a model of male sociopathy based on the premise
that sexual opportunism and manipulation are the key
features driving both the individual sociopath and the
evolution of sociopathy. Harpending and Sobus (1987)
posited a similar basis for the evolution and behavioral
manifestations of Briquet's hysteria in women, suggesting
that this syndrome of promiscuity, fatalistic dependency,
and attention-getting, is the female analogue, and homologue, of male sociopathy.
2.2.2. Delinquency. Childhood delinquency is a common
precursor of adolescent delinquency and adult criminal and sociopathic behavior (Loeber 1982; Loeber &
Dishion 1983; Loeber & Stouthamer-Loeber 1987; Patterson et al. 1989; Robins & Wish 1977); in fact, childhood
conduct disorder is a prerequisite finding in order to
diagnose adult antisocial personality (American Psychiatric Association 1987). As in the literature on adults, an
important distinction is frequently made between two
subtypes of conduct disorder in children: Lytton (1990),
for example, distinguishes between "solitary aggressive
type" and "group type"; Loeber (1990) distinguishes between "versatiles" and "property offenders"; and Strauss
& Lahey (1984) distinguish between "unsocialized' and
"socialized." I will argue that these subtypes are precursors of two types of adulthood antisociality (with "solitary
aggressive," "versatile," or "unsocialized" types leading to
primary sociopathy and "group," "property offender," or
"socialized" types presaging secondary sociopathy). I will
also argue that the differing life history patterns of these
two types of delinquents are reflections of two different
evolutionary mechanisms for maintaining ESSs in a population: mechanism 1 and mechanism 5, respectively (see
sect. 1.2).
Although more than half of juvenile delinquents outgrow their behavior (Gottesman & Goldsmith 1993; Lytton 1990; Robins et al. 1991), the frequency of juvenile
antisocial behaviors is still the best predictor of adult
antisocial behavior, and the earlier such behavior appears, the more likely it is to be persistent (Farrington
1986; Loeber & Stouthamer-Loeber 1987; Lytton 1990;
Robins et al. 1991; Stattin & Magnusson 1991; White et
al. 1990). The mean age at which adult sociopaths exhibited their first significant symptom is between 8 and 9
years; 80% of all sociopaths exhibited their first symptom
by age 11 (Robins et al. 1991). Over two-thirds of eventual
chronic offenders are already distinguishable from other
children by kindergarten (Loeber & Stouthamer-Loeber
1987). Thus, by evaluating the environments of juvenile
delinquents, we can fairly reliably reconstruct the childhood environments of adult sociopaths.
Studies of this sort consistently implicate several relevant environmental factors correlated with boyhood antisocial behavior: inconsistent discipline, parental use of
punishment as opposed to rewards, disrupted family life
(especially father absence, family violence, alcoholic parent, or mentally ill parent), and low socioeconomic status
(Cadoret 1982; Farrington 1986; 1989; Loeber & Dishion
1983; Lytton 1990; McCord 1986; Offord et al. 1991;
Patterson et al. 1989; Silverton 1988; van Dusen et al.
1983; Wilson & Herrnstein 1985). Aside from the fact that
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all of these variables are more likely to exist when one or
the other parent is sociopathic, and the child, hence,
genetically predisposed to sociopathy, behaviorist and
social learning models of the dynamics of early parentchild interactions (to be described in sect. 2.4.2) have
been fairly convincing in explaining how antisocial behaviors can be reinforced under such living conditions.
In line with the postulate that cheating strategies would
most likely be used by individuals who are at a competitive disadvantage, Hartup (1989), Hogan & Jones (1983),
Kandel et al. (1988), Loeber & Dishion (1983), Magid &
McKelvie (1987), McGarvey et al. (1981), and Patterson et
al. (1989) suggest that the common way in which high risk
familial and environmental factors contribute to delinquency is by handicapping children with respect to their
peers in terms of social skills, academic ability, and selfesteem. This noncompetitiveness then leads disadvantaged youths to seek alternative peer groups and social
environments in which they can effectively compete
(Dishion et al. 1991). If they are successful in the estimation of their new peer group, adopting this strategy may
lead to "local prestige" (Rowe, personal communication)
sufficient to commandeer resources, deter rivals, or gain
sexual opportunities within the new referent group (see
also Moffitt 1993). In other words, competitively disadvantaged youth may be trying to "make the best of a bad
job" (Cohen & Machalek 1988, p. 495; Dawkins 1980,
p. 344), by seeking a social environment in which they
may be less handicapped or even superior.
The correlates of delinquency in girls are essentially
the same as those for boys, although delinquency is less
common in girls (Lytton 1990; Robins 1986; White et al.
1990). Caspi et al. (1993) found that delinquency in girls,
as in boys, is arrived at via two different developmental
trajectories. One pattern includes a history of antisocial
behavior throughout childhood and a tendency to seek out
delinquent peers; based on previous research (White et
al. 1990), this life history trajectory is thought to lead to
persistent antisocial behavior in adulthood. The second
pattern is exhibited by girls who have few behavior
problems in childhood, but who, upon reaching menarche, exhibit more and more frequent antisocial behaviors.
The antisocial behavior of girls who show this latter
pattern is thought to be more a product of environmental
influence than that of girls who follow the first trajectory,
as this pattern is selectively exhibited by girls who (a) have
an early age of menarche and (b) are in coeducational
school settings. These girls, upon reaching early sexual
maturity, start associating with older male peers and
exhibiting some of the antisocial behaviors that are more
often displayed by older boys than by their younger
female peers (see Maccoby 1986); girls who follow this
trajectory are expected to "outgrow" their antisocial activities. Although the two subsets of delinquent girls would
be difficult to differentiate using a cross-sectional methodology, in accordance with the model presented here,
Caspi et al. (1993) consider their differing developmental
histories to be of theoretical importance for longitudinal
studies and of practical importance for early intervention
(see Moffitt 1993 for a similar scenario regarding boys.)
2.2.3. Moral development. Like the tendency to engage in
antisocial behavior, an individual's tendency to engage in
prosocial behavior seems to be fairly stable from an early
Mealey: The sociobiology of sociopathy
age (Rushton 1982). Yet the development of individual
differences in behavior has not been as well studied as the
presumably universal stages of cognition that underlie
changes in moral reasoning. Kohlberg's (1964) stage
model of moral development, for example, ties advances
in moral thinking to advances in reasoning ability and
attributes individual differences largely to differences in
cognitive ability. Although it is clear that both moral
reasoning and moral behavior covary with age (Rushton
1982) and may do so in a manner consistent with some
evolutionists' thinking (e.g., Alexander 1987), cognitive
models alone cannot explain the absence of moral behavior in sociopaths, who are not intellectually handicapped
with respect to the normal population.
Other developmental models posit the emergence of
empathy and the other social emotions as prerequisites
for moral behavior (see Zahn-Waxler & Kochanska 1988
for a review). Even very young children, it seems, are in a
sense biologically prepared to learn moral behavior, in
that they are selectively attentive to emotions - especially
distress - in others (Hoffman 1978; Radke-Yarrow &
Zahn-Waxler 1986; Zahn-Waxler & Radke-Yarrow 1982).
Hoffman (1975; 1977; 1982), for example, suggests that
the observation of distress in others triggers an innate
"empathic distress" response in the child, even before the
child has the cognitive capacity to differentiate "other"
from "self." Accordingly, any instrumental behavior that
serves to reduce the distress of the other also serves to
relieve the vicarious distress of the child. Thus, very
young children might learn to exhibit prosocial behavior
long before they are able to conceptualize its effect on
others.
In Hoffman's model, the motivation behind early prosocial behavior is the (egocentric) need to reduce one's
own aversive feelings of arousal and distress. As the child
ages, the range of cues and stimuli that can trigger the
vicarious distress increase through both classical and
operant conditioning. Eventually, when the child develops the cognitive ability to "role play," or take on
another's perspective, empathic distress turns to "sympathetic distress," which motivates the prosocial behavior
that is more likely to be interpreted as intentional, altruistic, and moral. Hoffman's model of prosocial behavior
dovetails nicely with Hirshleifer's (1987) "Guarantor"
and Frank's (1988) "Commitment" model of emotion (see
sect. 1.1): the reduction in anxiety that follows cooperative or prosocial behavior reinforces such behavior,
whereas the increase in anxiety which, through stimulus
generalization, follows acts or thoughts of antisocial behavior will punish and therefore reduce those acts and
thoughts.
Dienstbier (1984) reported an interesting series of
studies testing the role of anxiety and emotional arousal
on cheating. As expected, high arousal levels were associated with low cheating levels (and vice versa), but the
subjects' attribution of the cause of high arousal was also
important. When subjects were able to attribute their
arousal to a cause other than the temptation to cheat, they
found it much easier to cheat than when they had no other
explanation for their arousal level. Subjects were also less
willing to work to avoid punishment when they were able
to attribute their arousal to an external cause rather than
to an internal source of anxiety associated with the threat
of punishment. Dienstbier concluded that when a situa-
tion is perceived to be "detection-free," one's temptation
to cheat is either resisted or not, depending on the levels
of anxiety perceived to be associated with the temptation.
The ability to act intentionally in either a prosocial or
antisocial manner (or in the terms of game theory, cooperatively or deceptively), depends upon having reached a
certain level of cognitive development at which it is
possible to distinguish emotions of the self from emotions
of others; that is, the child must pass from empathic
responses to sympathetic responses (Dunn 1987; Hoffman 1975; 1977; 1984; Mitchell 1986; Vasek 1986). This
transition begins to occur some time during the second
year (Dunn 1987; 1988; Dunn et al. 1991; Hoffman 1975;
1982; Leslie 1987) when the child is beginning to develop
what has come to be called a "theory of mind" (Premack &
Woodruff 1978). [See also Gopnik: "How We Know Our
Minds" BBS 16(1) 1993 and Tomasello et al.: "Cultural
Learning" BBS 16(3) 1993.]
Having a theory of mind allows one to impute mental
states (thoughts, perceptions, and feelings) not only to
oneself, but also to other individuals. Humphrey (1976;
1983) suggests that this kind of awareness evolved in
humans because it was a successful tool for predicting the
behavior of others. Humphrey claims that the best strategists in the human social game would be those who could
use a theory of mind to empathize accurately with others
and thereby be able to predict the most adaptive strategy
in a social interaction (Byrne & Whiten 1988 call this
aptitude "Machiavellian intelligence.") [See also Whiten
& Byrne: "Tactical Deception in Primates" BBS 11(2)
1988.] Humphrey's model is something of a cognitive
equivalent of the evolutionary models of emotion discussed in section 1.1; they can probably be considered
complementary and mutually reinforcing. With regard to
sociopathy, the question is whether a strategist can be
successful using only the cognitive tool of a theory of
mind, without access to emotional, empathic information
which, presumably, sociopaths lack (Mealey 1992). In the
next section I will argue that this is exactly what a
sociopath does.
2.3. Personality theory
The models of normative moral development presented above are helpful but clearly insufficient to explain
sociopathy. Although some adoption studies and most
longitudinal studies report significant effects of social and
environmental risk factors on delinquency and criminality, the magnitude of that risk as a simple main effect is
rather small. Despite repeated exposure to inconsistent
and confusing reinforcement and punishment, most children who grow up with these risk factors do not turn out to
be sociopathic, whereas some children who do not experience such risk factors do. Studies have repeatedly shown
that the effect of the environment is much more powerful
for children at biological risk than for others. What is it
that makes high risk environmental features particularly
salient for those individuals who have a certain predisposing genotype?
2.3.1. The role of gene-environment interactions. Stimulated by the work of Rowe and Plomin (Dunn & Plomin
1990; Plomin & Daniels 1987; Rowe 1983a; 1983b; 1990a;
1990b; Rowe & Plomin 1981), evidence is accumulating
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
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Mealey: The sociobiology of sociopathy
that, unlike what has been traditionally assumed, the
most important environmental features and events that
influence personal development are not those that are
shared by siblings within a family (such as parenting style,
socioeconomic status, and schooling), but rather are idiosyncratic events and relationships which are difficult to
study systematically with traditional methods. Despite a
shared home, individual children will encounter different
microenvironments: their individual relationships with
their parents will differ, and their experiences on a day to
day, minute by minute basis will not overlap significantly.
In addition, there will be some environmental differences
which are due to genetic differences; children with different personalities, aptitudes, and body types will not only
seek out different experiences (Caspi et al. 1987; Rowe
1990a; Scarr & McCartney 1983), but will also attribute
different phenomenological interpretations to the same
experiences (Dunn 1992; Rowe 1983a; 1990b). For these
reasons, any two children will experience an (objectively)
identical environment in different ways; there is, in some
sense, no real validity to some of the operational measures we currently use to describe a child's environment
(Plomin & Daniels 1987; Rowe & Plomin 1981; Wachs
1992).
Although this may sound discouraging for those who
seek to apply psychological research to the prevention of
crime and delinquency - and most such efforts have, in
fact, been fairly unsuccessful (Borowiak 1989; Feldman
1977; Gottschalk et al. 1987; Patterson et al. 1989) - there
are reasons for optimism. Palmer (1983) suggests that the
"nothing works" conclusion is valid only in the sense that
no single intervention technique will be successful across
the board, and that targeting different strategies to different individuals should prove more successful. More and
more studies are suggesting that there are at least two
developmental pathways to delinquency and sociopathy
and that we need to address them separately (Caspi et al.
1993; Dishion & Poe 1993; Lytton 1990; McCord 1993;
Moffitt 1993; Patterson 1993; Quay 1990b; Simons 1993;
White et al. 1990). The evolutionary model presented
here makes specific predictions about the likely differential success of various intervention and prevention strategies for individuals arriving at their antisocial behavior via
different paths: although individuals of dissimilar genotypes may end up with similar phenotypes, different
environmental elements and experiences may be particularly salient for them. (This is a corollary of mechanism 5
for the maintenance of ESSs presented in sect. 1.2.)
As I will argue below, primary and secondary sociopathy seem to provide an excellent illustration of
the development of similar phenotypes from different
genotype-environment interactions. To the extent that
we understand it now, primary sociopaths come from one
extreme of a polygenic genetic distribution and seem to
have a genotype that disposes them "to acquire and be
reinforced for displaying antisociality" (Rowe 1990a,
p. 122). That genotype results in a certain inborn temperament or personality coupled with a particular pattern of
autonomic arousal which, together, seem to design the
individual (1) to be selectively unresponsive to those
environmental cues necessary for normal socialization
and moral development and (2) to actively seek the more
deviant and arousing stimuli within the environment.
Secondary sociopaths, on the other hand, are not as
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genetically predisposed to their behavior; rather they are
more responsive to environmental cues and risk factors,
becoming sociopathic "phenocopies" (after Raine 1993) or
"mimics" (after Moffitt 1993) when the carrying capacity
of the "cheater" niche grows. What are the predisposing
constitutional factors that place some individuals at high
risk?
2.3.2. The role of temperament. In a twin study, Rushton
et al. (1986) found evidence of substantial heritability of
self-reported measures of altruism, nurturance, aggressiveness, and empathy. Across twin pairs, altruism, nurturance, and empathy increased with age, whereas aggressiveness decreased; sex differences (in the expected
direction) were found for nurturance, empathy, and aggression; for all measures, the environmental contributions were determined to be individual rather than familial. Methodological considerations do not allow full
confidence in the numerical heritability estimates of this
study, but Eisenberg et al. (1990) conclude that it reports
true individual differences that are likely to be a result
of genetic differences in temperament, specifically sociability and emotionality.
More recently, two additional twin studies have confirmed the findings of Rushton et al. Emde et al. (1992)
reported significant heritabilities for empathy, behavioral
inhibition, and expressions of negative affect, while
Ghodsian-Carpey & Baker (1987) found significant heritabilities on four measures of aggressiveness in children.
Like the Rushton et al. study, both of these studies also
reported sex differences, and both confirmed the relative
importance of nonshared, as opposed to shared, environmental influences.
A fourth twin study (Rowe 1986) used a different set of
personality indices but went a step further in establishing
the link between temperament and antisocial behavior.
Rowe's analysis suggests that, especially for males, the
inherited factors correlated with one's genetic risk of
delinquency are the same as those that lead to the temperamental attributes of anger, impulsivity, and deceitfulness ("self-serving dishonesty with people with whom a
person ordinarily has affectional bonds," p. 528). It is
interesting to note that although Rowe found that common genetic factors related temperament and delinquency, environmental factors related academic nonachievement with delinquency.
These findings provide evidence for the two-pathway
model presented in section 1.2, in that such a geneenvironment interaction (1) would create at least two
possible routes to sociopathy or criminality, one primarily
heritable and one less so; and (2) in terms of the latter, less
heritable pathway, would set the stage for developmentally and environmentally contingent individual differences in antisocial behavior. In addition, in line with
previously mentioned studies and the proposed model,
the environmental factors Rowe found to be statistically
significant varied within families and were more significant for males than for females.
Most of the research into the relationship between
temperament, personality and sociopathy has been based
on the extensive work of Hans Eysenck (summarized in
Eysenck 1977 and 1983, Eysenck & Gudjonsson 1989,
and Zuckerman 1989). Eysenck first postulated and then
convincingly documented that sociopathy in particular
Mealey: The sociobiology of sociopathy
and antisocial behavior in general are correlated with high
scores on all three of the major personality dimensions of
the Eysenck Personality Questionnaire: "extraversion"
(versus introversion), "neuroticism" (versus emotional
stability), and "psychoticism" (versus fluid and efficient
superego functioning - not synonymous with psychotic
mental illness; Zuckerman (1989) suggests that this scale
would be better called "psychopathy"). All three of these
dimensions exhibit substantial heritability, and since psychoticism is typically much higher in males than females,
it is a likely candidate for one of the relevant sex-limited
traits that fits Cloninger's two-threshold risk model explaining the sex difference in expression of sociopathy.
In trying to explain the proximate connections between
temperament, delinquency, sociopathy, and criminal behavior, Eysenck and his colleagues devised the "General
Arousal Theory of Criminality" (summarized in Eysenck
& Gudjonsson 1989), according to which the common
biological condition underlying all of these behavioral
predispositions is the inheritance of a nervous system that
is relatively insensitive to low levels of stimulation. Individuals with such a physiotype, it is argued, will be
extraverted, impulsive, and sensation seeking, because
under conditions of relatively low stimulation they find
themselves at a suboptimal level of arousal; to increase
their arousal, many will participate in high-risk activities
such as crime (see also Farley 1986 and Gove & Wilmoth
1990). In general support of this model, Ellis (1987)
performed a meta-analysis which found that both criminality and sociopathy were associated with a variety of
indicators of suboptimal arousal, including childhood
hyperactivity, recreational drug use, risk taking, failure to
persist on tasks, and preference for wide-ranging sexual
activity.
Additional confirmation of the arousal model comes
from Zuckerman, who found a similar pattern of behaviors associated with his measure of sensation seeking.
(The following summary is derived from Daitzman &
Zuckerman 1980; Zuckerman 1979; 1983; 1984; 1985;
1990; 1991; and Zuckerman et al. 1980). In addition to
seeking thrill and novelty, sensation seekers describe "a
hedonistic pursuit of pleasure through extraverted activities including social drinking, parties, sex, and gambling," "an aversion to routine activities or work and to
dull and boring people," and "a restlessness in an unchanging environment" (Zuckerman et al. 1980, p. 189).
In college students, sensation seeking is correlated with
the Pd (Psychopathic Deviate) scale of the Minnesota
Multiphasic Personality Inventory; among prisoners it
can be used to distinguish primary psychopaths from
secondary psychopaths and nonpsychopathic criminals
(see also Fagan & Lira 1980). Zuckerman also shows that
sensation seeking as a temperament appears at an early
age (3-4 years), exhibits a high degree of heritability,
correlates negatively with age in adults, and exhibits sex
differences, with higher scores more often in males.
Because it shows a relationship with both sex and age,
sensation seeking (and its presumed underlying hypoarousal) may also be a good candidate for a trait that can
explain the distribution and expression of sociopathy (see
also Baldwin 1990).
Gray (1982; 1987), and Cloninger (Cloninger 1987a;
Cloninger et al. 1993) have proposed updated versions of
the Eysenck model in which the three personality factors
are rotated and renamed to more clearly correspond to
known neural circuitry. Gray names the three systems:
the approach or behavioral activation system, the behavioral inhibition system, and the fight/flight system; Cloninger names them "novelty-seeking," "harmavoidance," and "reward-dependence." The three factors
explain the same variance in personality as Eysenck's
original factors and have been shown to be independent
and highly heritable (Cloninger 1987a). In addition to
mapping more closely to known neural systems, these
three factors are also proposed to correspond to differential activity of three neurochemicals: dopamine for behavioral activation (or novelty seeking), serotonin for behavioral inhibition (or harm avoidance), and norepinephrine
for fight/flight (or reward dependence); see Charney et al.
1990; Cloninger 1987a; Depue & Spoont 1986; Eysenck
1990; and Raine 1993 for partial reviews.
2.3.3. The role of physiology. Using Cloninger's terminology, sociopaths are individuals who are high on novelty
seeking, low on harm avoidance, and low on reward
dependence. Thus, we should expect them to be high on
measures of dopamine activity, low on measures of serotonin activity, and low on measures of norepinephrine
activity; data suggest that they are.
Zuckerman (1989) reports that sensation seeking is
negatively correlated with levels of dopamine-betahydroxylase (DBH), the enzyme that breaks down dopamine, and that extremely low levels of DBH are associated with undersocialized conduct disorder and
psychopathy. With respect to the two pathway model,
boys with socialized conduct disorder (those with fewer,
later-appearing symptoms and who are posited to be at
risk for secondary, as opposed to primary, sociopathy) had
high levels of DBH.
In addition, extraverts and delinquents are reported to
have lower than average levels of adrenaline (epinephrine) and norepinephrine under baseline circumstances;
Magnusson (1985, as cited by Zuckerman 1989) reports
that urinary epinephrine measures of boys at age 13
significantly predicted criminality at ages 18-25. High
sensation seekers, criminals, and other individuals scoring high on measures of impulsivity and aggression also
have significantly lower levels than others of the serotonin
metabolite, 5-HIAA (Brown et al. 1982; Brown et al. 1979;
Depue & Spoont 1986; Kruesi et al. 1992; Muhlbauer
1985; Raine 1993; Zuckerman 1989; 1990). These are not
small effects: Raine (1993) reports an average effect size
(the difference between groups divided by the standard
deviation) for serotonin of 0.75, and for norepinephrine of
0.41; Brown et al. (1979) reported that 80% of the variance
in aggression scores of their sample was explained by
levels of 5-HIAA alone; Kruesi et al. reported that knowing 5-HIAA levels increased the explained variance of
aggression at a two yearfollow up from 65% (using clinical
measures only) to 91% (clinical measures plus 5-HIAA
measures).
Levels of monoamine oxidase (MAO) - an enzyme that
breaks down the neurotransmitters serotonin, dopamine,
epinephrine, and norepinephrine - are also low in antisocial and sensation-seeking individuals (Ellis 1991b;
Zuckerman 1989). Individual differences in platelet MAO
appear shortly after birth and are stable (Raine 1993;
Zuckerman 1989; 1990); Zuckerman reports an estimated
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
531
Mealey: The sociobiology of sociopathy
heritability of 0.86. Recently, a mutant version of the gene
coding for MAO-A, the version of MAO specific to serotonin, has been identified in an extended family in which
the males show a history of repeated, unexplained outbursts of aggressive behavior (Brunner et al. 1993; Morell
1993); urinalysis indicated that the MAO-A is not functioning normally in the affected men.
Results of psychophysiological studies also report significant differences between sociopaths and others. Reviews of this literature can be found in Eysenck & Gudjonsson (1989), Mednick et al. (1987), Raine (1989), Raine
(1993), Raine & Dunkin (1990), Trasler (1987), and
Zuckerman (1990). Among the findings are: high sensation seekers and sociopaths are more likely than lows and
normals to show orienting responses to novel stimuli of
moderate intensity, whereas lows and normals are more
likely to show defensive or startle responses; criminals
and delinquents tend to exhibit a slower alpha (resting)
frequency in their electroencephalogram (EEG) than
age-matched controls; high sensation seekers and delinquents differ from lows and nondelinquents in the amplitude and shape of cortical evoked potentials; extraverts
and sociopaths show less physiological arousal than introverts and normals in response to threats of pain or punishment and more tolerance of actual pain or punishment;
and delinquents (though not necessarily adult criminals)
tend to have a lower baseline heart rate than nondelinquents.
The importance of the role of these psychophysiological
factors as significant causes, not just correlates, of sociopathy is strengthened by evidence that (a) these measures of
autonomic reactivity are just as heritable as the temperament with which they are associated (Gabbay 1992;
Zuckerman 1989), and that (b) the same physiological
variables that differentiate identified sociopaths, delinquents, and criminals from others can also significantly
predict later levels of antisocial behavior in unselected
individuals (Loeb & Mednick 1977 using skin conductance; Raine et al. 1990a using EEG, heart rate, and skin
conductance; Raine et al. 1990b using evoked potentials;
Satterfield 1987 using EEG; and Volavka et al. 1984 using
EEG). As for the reports on neurochemistry, these effects
are not small; Raine (1993) reports that for heart rate, the
average effect size across ten studies was 0.84.
Another important physiological variable in the distribution of sociopathic behavior is testosterone. Testosterone (or one of its derivatives) is a likely trigger for
the sex-limited activation of genes required by the twothreshold model presented earlier. The mechanism of
action of steroid hormones is to enter the nucleus of the
cell and interact with the chromosomes, regulating gene
expression. This differential activity of the genes leads to
some of the individual, age, and sex differences we see in
temperament, specifically, psychoticism, aggression, impulsivity, sensation-seeking, nurturance, and empathy
(Ellis 1991b; Zuckerman 1984; 1985; 1991; Zuckerman et
al. 1980). Variation in testosterone levels also parallels the
age variation in the expression of sociopathic behavior and
is correlated with such behavior in adolescent and adult
males (Archer 1991; Dabbs & Morris 1990; Daitzman &
Zuckerman 1980; Ellis & Coontz 1990; Gladue 1991;
Olweus 1986; 1987; Rubin 1987; Schalling 1987; Susman
et al. 1987; Udry 1990; Zuckerman 1985). Testosterone is
thus likely to play a dual role in the development of
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
sociopathy, just as it does in the development of other sex
differences: one as an organizer (affecting traits) and one
as an activator (affecting states).
Udry (Drigotas & Udry 1993; Halpern et al. 1993),
unable to replicate his own 1990 study suggesting an
activating effect of testosterone, has suggested that the
correlation between testosterone and aggression might
be due to a physiosocial feedback loop; he posits that boys
with high, early levels of testosterone mature faster and,
being bigger, are more likely to get into fights. Since
levels of testosterone, adrenaline, and serotonin have
been shown to fluctuate in response to social conditions
(Archer 1991; Kalat 1992; McGuire et al. 1983; Olweus
1987; Raleigh et al. 1991; Raleigh et al. 1984; Schalling
1987), this sociophysiological interaction creates a positive feedback loop: those who start out with high levels of
testosterone and sensation seeking (and low levels of
adrenaline, serotonin, and MAO) are (1) more likely than
others to initiate aggressive behavior and (2) more likely
to experience success in dominance interactions, leading
to (3) a greater probability of experiencing further increases in testosterone, which (4) further increases the
likelihood of continued aggressive behavior.
Another example of a sociophysiological feedback loop
comes from Dabbs and Morris (1990), who found significant correlations between testosterone levels and antisocial behavior in lower class men but not in upper class
men. They explained this by positing that upper class
men are more likely, because of differential socialization,
to avoid individual confrontations. If this is true, it would
mean that upper class men are, because of their socialization, specifically avoiding the types of social encounters
that might raise their testosterone (and, in turn, their
antisocial behavior). This interpretation is supported by
the finding (in the same study) that significantly fewer
upper class than lower class men had high testosterone
levels. Thus, it is possible that upper class socialization
may mitigate the influence of testosterone. An alternative explanation - that the aggressive behavior associated
with higher testosterone levels leads to downward social
mobility - also suggests a recursive sociophysiological
interaction.
Raine (1988) has argued that since upper class children
are less likely than lower class children to suffer the
environmental risks predisposing one toward sociopathic
behavior, when such behavior is seen in upper class
individuals, it is likely to be the result of a particularly
strong genetic predisposition. Evidence supporting this
has been reported by three independent studies. Wadsworth (1976) found physiological indicators of hypoarousal among upper-class, but not lower-class, boys who
subsequently became delinquent. Raine (Raine 1988;
Raine & Dunkin 1990; Raine & Venables 1981; 1984)
found indicators of hypoarousal in his upper-class antisocial subjects, but the reverse in his lower-class subjects.
Satterfeld (1987) found that of his lower-class subjects,
those in a biological high-risk group were seven times
more likely to have been arrested than those in his control
group, whereas among his middle- and upper-class subjects, the rate was 25 and 28 times, respectively. This
outcome was a result of lower rates of criminal activity in
the control groups of the middle- and upper-class subjects
as compared to the lower-class controls; that is, almost all
of those who had been arrested from the middle and
Mealey: The sociobiology of sociopathy
upper class were biologically at high risk, but this was not
true for the lower class subjects. The implications of these
findings are of tremendous import, as they suggest that
(1) the effect of the social environment might be considerably larger than suggested by adoption studies and
(2) there might be different etiological pathways to sociopathy and therefore different optimal strategies for its
prevention or remediation, depending upon what kind of
social and environmental background the individual has
experienced.
Adoption studies show that the environment clearly plays
an important role in the etiology of sociopathy, but that its
effects are different for individuals of different genotypes.
As mentioned in section 2.3.1, some of this difference is
likely to be a result of gene-environment correlations, in
that different environments are sought by individuals of
different genotypes; some will be a result of differences in
the interpretation of the same environment by individuals of different genotypes; and some will be a result of
differences in environment impinging upon people because of differences in their genotype (e.g. discriminating
parental treatment of two children differing in temperament). In nonadoptive families, gene-environment correlations will be even stronger because parents with
certain personality types will provide certain environments for their children. These differential effects of
environment on individuals of varying genetic risk for
sociopathy become readily apparent when we examine
the effect of the interaction between physiotype and
conditioning on the process of socialization.
result of their behavior or an indirect result such as
parental punishment.
Despite continuing problems with operational definitions, recent research suggests that there might be distinguishable differences in learning between primary and
secondary sociopaths, or children with unsocialized
versus socialized conduct disorder (Gray 1987; Newman
et al. 1992; Newman et al. 1985; Quay 1990b). Primary
sociopaths, with their inability to experience the social
emotions, exhibit deficits on tasks which typically induce
anxiety in others, specifically, passive avoidance tasks,
approach-avoidance tasks, and tasks involving punishment, but they can learn well under other conditions
(Newman et al. 1992; Patterson & Newman 1993; Raine
1993; Raine et al. 1990b). Secondary sociopaths and
extraverts, on the other hand, have normal levels of
anxiety and responses to punishment, but they may be
especially driven by high reward conditions (Boddy et al.
1986; Derryberry 1987; Newman et al. 1990).
Primary sociopaths, with diminished ability to experience anxiety and to form conditioned associations between antisocial behavior and the consequent punishment, will be unable to progress through the normal
stages of moral development. Unlike most children who
are biologically prepared to learn empathy, they are
contraprepared to do so, and will remain egoistic unable to acquire the social emotions of empathy, shame,
guilt, and love. They present at an early age with "unsocialized" conduct disorder. Secondary sociopaths, with
normal emotional capacities, will present, generally at a
later age, with "socialized" conduct disorder (Loeber
1993; Patterson 1993; Simons 1993). What socialization
processes contribute to their development?
2.4.1. Conditioning. There is evidence that individuals
with a hypoaroused nervous system are less sensitive than
most people to the emotional expression of other individuals and to social influences in general (Eliasz & Reykowski 1986, Eysenck 1967 as cited in Patterson and
Newman 1993). They are also less responsive to levels and
types of stimuli that are normally used for reinforcement
and punishment (Eliasz 1987); as a result, they are handicapped in learning through autonomic conditioning although they exhibit no general intellectual deficit (e.g.,
Eysenck 1977; Gorenstein & Newman 1980; Hare &
Quinn 1971; Lytton 1990; Mednick 1977; Newman et al.
1985; Raine 1988; Ziskind et al. 1978; Zuckerman 1991).
One of the posited consequences of this learning deficit
is a reduced ability to be socialized by the standard
techniques of reward and punishment that are used (especially in the lower classes and by uneducated parents) on
young children. In particular, hypoaroused individuals
have difficulty inhibiting their behavior when both reward and punishment are possible outcomes (Newman
1987; Newman & Kosson 1986; Newman et al. 1985;
Patterson & Newman 1993; Zuckerman 1991); in situations when most people would experience an approachavoidance conflict, sociopaths and extraverts are more
likely to approach (see also Dienstbier 1984). Because of
their high levels of sensation seeking, children with a
hypoaroused nervous system will be more likely than
other children to get into trouble and when they do, they
will be less likely to be affected by, and learn from, the
consequences, whether those consequences are a direct
2.4.2. Social learning. In sect. 2.2.1, it was noted that a
cheating strategy is predicted to develop when a male
(especially) is competitively disadvantaged, and that
criminal behavior (especially in males) is clearly related to
factors associated with disadvantage. These factors are:
large numbers of siblings, low socioeconomic status, urban residency, low intelligence, and poor social skills.
How, in a proximate sense, do these variables contribute
to the development of secondary sociopathy? Path models
suggest a two-stage process involving a variety of cumulative risk factors (Dishion et al. 1991; Loeber 1993;
McGarvey et al. 1981; Moffitt 1993; Patterson et al. 1991;
Simons 1993; Snyder et al. 1986; Snyder & Patterson
1990).10
In the first stage, disrupted family life, associated with
parental neglect, abuse, inconsistent discipline, and the
use of punishment as opposed to rewards, are critical
(Conger 1993; Feldman 1977; Luntz & Widom 1993;
McCord 1986; Patterson et al. 1989; Simons 1993; Snyder
et al. 1986; Wilson & Herrnstein 1985). Poor parenting
provides the child with inconsistent feedback and poor
models of prosocial behavior, handicapping the child in
the development of appropriate social, emotional, and
problem-solving skills. This pattern is found most frequently in parents who are themselves criminal, mentally
disturbed, undereducated, of low intelligence, or socioeconomically deprived (Farrington 1986; McCord 1986;
McGarvey et al. 1981), leading to a cross-generational
cycle of increasing family dysfunction (e.g., Jaffe et al.
1992; Luntz & Widom 1993).
2.4. Learning theory
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
533
Mealey: The sociobiology of sociopathy
In the second stage, children with poor social skills find
themselves at a disadvantage in interactions with age
mates; rejected by the popular children, they consort
with one another (Dishion et al. 1991; Hartup 1989;
Kandel et al. 1988; Loeber & Dishion 1983; Patterson et
al. 1989; Snyder et al. 1986). In these socially unskilled
peer groups, which will also include primary sociopathic
or unsocialized conduct disorder children, delinquent,
antisocial behavior is reinforced and new (antisocial) skills
are learned (Maccoby 1986; Moffitt 1993). Antisocial behavior may then escalate in response to, or as prerequisite
for, social rewards provided by the group, or as an attempt
to obtain the perceived social (and tangible) rewards
which often accompany such behavior (Moffitt 1993). As
the focus of the socialization process moves outside the
home, parental monitoring becomes more important
(Conger 1993; Dishion etal. 1991; Forgatch 1991; Simons
1993; Snyder et al. 1986; Snyder & Patterson 1990), as
does the availability of prosocial alternatives for the socially unskilled adolescent (Apter 1992; Farrington 1986;
Moffitt 1993).
The development of secondary sociopathy appears to
depend much more on environmental contributions than
does primary sociopathy. Since it is secondary sociopathy
which, presumably, has increased so rapidly and so recently in our culture, what can social psychologists contribute to our understanding of the sociocultural factors
involved in its development?
2.5. Social psychology
2.5.1. Machiavellianism. First, the use of antisocial strategies is not restricted to sociopaths. The majority of people
who are arrested are not sociopathic and many people
exhibit antisocial behavior that is infrequent enough or
inoffensive enough to preclude arrest. Some antisocial
behavior is even considered acceptable if it is expressed in
socially approved circumstances. Person (1986), for example, relates entrepreneurism to psychopathy, whereas
Christie (1970) notes that people who seek to control and
manipulate others often become lawyers, psychiatrists, or
behavioral scientists; Jenner (1980) also claims that "subtle, cynical selfishness with a veneer of social skills is
common among scientists" (p. 128).
Christie (see Christie & Geis 1970) developed a scale
for measuring this subclinical variation in antisocial personality; he called it the "Machiavellianism" or "Mach"
scale. One's Mach score is calculated by compiling answers to Likert-format queries of agreement or disagreement with statements like "humility not only is of no
service but is actually harmful," "nature has so created
men that they desire everything but are unable to attain
it," and "the most important thing in life is winning."
Adults who score high on the Mach scale express "a
relative lack of affect in interpersonal relationships," "a
lack of concern with conventional morality," "a lack of
gross psychopathology," and "low ideological commitment" (Christie & Geis 1970, pp. 3-4); children who score
high on Machiavellianism have lower levels of empathy
than their age mates (Barnett & Thompson 1984).
High Machs have an "instrumental cognitive attitude
toward others" (Christie & Geis 1970, p. 277) and, because they are goal oriented as opposed to person oriented, they are more successful in face-to-face bargaining
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situations than low Machs. High Machs "are especially
able communicators, regardless of the veracity of their
message" (Kraut & Price 1976). In a related vein, high
Machs, like sociopaths, are more resistant to confession
after cheating than are low Machs, and they are rated as
being more plausible liars (Bradley & Klohn 1987;
Christie & Geis 1970); like sociopaths, high Machs are
often referred to as "cool." According to Christie, "If
Machiavellianism has any behavioral definition. . . selfinitiated manipulation of others should be at its core"
(p. 76). One can thus easily think of Machiavellianism as a
low-level manifestation of sociopathy. It even shows a sex
difference consistent with the two threshold model
(Christie & Geis 1970), an age pattern consistent with age
variation in testosterone levels (Christie & Geis 1970),
significant positive correlations with Eysenck's psychoticism and neuroticism scales (Allsopp et al. 1991), and a
correlation with serotonin levels (Madsen 1985).
In one study, Geis & Levy (1970) found that high Machs
(who were thought to use an "impersonal, cognitive,
rational, cool" approach with others), were much more
accurate than low Machs (who were thought to use a
"more personal, empathizing" approach) at assessing how
other "target" individuals answered a Machiavellian attitudes questionnaire. Even more interesting is the result
(from the same study) that the high Machs achieved their
accuracy by using a nomothetic or actuarial strategy: they
guessed that everyone was at about the average level,
without discriminating between individuals based on differences they had had an opportunity to observe during a
previous experimental session. In addition, their errors
tended to be random, which would fit with reports by
Eliasz & Reykowski (1986) and Damasio et al. (1990), who
found that hypoaroused and antisocial individuals are less
attentive to social and emotional cues than others. Low
Machs, on the other hand, used an idiographic approach,
and although they successfully differentiated between
high scorers and low scorers, they grossly underestimated
the scores of both, guessing at a level that was more
reflective of their own scores than those of the population
at large.
This study suggests two things: (1) Basing one's playing
strategies on an "impersonal, cognitive, rational, cool"
approach to others might be more accurate in the long run
than using a "personal, empathizing" approach (at least in
those situations where cooperative long-term partnerships are not possible) and (2) the errors made by those
who use the personal, empathizing approach are of the
kind more likely to result in playing the cooperation
strategy when the cheating strategy would be more appropriate (rather than vice versa). Thus, the personal,
empathizing approach is likely to make one susceptible to
being exploited by others who use the impersonal cognitive approach; indeed, high Machs outcompete low
Machs in most experimental competitive situations
(Christie & Geis 1970; Terhune 1970).
As I have argued elsewhere (Mealey 1992), the common assumption that an empathy-based approach to predicting the behavior of others is better than a statistical
approach is not necessarily correct; this belief may itself
be an emotion-based cognitive bias. To have such a bias
may be beneficial, however, for the same reason that
emotional commitment biases are beneficial: in situations
where voluntary, long-term coalitions can be formed, the
Mealey: The sociobiology of sociopathy
personal, empathizing (and idealistic) low Machs might
outperform the more impersonal, cognitive (and realistic)
high Machs, since low Machs would be more successful
than high Machs in selecting a cooperator as a partner.
Although two studies (Hare & Craigen 1974 and
Widom 1976a) report on the strategy of sociopaths in
Prisoner's Dilemma-type settings, in both studies the
sociopaths were paired with one another; thus, we do not
have a measure of the strategy sociopaths use against
partners of their own choosing or in situations with random, rotating partners. 11 I would predict that in such
settings, sociopaths, like Geis & Levy's high Mach subjects, would be less proficient than others in distinguishing between high and low Mach partners, and would thus
be at a disadvantage in iterated games with a chosen
partner; on the other hand (again like high Mach subjects), they should perform at better than average levels
when playing with randomly assigned, rotating partners.
Widom (1976b) found that when asked to guess how
"people in general" would feel about different social
situations sociopaths guessed that others would feel essentially the same way that they do, whereas control
subjects guessed that others would feel differently. As in
the Geis & Levy study, both groups were wrong, but in
different ways: the sociopaths underestimated their differences from others, whereas the control subjects substantially overestimated their differences from others,
suggesting that sociopaths (like high Machs) were using a
nomothetic approach to prediction, whereas controls (like
low Machs) were using an idiographic approach.
Machiavellianism and the related propensity to use
others in social encounters has generally been looked
upon as a trait. An alternative perspective, however,
acknowledges both the underlying variation in personality and the situational factors that are relevant to an
individual's behavior at any given moment (e.g., Barber
1992). In line with mechanism 5 for maintaining ESSs
(presented in sect. 1.2), Terhune (1970) says "actors bring
to the situation propensities to act in a certain general
way, and within the situation their propensities interact
with situational characteristics to determine their specific
behavior" (p. 229).12 This brings us to the last question:
beyond the constitutional and environmental variables
that contribute to the development of individual differences in personality and antisocial behavior, what can
social psychology tell us about the within-individual situational factors that encourage or discourage cheating strategies, and how can these be explained?
2.5.2. The role of mood. Although mood and emotion are
not identical concepts, they are clearly related. 13 Mood
might be thought of as a relative of emotion which clearly
varies within individuals but is perhaps less an immediate
response to concrete events and stimuli and more a
generalized, short- to mid-term response to the environment. As such, the role of mood must be addressed by any
model that relies so heavily on the concepts of emotion,
emotionality, and emotionlessness, as determinants of
behavior.
Positive mood and feelings of success have been demonstrated to enhance cooperative behavior (Cialdini et al.
1982; Farrington 1982; Mussen & Eisenberg-Berg 1977).
If, as Nesse (1991) has argued, positive mood is a reflection not only of past success, but also of anticipation of
future success, the facilitation of cooperation by positive
mood could be seen as part of a long-term strategy by
individuals who feel they can afford to pass up possible
short-term gains for the sake of establishing a cooperative
reputation.
Sad affect and feelings of failure can also affect strategy
in social interactions. To the extent that sadness and
feelings of failure follow losses of various sorts, individuals
in these circumstances should be expected to be egoistic
and selfish. In children, this is typically what is found
(Baumann et al. 1981; Mussen & Eisenberg-Berg 1977).
In some children, and more consistently in adults, on the
other hand, sadness and feelings of failure can facilitate
prosocial behavior. Mussen & Eisenberg-Berg (1977) suggest that this is a result of a deliberate effort: to enhance
one's (diminished) reputation among others; Baumann et
al. (1981) and Cialdini et al. (1982) suggest that it is a result
of a deliberate effort to relieve negative affect based on
prior experience that prosocial behavior often has a positive, self-gratifying effect.
If sadness is profound, that is, if one is depressed and
experiencing the cognitive biases and selective attention
associated with depression (Mineka & Sutton 1992; Nesse
1991; Sloman 1992), one would be expected to desist from
all social interaction, being neither antisocial nor prosocial, but asocial (Nesse 1991; Sloman 1992). In this view,
the lethargy and anhedonia associated with depression
could be considered to be facultative lapses in the emotions or moods which typically motivate a person toward
social interaction.
Hostility can also lead to cognitive biases and selective
attention to relevant social stimuli. Dodge and Newman
(1981) show that aggressiveness in boys is associated with
the over-attribution of hostile intent to others. The authors conclude that such attributions lead to increased
"retaliatory" aggression by the hostile individuals, fueling
a cycle of true hostility and retaliation by all parties. It is
also abundantly clear that anger and hostility, once expressed, do not lead to catharsis, but to amplified feelings
and outward expressions of that anger (Tavris 1982).
Guilt, which often follows selfish behavior, typically
results in an increase in subsequent prosocial behavior
(Cialdini et al. 1982; Hoffman 1982); Hoffman calls this
"reparative altruism." Guilt can easily be seen as one of
Hirshleifer's (1987) or Frank's (1988) emotional commitment devices, compelling one to perform prosocial behavior as a means of reestablishing one's tarnished
reputation.
Cialdini et al. (1982) also report that prosocial behavior
increases after observing another's transgression. They
explain this phenomenon within the context of what they
call the "negative relief model: prosocial behavior is
performed as a means of alleviating negative feelings in
general (including direct or vicarious guilt, sympathy,
distress, anxiety, or depression). Like Hoffman's, this
model postulates that the reinforcing power of (relief
provided by) prosocial behavior is learned during
childhood.
Since guilt, anxiety, and sympathy are social emotions
that primary sociopaths rarely, if ever, experience, there
is no reason to expect that they might moderate their
behavior so as to avoid them. On the other hand, there is
no reason to expect that sociopaths do not experience
fluctuations in mood (such as depression, optimism, or
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
535
Mealey: The sociobiology of sociopathy
anger) in response to their changing evaluation of their
prospects of success and failure. To the extent that we can
manipulate the sociopath's mood, therefore, we might be
able to influence his behavior.
2.5.3. Cultural variables. Competition, in addition to being one of the most important variables in determining
long-term life strategy choices, is also one of the more
important situational variables influencing the choice of
immediate strategy. Competition increases the use of
antisocial and Machiavellian strategies (Christie & Geis
1970) and can counteract the increase in prosocial behavior that generally results from feelings of success (Mussen
& Eisenberg-Berg 1977). Some cultures encourage competitiveness more than others (Mussen & EisenbergBerg 1977; Shweder et al. 1987) and these differences in
social values vary both temporally and crossculturally.
Across both dimensions, high levels of competitiveness
are associated with high crime rates (Farley 1986; Wilson
& Herrnstein 1985) and Machiavellianism (Christie &
Geis 1970).
High population density, an indirect form of competition, is also associated with reduced prosocial behavior
(Farrington 1982) and increased antisocial behavior (Ellis
1988; Robins et al. 1991; U.S. Department of Justice
1993; Wilson & Herrnstein 1985) - especially in males
(Wachs 1992; see sect. 3.2.1 and references therein for
ultimate, game theoretic explanations why this might
occur; see Draper 1978; Foster 1991; Gold 1987; Siegel
1986; and Wilson & Daly 1993 for a variety of proximate
explanations). Fry (1988) reports large differences in the
frequency of prosocial and antisocial behaviors in two
Zapotec settlements equated for a variety of socioecological variables; the one major difference - thought
perhaps to be causal - was in land holdings per capita,
with the higher levels of aggression found in the community with the smaller per capita land holdings.
Last, but not least, is the relatedness or similarity of the
interactors to their partners in an interaction. Based on
models of kin selection and inclusive fitness, individuals
should be more cooperative and less deceptive when
interacting with relatives who share their genes, or relatives who share investment in common descendents.
Segal (1991) reported that identical twins cooperated
more than fraternal twins in playing the Prisoner's Dilemma. Barber (1992) reported that responses on an
altruism questionnaire were more altruistic when the
questions were phrased so as to refer to relatives (as
opposed to "people" in general), and that Machiavellian
responses were thereby reduced. Rushton (1989; Rushton et al. 1984) presents evidence that people also cooperate more with others who are similar to them even though
not genetically related. There are a variety of plausible
evolutionary explanations for this behavior (see Mealey
1984; Pulliam 1982; and BBS commentary on Rushton
1989).
3. Integration, implications, and conclusions
3.1. Integration: Sociopathy as an ESS leads to two
types of sociopaths
3.1.1. Primary sociopathy. I have thus far argued that
some individuals seem to have a genotype that disposes
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
them "to acquire and be reinforced for displaying antisociality" (Rowe 1990a, p. 122). The genotype results in a
certain inborn temperament or personality, coupled with
a particular pattern of autonomic hypoarousal that, together, design the child to be selectively unresponsive to
the cues necessary for normal socialization and moral
development. This scenario is descriptive of mechanism 1
(sect. 1.2) of maintaining ESSs in the population; it
describes the existence of frequency-dependent, genetically based individual differences in employment of life
strategies. I accordingly suggest that there will always be
a small, cross-culturally similar, and unchanging baseline
frequency of sociopaths: a certain percentage of sociopaths - those individuals to whom I have referred as
primary sociopaths - will always appear in every culture,
no matter what the sociocultural conditions. Those individuals will display chronic, pathologically emotionless,
antisocial behavior throughout most of their lifespan and
across a variety of situations, a phenotype that is recognized (according to Robins et al. 1991, p. 259) "by every
society, no matter what its economic system, and in all
eras".14 Since it is a genetically determined strategy,
primary sociopaths should be equally likely to come from
all kinds of socioeconomic backgrounds; on the other
hand, since they constitute that small group of individuals
whose physiotype makes them essentially impervious to
the social environment almost all sociopaths from the
upper classes will be primary sociopaths.15
Of course, because they are not intellectually handicapped, these individuals will progress normally in terms
of cognitive development and will acquire a theory of
mind. Theirs, however, will be formulated purely in
instrumental terms, without access to the empathic understanding that most of us rely on so much of the time.
They may become excellent predictors of others' behavior, unhandicapped by the vagaries and "intrusiveness" of
emotion, acting, as do professional gamblers, solely on
nomothetic laws and actuarial data rather than on hunches
and feelings. In determining how to "play" in the social
encounters of everyday life, they will use a pure costbenefit approach based on immediate personal outcomes,
with no "accounting" for the emotional reactions of the
others with whom they are dealing. Without love to
"commit" them to cooperation, anxiety to prevent "defection, " or guilt to inspire repentance, they will remain free
to continually play for the short-term benefit in the
Prisoner's Dilemma.
3.1.2. Secondary sociopathy. At the same time, because
changes in gene frequencies in the population would not
be able to keep pace with the fast-changing parameters of
social interactions, an additional, fluctuating proportion
of sociopathy should be a result of mechanism 5 for
maintaining ESSs, which allows for more flexibility in
the ability of the population to track the frequencydependent nature of the success of the cheating strategy.
Mechanism 5 (genetically based individual differences in
response to the environment, resulting in differential use
by individuals of environmentally contingent strategies)
would explain the development and distribution of what I
have referred to as secondary sociopathy. Secondary sociopathy is expressed by individuals who are not extreme
on the genetic sociopathy spectrum, but who, because of
their exposure to environmental risk factors, pursue a life
Mealey: The sociobiology of sociopathy
strategy that involves frequent, but not necessarily emotionless cheating. Unlike primary sociopaths, secondary
sociopaths will not necessarily exhibit chronic antisocial
behavior, because their strategy choices will be more
closely tied to age, fluctuation in hormone levels, their
competitive status within their referent group, and
changing environmental contingencies. Since secondary
sociopathy is more closely tied to environmental factors
than to genetic factors, secondary sociopaths will almost
always come from lower class backgrounds and their
numbers could vary substantially across cultures and
time, tracking environmental conditions that favor or
disfavor the use of cheating strategies.
The existence of this second etiological pathway to
sociopathy explains the fact that cultural differences are
correlated with differences in the overall incidence of
antisocial behavior (Ellis 1988; Farley 1986; Gold 1987;
Robins et al. 1991; Wilson & Hermstein 1985). It also
explains why, as the overall incidence of sociopathy increases, the discrepancy in the ratio of male to female
sociopaths decreases (Robins et al. 1991): since secondary
sociopathy is less heritable than primary sociopathy (according to this model), the effect of sex-limited genes (like
that of all the genes contributing to the spectrum) should
be less important for the development of secondary
sociopathy, resulting in less of a sex difference. Rased on
this model, I would predict that, unlike what we find for
primary sociopathy (see sect. 2.1.3), we should find no
differential heritability between the sexes for secondary
sociopathy (even though there will still be a sex difference
in prevalence).
3.2. Implications of the two-pathway model
Terliune (1970) suggests that choice of strategy in experimental game situations (and, presumably, real-life settings as well) depends upon two things: (1) cognitive
expectations regarding others (i.e., a theory of mind) and
(2) motivational/emotional elements such as hopes and
fears. Since primary sociopaths have a deficit in the realm
of emotional motivation, they presumably act primarily
upon their cognitive expectations of others; to the extent
that they do act upon emotions, it is most likely to be upon
mood and the primary emotions (like anger and fear)
rather than upon the social and secondary emotions (like
love and anxiety). Thus, the extent to which a society will
be able to diminish the antisocial behavior of primary
sociopaths will depend upon two things: (1) its influence
on the sociopath's cognitive evaluation of its own reputation as a player in the Prisoner's Dilemma, and (2) the
primary emotion- or mood-inducing capacity of the stimuli it utilizes in establishing the costs and benefits of
prosocial versus antisocial behavior.
Manipulations of these two variables will also influence
the numbers of secondary sociopaths by changing the size
of the adaptive niche associated with antisocial behavior.
In addition, since the development of secondary sociopathy is more influenced by the social environment than is
the development of primary sociopathy, and since secondary sociopaths are not devoid of social emotions, changing
patterns in the nurturing and socialization of children and
in the socialization and rehabilitation of delinquents and
adult criminals is an additional, viable possibility for
reducing the overall prevalence of antisocial behavior.
3.2.1. Minimizing the impact of primary sociopaths: Society as a player in the Prisoner's Dilemma. Sociopaths'
immediate decisions are based in part on their ability to
form a theory of mind, and to use those expectations of
others' behavior in a cost benefit analysis to assess what
actions are likely to be in their own self-interest. (This is
true for both primary and secondary sociopaths.) The
outcome of such analyses is therefore partially dependent
on the sociopath's expectations of the behavior of other
players in the game. I would argue that an entire society
can be seen as a player and that the past behavior of that
society will be used by the sociopath in forming the
equivalent of a theory of mind to predict the future
behavior of that society.
Like an individual player, a society will have a certain
probability of detecting deception, a more-or-less accurate memory of who has cheated in the past, and a certain
proclivity to retaliate or not, based upon a cheater's past
reputation and current behavior. Since the sociopath is
using a rational and actuarial approach to assess the costs
and benefits of different behaviors, it is the actual past
behavior of the society which will go into his calculations,
rather than risk assessments inflated from the exaggerated fears or anxieties that most people feel in anticipation
of being caught or punished. Thus, to reduce antisocial
behavior, a society must establish and enforce a reputation for high rates of detection of deception and identification of cheaters, and a willingness to retaliate. In
other words, it must establish a successful strategy of
deterrence.
Game theory models by Axelrod and others have shown
that the emergence, frequency, and stability of social
cooperation is subject to an abundance of potential deterrent factors (Axelrod 1984; Axelrod & Dion 1988; Axelrod
& Hamilton 1981; Boyd 1988; Boyd & Richerson 1992;
Dugatkin & Wilson 1991; Feldman & Thomas 1987;
Heckathorn 1988; Hirshleifer & Coll 1988; Nowak &
Sigmund 1993; Vila & Cohen 1993). Among these are:
group size (as it decreases, cooperation increases); nonrandom association of individuals within the population
(as it increases, cooperation increases); the probability of
error in memory or recognition of an individual (as it
decreases, cooperation increases); the effect of a loss on a
cooperator (as it decreases, cooperation increases); the
effect of a gain on a defector (as it decreases, cooperation
increases); the frequency of punishment against defectors
(as it increases, cooperation increases); the cost of punishment for the punished (as it increases, cooperation increases); and the cost of punishment for the punishers (as
it decreases, cooperation increases).16
Recent game-theoretic models are coming closer and
closer to the complexity of real-world, human social
interactions on a large scale by examining the role of
culture and technology in expanding society's collective
memory of individual players' past behavior, broadcasting
the costs and benefits of cooperation and defection, and
the development and application of new socialization,
deception-detection, and punishment techniques (see
especially Dugatkin 1992; Hirshleifer & Rasmusen 1989;
Machalek & Cohen 1991). These models begin to provide
useful strategies for the real-world prediction and reduction of cheating strategies and antisocial behavior. (See
also Axelrod 1986; Bartol 1984; Ellis 1990a; Eysenck &
Gudjonsson 1989; Farrington 1979; Feldman 1977; MaBEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
537
Mealey: The sociobiology of sociopathy
chalek & Cohen 1991; and Wilson & Herrnstein 1985 for
some nonquantitative models and tests that incorporate
some of these variables in their explanation of the socialization, punishment, and deterrence of crime.)
Since neither secondary nor primary sociopaths have a
deficit in the ability to perform accurate cost-benefit
analyses, increasing the probabilities of criminal detection, identification, and punishment can also reduce
crime; a society must therefore establish a reputation for
willingness to retaliate. (The National Research Council
[1993] reports that a 50% increase in the probability of
incarceration for any single crime reduces subsequent
crime twice as much as does doubling incarceration duration [p. 294]). Harsher penalties can also be deterring, but
only if they are reliably meted out.
Another key is in making the costs of cheating salient.
Generally speaking, antisocial and uncooperative behaviors increase as the costs become more diffuse or removed
in time, and prosocial and cooperative behaviors decrease
as the benefits become more diffuse or removed in time
(Bartol 1984; Low 1993; Ostrom 1990). For primary sociopaths, this is even more so, since their sensation-seeking
physiotype makes them particularly unable to make decisions based on nonimmediate consequences. Although
able to focus attention on interesting tasks for short
periods, the sociopath cannot perform well under conditions of delayed gratification (Pulkkinen 1986) and is more
motivated to avoid immediate costs than by threats or
avoidance of future punishments (Christie & Geis 1970;
Forth & Hare 1989; McCord 1983; Raine 1988; 1989).
Costs associated with social retaliation must therefore not
only be predictable, but swift, and the swiftness itself
must also be predictable.
Another factor the sociopath will use to "compute" the
potential value of an antisocial action is the cost-benefit
ratio of the alternatives (Piliavin et al. 1986). For the
sociopath, money and other immediate tangible rewards
are more motivating than social reinforcers (such as
praise) or promises of future payoff, and visual stimuli are
more salient than auditory stimuli (Chesno & Kilmann
1975; Forth & Hare 1989; Raine 1989; Raine & Venables
1987; Raine et al. 1990b; Zuckerman 1990). Thus, alternatives to crime must be stimulating enough and rewarding
enough to preferentially engage the chronically hypoaroused sensation seeker. This will be a difficult task to
achieve, but it will be more successful if we can effectively
distinguish primary from secondary sociopaths. Primary
sociopaths, with their emotional, but not intellectual
deficit, will be competent on some tasks on which secondary sociopaths, with deficits in social skills, emotion
regulation and problem solving, will not. Possibilities
might include: novelist, screenplay writer, stunt man,
talk show host, disk jockey, explorer, treasure hunter,
race car driver, or skydiving exhibitionist. Given that
primary sociopaths will always be with us in low numbers,
it would be a wise social investment to create - even on an
individual basis, if necessary - a number of exciting, highpayoff alternatives for them, in order to minimize the
number who may otherwise cause pain and destruction.
Distinguishing between primary and secondary sociopaths is also critical for decisions about confinement and
rehabilitation. Quinsey & Walker (1992) cite examples
where recidivism rates went up for psychopaths, but
down for nonpsychopaths, after they were exposed to the
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
same kind of "treatment." Recidivism is much greater in
primary sociopaths than in secondary sociopaths (Hare et
al. 1992), and sometimes the only response is prolonged
incapacitation (until they literally "grow out of it"). A
recent international meeting of experts concluded that
"treatment" programs dealing with primary sociopaths
should be "less concerned with attempts to develop empathy or conscience than with intensive efforts to convince them that their current attitudes and behavior
(simply) are not in their own self-interest" (Hare 1993,
p. 204).
3.2.2. Minimizing the prevalence of secondary sociopathy: Society as a socializing agent and mood setter.
Given that secondary sociopaths have a different life
history and are more responsive to environmental influences than primary sociopaths, social changes can be
designed to minimize not only their impact, but their
incidence. Loeber (1990) argues that each generation in
our society is being raised with an increasing number
of environmental risk factors, leading to increasing
generation-wide deficits in impulse control. He makes
specific suggestions to screen for high-risk children and
institute early intervention, noting that different interventions are likely to be more or less effective given
different risk factors in the child's or adolescent's life
history (see also U.S. Department of Justice 1993).
One possible intervention is parent training (see Magid
& McKelvie 1987 and Dumas et al. 1992 for reviews
and programmatic suggestions). Laboratory experiments
show that antisocial behaviors can be reduced and prosocial behaviors reinforced by appropriate use of modelling, induction, and behavioral modification techniques
(Feldman 1977; Gelfand & Hartmann 1982; Grusec 1982;
Kochanska 1991; 1992; Mussen & Eisenberg-Berg 1977;
Radke-Yarrow & Zahn-Waxler 1986; Rushton 1982). Recent longitudinal studies in natural settings suggest that
the positive effects of good parenting, especially parental
warmth and predictability, may be long lasting (Kochanska 1991; 1993; Kochanska & Murray 1992; McCord
1986).
The cause and effect relationship between parental
behavior and child behavior, however, is not likely to be
one-way. Children of different gender, temperaments,
and even social classes, respond differentially to different
socialization techniques (Dienstbier 1984; Kochanska
1991; 1993; Kochanska & Murray 1992; Lytton 1990;
McCord 1993; Radke-Yarrow & Zahn-Waxler 1986), and,
to some extent, difficult children elicit poor parenting
(Bell & Chapman 1986; Buss 1981; Eron et al. 1991; Lee
& Bates 1985; Lytton 1990; Snyder & Patterson 1990). It
is easy for parents of difficult children to lose heart, and in
so doing, become even less effective (Patterson 1992). For
example, studies cited in Landy & Peters (1992) found
that mothers of aggressive children, like other mothers
with a low sense of personal power, tend to give weak,
ineffectual commands to their children.
This lack of "goodness of fit" between parental style and
the needs of the child is probably an important factor in
the exacerbation of conduct disorder (Landy & Peters
1992; Lee & Bates 1985; Moffitt 1993; Wachs 1992).
Parents need help in identifying high-risk children, and
they need instruction in how to take a practical, assertive
approach with them (see Garmezy 1991; Magid &
Mealey: The sociobiology of sociopathy
McKelvie 1987), while using a more inductive, empathic
approach with their other children (see Kochanska 1991;
1993; Kochanska & Murray 1992).
Social workers, health care providers, and employees
of the criminal justice system also need to be able to
distinguish between children with different risk factors
and life histories and to respond accordingly. Palmer
(1983) argues that agents should be individually matched
with each client/offender based on style and personality
characteristics to prevent high Mach and sociopathic
offenders from taking advantage of low Mach employees.
At a broader level, many sociocultural aspects of modern society seem to contribute to antisocial behaviors and
attitudes (Moffitt 1993; National Research Council 1993).
As a society gets larger and more competitive, both
theoretical models (sect. 3.2.1) and empirical research
(sect. 2.4.2) show that individuals become more anonymous and more Machiavellian, leading to reductions in
altruism and increases in crime. Social stratification and
segregation can also lead to feelings of inferiority, pessimism, and depression among the less privileged, which
can in turn promote the use of alternative competitive strategies, including antisocial behavior (Magid &
McKelvey 1987; Sanchez 1986; Wilson & Daly 1993).
Crime may be one response to the acquisition of an
external locus of control (Raine et al. 1982) or learned
helplessness. Learned helplessness and other forms of
depression have been associated with reduced levels of
serotonin (Traskman et al. 1981); since reduced levels
of serotonin have also been shown to be related to increased aggression, it is likely that physiological changes
mediate these psychological and behavioral changes. The
neurochemical pathway involved in learned helplessness
(identified by Petty & Sherman 1982) appears to be the
same one identified by Gray (1982; 1987) and Cloninger
(1987a) with mediation of behavioral inhibition/harm
avoidance, and by Charney et al. (1990) with anxietymediated inhibition.
Crime may also function to obtain desirable resources,
increase an individual's status in a local referent group, or
provide the stimulation that the more privileged find in
more socially acceptable physical and intellectual challenges (e.g., Apter 1992; Farley 1986; Farrington 1986;
Lyng 1990; Moffitt 1993). According to Apter, "the vandal
is a failed creative artist," a bored and frustrated sensation
seeker who "does not have the intellectual or other skills
and capacities to amuse or occupy himself" (1992, p. 198).
Thus, in addition to making the costs of antisocial behavior greater, strong arguments can be made for providing
early social support for those at risk, and for developing
alternative, nonexploitative, sensation-seeking ventures
that can meet the psychological needs of disadvantaged
and low-skilled individuals.
3.3. Conclusions
A review of the literature in several areas supports the
concept of two pathways to sociopathy:
(la) "Primary sociopaths" are individuals of a certain
genotype, physiotype, and personality who are incapable
of experiencing the secondary, "social" emotions that
normally contribute to behavioral motivation and inhibition; they fill the ecological niche described by game
theorists as the "cheater strategy," and, as the result of
frequency-dependent selection, will be found in low frequency in every society.
(lb) To minimize the damage caused by primary sociopaths, the appropriate social response is to modify the
criminal justice system in ways that obviously reduce the
benefits and increase the costs of antisocial behavior,
while simultaneously creating alternatives to crime that
could satisfy the psychophysiological arousal needs of the
sociopath.
(2a) "Secondary sociopaths" are individuals who use
an environmentally contingent, facultative cheating strategy not as clearly tied to genotype; this strategy develops
in response to social and environmental conditions related to disadvantage in social competition and will thus
covary (across cultures, generations, and even within an
individual lifetime), with variation in immediate social
circumstances.
(2b) To reduce the frequency of secondary sociopathy,
the appropriate social response is to implement programs
that reduce social stratification, anonymity, and competition, intervene in high-risk settings with specialized parent education and support, and increase the availability of
rewarding, prosocial opportunities for at-risk youth.
Since the genetics and life histories of primary and
secondary sociopaths are so different, successful intervention will require differential treatment of different
cases; we thus need to encourage the widespread adoption of common terminology and diagnostic criteria.
ACKNOWLEDGMENTS
I would like to thank Mr. Rainer Link, who helped me get
started on this project, and who collaborated with me on the first
version and first public presentation of the model (Link &
Mealey 1992). I would also like to extend thanks to the many
individuals who provided useful comments during the revision
process: J. D. Baldwin, David Buss, Patricia Draper, Lee
Dugatkin, Lee Ellis, Hans Eysenck, David Farrington, Hill
Goldsmith, Henry Harpending, James Kalat, John Loehlin,
Michael McGuire, Randy Nesse, Jaak Panskepp, David Rowe,
Sandra Scarr, Nancy Segal, Chuck Watson, David S. Wilson,
and four anonymous BBS reviewers.
NOTES
1. As of January 1, 1996 Linda Mealey will be at the Dept. of
Psychology, University of Queensland, Brisbane, Australia
4702. Email: lmealey@psych.uq.edu
2. Plutchik's eight primary emotions are: anger, fear, sadness, disgust, surprise, joy, acceptance, and anticipation.
Others posit a few more (Izard 1977; 1991) or fewer (Ekman
1971; Panksepp 1982) but what is basically agreed is that primary
emotions are those which can be found in other mammals, are
hard-wired in the brain, are reflexively produced in response to
certain stimuli, are associated with certain, sometimes speciesspecific, physiological responses (e.g., piloerection, changes in
heart rate, facial expressions), and, in humans, are found crossculturally and at an early age (see Ortony & Turner 1990 for a
dissenting opinion).
Note that the "social emotions," including love, guilt, shame,
and remorse, do not meet the above criteria, and are not
considered to be primary emotions by most authors (see Izard
1991 for another perspective). Although distinctly human, the
social emotions seem to involve a critical element of learning, and, central to the argument I will be making, are not
panhuman.
3. According to Plutchik, cognitive processes themselves
evolved "in the service of emotions," "in order to make the
evaluations of stimulus events more correct and the predictions
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Mealey: The sociobiology of sociopathy
more precise so that the emotional behavior thatfinallyresulted
would be adaptively related to the stimulus event" (1980,
p. 303). This model of the relationship between emotion and
cognition is somewhat similar to Bigelow's (1972), which postulates that intelligence evolved as a result of the need to control
the emotions (especially the aggressive emotions), in the service
of sociality, and Humphrey's (1976; 1983), which claims that selfawareness evolved because it was a successful toolforpredicting
the behavior of others.
4. See Draper (1978) and the 1986 special issue of Ethology
and Sociobiology on ostracism for further discussion of the role
of shunning with specific reference to human societies; see
Hirshleifer & Rasmusen (1989) for a game theoretic model of
shunning; and see Nathanson (1992) for the importance of the
social emotion shame.
5. The wealth of literature on strategies that people use to
detect deception in interpersonal interactions, as well as the
technologies that have been developed in order to further
enhance that ability in less personal social interactions, are
indicators of the importance we bestow on such ability (see
Zuckerman et al. 1981, Mitchell & Thompson 1986, and especially Ekman 1992).
6. Although the data are overwhelming, the particular articles cited in this section should not be considered to be independent reports, since most of the reviews cited overlap substantially in their coverage and many authors or teams report their
findings more than once in a series of updates. Interested
readers should direct themselves to the most recent publications; however, older publications do contain some information
not presented in the updates and are thus included for thoroughness and ease of reference.
7. Twin study methods yield estimates of what is termed
"broad heritability," which includes both "additive" genetic
factors (i.e., the summed effect of individual genes on the
phenotype) and "non-additive" genetic factors (i.e., the phenotypic effects of dominance interactions between homologous
alleles on paired chromosomes, and the epistatic interactions
between nonhomologous genes throughout the genome). Adoption study methods, on the other hand, yield estimates of what is
termed "narrow heritability," which is only the additive genetic
component. The additive component is that which can be
selected for (or against) as it is transmitted from generation to
generation, whereas the nonadditive effect is unique to each
individual genotype and is broken and reshuffled with every
episode of sexual recombination. Because of this difference,
twin studies typically yield higher heritability estimates than
adoption studies. [See also Plomin & Daniels: "Why Are Children in the Same Family So Different from One Another?" BBS
10(1) 1987; Plomin & Bergeman: "The Nature of Nurture" BBS
14(3) 1991; and Wahlsten: "Insensitivity of the Analysis of
Variance to Heredity-Environment Interaction" BBS 13(1) 1990.]
Another difference between the twin methodology and the
adoption methodology is that twin studies generally provide
heritability coefficients which estimate the proportion of the
total explained variance accounted for by genetic factors,
whereas adoption studies provide heritability coefficients which
estimate the proportion of the total variance (including measurement error) that is accounted for by additive genetic factors.
Since measurement error is so large when assessing criminality,
adoption studies tend to yield both smaller and more varied
heritability estimates than do twin studies.
A third difference is that twin studies yield heritability estimates for members of a particular generational cohort, usually
tested at the same age, whereas adoption studies necessarily
regress measures from one generation to another. This leads to
two problems in interpreting heritability estimates derived
from adoption studies that are not germane to twin studies. The
first is that heritability can change across generations - even in
the absence of genetic change - due to changes in the environment; this effect cannot be assessed in either twin or adoption
540
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
studies, but is only a limitation of generalizability for the former,
whereas it is conflated in the latter. The second is that heritability can also be different at different ages. Huesmann et al.
(1984), for example, report that the correlation between children's aggression level and their parents' aggression level when
measured at the same age, is greater than the correlation
between the child's own aggression level at one age and at a later
age. This phenomenon also results simply in limited generalizability of heritability estimates derived from twin studies, but
yields conflated estimates from adoption studies.
The heritability of 0.6 reported herein is an estimate of broad
heritability as derived directly from twin studies; similar estimates can also be calculated indirectly from adoption study data
after accounting for measurement error, but cohort effects
cannot be separated out. See Loehlin (1992) for a general
discussion of twin and adoption methodologies and Emde et al.
(1992) and Raine (1993) for further discussions of the relevance
of methodological considerations as they pertain to interpretation of the specific studies summarized herein.
8. There is also evidence that at least one form of alcoholism
belongs to the sociopathy spectrum: Type II alcoholism, which is
also much more prevalent in men than women and seems to be
transmitted in the same way (Bohman et al. 1981; Cadoret 1986;
Cloninger et al. 1978; 1981; McGue et al. 1992; Stabenau 1985;
Zucker & Gomberg 1986). Type II alcoholism is characterized by
early onset, frequent violent outbursts, EEG abnormalities, and
several of the personality attributes that are often seen in sociopathy: impulsivity, extraversion, sensation seeking, aggressiveness, and lack of concern for others (Cloninger 1987b; Tarter 1988).
9. The interesting phenomenon of differential heritability of
traits across the sexes can occur, as in this case, as a result of
differential (sex-limited) expression of the same genes or, as it
does with Type I alcoholism (a milder, nonviolent form), as a
result of differential environmental experiences of the sexes
(Cloninger et al. 1978). Since heritability is measured as a
proportion, the value of a heritability estimate will change
whenever the numerator (variance in a trait due to genetic
variance) or the denominator (total variance in the trait
changes). Since the denominator (total variance) is composed of
both genetic and environmental variance, changes in either will
change the heritability. This method of defining heritability also
explains some other apparent paradoxes, such as how two
populations (e.g., racial groups or two successive generations of
a single group) could have exactly the same genotypic variation
with respect to a trait, but because of differences in their
environments, exhibit differential phenotypes and differential
heritability of the trait.
10. Like the behavior-genetic studies cited in section 2.1,
these studies provide overwhelming data, but should not be
considered as independent reports, because many overlap or
update earlier work. Methodologically, although path models
and the longitudinal studies from which they are derived have
excellent ecological validity, they are correlational; although
they improve upon cross-sectional designs by noting which
factors precede others developmentally, they cannot completely
sort out cause and effect - especially in the earliest stages of
parent-child interactions.
11. The strategy of sociopaths against one another, although
not a test of the current model, is still interesting in its own
right. In the Hare and Craigen (1974) modified Prisoner's
Dilemma, the majority of sociopaths, in their turn, chose from
amongfive"plays," the choice that minimized their own pain (an
electric shock) for that trial, but that maximized their partner's.
Since partners took turns in selecting from the samefive"plays,"
this strategy actually maximized pain over the long run. The
alternative, pain-minimizing strategy, involved giving both oneself and one's partner a small shock - a choice that most subjects
declined to use. This result seems to confirm the sociopath's
inability to consider anything other than the immediate consequences of an act, as well as the ineffectiveness of delayed
Commentaryi'Mealey: The sociobiology of sociopathy
punishment or threat of punishment as a deterrent. In the
Widom (1976a) study, sociopaths did not, in general, "defect"
more often than the controls, but in the condition when subjects
were informed of their partner's move on the previous trial,
sociopaths were much more likely than controls to "defect" after
a mutual cooperation. On this measure, at least, the sociopaths
seemed to demonstrate an inability to "commit" to an ongoing
cooperative relationship. [See also Caporael et al.: "Selfishness
Examined" BBS 12(4) 1989.]
12. Terhune (1980) reports that personality is the most important factor for strategy choice within the setting of single-trial
Prisoner's Dilemma interactions. In multiple-trial interactions,
however, when players have the opportunity to learn one another's dispositions, situational factors are more important for
determining play (see Frank et al. 1993). This is consistent with
the idea that the establishment of reputation is a key goal, even
for players who on a single trial would choose not to cooperate.
For more on the idea that establishing a certain reputation
within one's referent group is a conscious goal and how that
might play a role in the development of antisocial behavior, see
Hogan and Jones (1983) and Irons (1991).
13. For some of the debate on this issue see the series of
comments and replies following Nesse (1991) in the electronic
journal Psycoloqtty. The comments specifically addressing the
relationship between mood and emotion are: Morris (1992),
Nesse (1992a), Plutchik (1992), and Nesse (1992b).
14. While searching for data to test this prediction, I came
across only the Robins et al. (1991) reference in support of it, and
one reference in an introductory psychology text (Wade & Tavris
1993) against it. The latter stated that antisocial personality
disorder "is rare or unknown in small, tightly knit, cooperative
communities, such as the Inuit, religious Hutterites, and Israelis raised on the communal plan of the kibbutz" (p. 584).
Contact with Dr. Tavris allowed me to follow up on the sources
from which the latter statement was derived (Altrocchi 1980;
Eaton & Weil 1953; and Montagu 1978). My conclusion (which
is shared by Dr. Tavris in personal communication) is that the
absence or rarity of sociopathy in these small, tightly knit
societies is not a result of the creation of a social system in which
sociopaths never develop; rather, it is that secondary sociopaths
do not develop (keeping total numbers at the low baseline) and
that primary sociopaths emigrate.
Small, closely knit societies have all the properties that game
theoretic models indicate will reduce (but not eliminate) the
incidence of the cheater strategy (see sect. 3.2.1). One of the
most important of these features is size per se; the cheater
strategy cannot be used repeatedly against the same interactors
and remain successful (see sect. 1.2). Thus, in small societies,
sociopaths are likely to do their damage, acquire a reputation,
and leave - to avoid punishment and move on to greener
pastures. This "roving strategist" model (Dugatkin 1992; Dugatkin & Wilson 1991; Harpending & Sobus 1987) allows for
both the evolution and the maintenance of a low baseline of
successful sociopaths even in small groups (like those in which
we presumably evolved).
15. Despite being a genetically based strategy, because primary sociopathy is the endproduct of the additive and interactive effects of many genes, we will not be able to predict or
identify individual sociopaths by knowledge of their genotype.
We will, however, be able to predict which children will be at
risk, given their genetic background, the same way we predict
which children will be at risk given their familial and sociocultural background. We will also be alerted to the need to
differentiate between diagnoses of primary sociopathy and secondary sociopathy (and our consequent approaches to them)
based upon knowledge of an already identified sociopath's genetic and environmental background.
16. Axelrod (1986) and Boyd and Richerson (1992) also consider the extension of punishment not only to cheaters, but to
those cooperators who do not, themselves, punish cheaters. The
presence of this strategy can lead to an ESS of practically any
behavior, regardless of whether there is any group benefit
derived from such cooperation. Clearly this extension of the
model has some analogues with totalitarian regimes and ingroups of a variety of sorts.
Open Peer Commentary
Commentary submitted by the qualified professional readership of this
journal will be considered for publication in a later issue as Continuing
Commentary on this article. Integrative overviews and syntheses are
especially encouraged.
Testing Mealey's model: The need to
demonstrate an ESS and to establish the
role of testosterone
John Archer
Department of Psychology, University of Central Lancashire, Preston,
Lanes, PR1 2HE, England. j.archer@uk.ac.uclan.p1
Abstract: Two specific aspects of Mealey's model are questioned: (1) the
application of the concept of Evolutionarily Stable Strategy to all
alternative strategies, including those that involve reduced lifetime
reproductive success; and (2) the evidence for the dual role of testosterone, which is based mainly on studies of a modulating effect on
aggression.
The case presented for sociopathy being an ESS (evolutionarily
stable strategy) is a subset of the general case where a minority of
individuals are successful because the majority behave in a
different way. It is true that those who do not follow the rules of
the majority may have evolved to behave like this, forming a
genuine equilibrium or ESS in the population, in which case the
two forms will have equivalent lifetime reproductive success
(Dominey 1984). This would fit the case argued for primary
sociopathy. An alternative is that the minority strategy is the
best available in the current circumstances; in this case, lifetime
reproductive success may be much lower than that of the
dominant strategy (Dominey 1984) and is not an ESS. This
would appear to fit the case argued for secondary sociopathy.
To pursue the argument that primary sociopathy is a genuine
ESS, a cross-cultural comparison would seem to be necessary.
Cross-cultural psychologists have described different cultures in
terms of individualism-collectivism (Triandis et al. 1988). In a
traditional collectivist society there will presumably be a greater
value placed on a reputation for cooperation, so that cheats will
be shunned. In hunter-gatherer societies this would presumably apply to a greater extent, and such circumstances will be
nearer the social arrangements of evolving hominids. In contrast, among individualist modern industrial societies, with
looser family networks and many more interactions with
strangers, the opportunities for cheating - Machiavellianism
and sociopathy - will be much greater.
It would therefore be instructive to compare societies that
differ in terms of the extent to which cheats would be expected
to prosper. We would need to show that there is indeed a stable
proportion of sociopaths in traditional societies, and that they
are on average as reproductively successful as nonsociopaths, for
the ESS explanation to be established.
Mealey suggested a dual role for testosterone. It is first
described as "a likely candidate for the role of trigger of the sexlimited activation of genes required by the two-threshold
model" (sect. 2.3.3., para. 7). This action is described as an
organizer role that affects traits, and contrasted with a second
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
541
Commentaryl Mealey: The sociobiology of sociopathy
role for the hormone, as "an activator (affecting states)." Some
clarification is required. When considering hormonal influences, the term "organizer" usually implies a neonatal influence
(of testosterone). However, Mealey used an analogy with secondary sexual characteristics such as beard and breast development: genes for these are present in both sexes but are only
activated by the appropriate hormonal environment. I presume
from this analogy that the term organizer was intended to refer
to an interaction between particular genes and testosterone at
puberty to produce a certain pattern of behavior, namely, a
threshold effect as opposed to a modulating effect, which varied
according to the level of testosterone. This would, however, be
difficult to reconcile with the claim that the tendency to engage
in antisocial behavior is stable from early childhood.
In support of a dual role for testosterone, Mealey cited studies
of adults (mainly men) linking testosterone with aggressive behavior, and with other forms of antisocial behavior and sensation
seeking. The evidence on these topics, of which most concerns
aggressiveness, is almost entirely correlational. It therefore fails
to address the issue of a threshold effect, only a modulating one.
Overall, there is a correlation between various measures of
human aggressive behavior and testosterone level of 0.38 (Archer 1991), providing suggestive evidence for a modulating role
for the hormone. Since successful aggressive and competitive
behavior can lead to increased testosterone levels, alternative
interpretations are possible, in particular, that testosterone
levels are largely the result of status-related and other activities
(Kemper 1990).
One difficulty with suggesting any major causal role for
testosterone in aggression — even with the addition of positive
feedback to take account of the two-way link - is that aggressiveness as a trait shows stability from childhood to adulthood (e.g.,
Olweus 1979). Thus, the onset of testosterone secretion at
puberty must interact with an already-established disposition.
Animal studies have shown that past learning associated with
aggression can override the effect of testosterone on aggressiveness. To understand the sequential effects of all possible influences, including hormonal ones, on a particular behavioral trait
requires a developmental perspective (Archer 1994). This is
more complex than the causal model adopted by Mealey.
Mealey stated that, "Variation in testosterone levels also
parallels the age variation in the expression of sociopathic
behavior and is correlated with such behavior in adolescent and
adult males" (sect. 2.3.3., para. 7). The phrase "such behavior" is
misleading as it apparently widens the scope of the discussion,
but the references that followed mostly concerned aggressiveness. It is not clear how this is related to other attributes known
to be associated with testosterone, such as impulsiveness and
sensation seeking, or indeed to sociopathy itself. To test the
specific hypothesis put forward by Mealey we would need to
establish that testosterone is primarily associated with sociopathy rather than with aggressiveness or some other disposition.
Abstract: Mealey's excellent target article rests on several assumptions
that may be questioned, including the overarching assumption that
sociopathy reflects the failure of a small minority of males to cooperate
with the larger group. I suggest that violent competition in ancestral
bands - and not "cheating" in the "game" of cooperation - was the
primary evolutionary precursor of sociopathy. Today's violent sociopath
is far more a "warrior hawk" than a failed cooperator.
primary and secondary sociopathy, and in generating thoughtful
and convincing recommendations for reducing sociopathic behavior and crime in general. Her excellent target article, however, rests on several implicit assumptions that can be questioned. Among these are the notion that both ancestral and
current forms of social organization are overwhelmingly
cooperation-based, that sociopathic outputs emanate primarily
from rational cost-benefit calculations, that intelligence is not a
major mediator of such calculations, and that game theory is the
appropriate metaphor for understanding and predicting sociopathic behavior. Space does not allow me to challenge all these
issues, so I will focus primarily on Mealey's assumption that
sociopathy is largely a failure of a small minority of human males
to cooperate with the larger group.
I will proceed on the basic assumption that violent competition in ancestral bands - and not "cheating" in the "game" of
cooperation - was the primary evolutionary precursor of sociopathy. It is likely that aggressive intermale competition for status,
females, and other resources was prevalent in the EEA (environment of evolutionary adaptation), and some degree of predatory violence was required in the seek and kill aspects of hunting
large game animals. Moreover, it is likely that violent interband
competition was present in the earliest phases of human evolution and a given group's number of healthy, adventurous, and
potentially violent young men was a major key to survival. The
absence of a contingent of "dawn warriors" (Bigelow 1969) in
readiness would be disastrous in the event of attack by an
invading party composed entirely of such men.
Although it is fashionable to ascribe intergroup conflict,
crime, and male violence in general to the exigencies of modernity, the common ancestor to the primate and human line has
been described as sexually dimorphic, socially aggregated into
closed networks, and prone to hostile intergroup encounters
among males (Wrangham 1987). In this scenario, the issue of
"cheaters" in the game of cooperation is transformed into the
game of "hawks" and "doves" (Maynard Smith 1982), where
doves always lose when one strategy is pitted against the other
(Archer 1988). Within groups, doves fare better against doves
than hawks do against each other, and a frequency-dependent,
evolutionary stable point is theoretically possible where the
competing classes are roughly equal in their respective fitnesses. But the fact remains that doves always lose in head-tohead competition with hawks. The only solution is for the
cooperating majority of doves to be protected by a subclass of
"warrior hawks" that is capable of repelling invasion by a similar
contingent from another group. The price that each group pays
is having to tolerate, within groups, the disruptiveness, callousness, bullying, and manipulativeness of the warrior class.
What might be the defining characteristics of these warrior
hawks? Mealey has done an excellent job of enumerating them
in detail: coldness, detachment, manipulativeness, egocentrism, absence of social emotions, lack of empathy, fearlessness,
deceptiveness, life history of "predatory social interactions,"
orientation to the present, strong need for respect and reputation among peers, extraversion, frequent "cheating," adventurousness and risk taking, impulsiveness, pleasure seeking,
resistance to socialization, moral deficiency, minimal responsiveness to reward/punishment, approach orientation, high
Machiavellianism, and proneness to anger and aggression.
These are exactly the traits necessary on the battlefield, where
an inherent "delight in destruction" fuses with a desire to
eradicate the hated enemy, and the warrior's "hunting impulses
are released to seek the most dangerous of all beasts" (Gray
1970, p. 149). In deadly combat with hated and feared enemies,
the traits of the warrior reach fruition and the most brutal
warrior hawks have the advantage over their less "sociopathic"
adversaries; it is in peacetime within groups that the suite of
warrior traits becomes problematic.
Mealey has performed a great service in reviewing current
knowledge on sociopathy, in carefully distinguishing between
As Bigelow (1972) tells us, intergroup conflict has been with
us since the dawn of humanity, and indeed, the success of the
The sociopath: Cheater or warrior hawk?
Kent G. Bailey
Department of Psychology, Virginia Commonwealth University, Richmond,
VA 23284-2018
542
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Commentaryi'Mealey: The sociobiology of sociopathy
human line was predicated on potential for aggressive group
response, when necessary, within the context of in-group solidarity, cooperation, and intelligent self-control. It is particularly
important that a social group keep its young, aggressive, and
impulsive males - that is, potential warrior hawks - under
control. From this perspective, violent, predatory crime in
modern contexts reflects a loss of control over potential warriors
who come to treat in-group members, even their neglectful and
abusive family members, as outsiders and enemies to exploit,
abuse, and even destroy in extreme cases. This they do with a
coldness and callousness that is impossible for the cooperative
doves in the group to understand. Moreover, cooperative, lawabiding doves often further their own exploitation by providing
sympathy and forgiveness to those who prey upon them without
empathy or remorse.
At several points, Mealey refers to sociopathy as abnormal
and pathological, and psychiatry has traditionally classified it as
a disorder. Certainly, the behavior of the sociopath is undesirable and destructive in peaceful and cooperative contexts, but
the issue of abnormality is problematic. The sociopath does not
see himself as abnormal, psychiatric symptoms are minimal or
absent, and any pathology is social and interactive rather than
internal (MacMillan & Kofoed 1984). Reasoning backwards to
the EEA, the warrior traits that underlie violent crime today
were not only normal in ancestral contexts but were necessary
for survival in an atmosphere of occasional interband conflict.
Presumably, in the small group atmosphere dominated by older
males past their warrior prime, young males were limited in the
antisocial options available to them in the in-group context. As
Mealey implies in her argument, such natural controls over
male adventurism and exploitation are absent today in our
crowded, poverty-ridden, and socially decaying urban environments, and it is no surprise that rootless and discouraged young
men will sometimes fall back on the ancient warrior hawk
option.
Mealey is right that sociopathy does involve cheating in cooperative contexts, but the overriding concern of most people in modern
society is not with lying, misrepresentation, and loss of material
resources; rather, our foremost concern is fear of violent crime in
the form of gang warfare and drive-by shootings, carjackings,
muggings, burglary and forcible entry, stranger rape, child abuse/
molestation, and various other threats to life and limb. Our
concern is heightened by knowledge that the "warriors without
portfolio" in our midst are becoming more numerous, more
violent, more addicted to substances that exacerbate their
violent traits, and increasingly alienated from family, community, cultural tradition, educational opportunities, and other
constraining influences. For many of today's warrior hawks,
anyone not in their socially marginal peer group is the "enemy"
and the status of hero is conferred upon those with the most
"wins" on the urban battlefield.
Continua outperform dichotomies
John D. Baldwin
Department of Sociology, University of California at Santa Barbara, Santa
Barbara, CA 93106. baldwin@alishaw.uscb.edu
Abstract: Mealey's data do not support her dichotomous model of
primary and secondary sociopathy; this data supports the view that there
is a continuum of degrees of sociopathy, from zero to the maximal
manifestation. There are multitudes of factors that can contribute to
sociopathy - from both genetic and environmental sources - and the
countless different mixes of them can produce multiple degrees and
variations of sociopathic behavior.
Mealey does a good job detailing many facets of sociopathy,
including the role of sensory stimulation as a powerful reinforcer
for people with hypoaroused nervous systems. The role of
empathy - or lack thereof - is of central importance, too. I
applaud her attention to both nature and nurture, though I
detect a favoritism for nature instead of a balanced respect for
both nature and nurture (see below).
In the introduction, Mealey states that she hopes "to convince
the reader that the distinction between primary and secondary
sociopaths is an important one because there are two different
etiological paths to sociopathy." I argue that her data do not
support a dichotomous model as well as they support the view
that there is a continuum of degrees of sociopathy, from zero to
the maximal manifestation. Mealey correctly notes that there
can be multiple genetic and multiple environmental inputs that
operate together, and it seems to me that this approach clearly
suggests that different mixtures of genetic and environmental
inputs can allow people to fall at many different positions on a
continuum of sociopathy.
First, in section 2.1.3, Mealey argues that sociopathy is
polygenetic and that each individual can inherit a different
"dose" or "genetic load" of the genes that predispose one for
sociopathy. In these pages, it is artificial to argue for a twothreshold model. Her suggested thresholds are merely hypothetical constructs that must be introduced if she hopes to create
a dichotomous model rather than a continuum model. I believe
that the many different possible genetic loadings of the polygenetic inputs can lead to numerous different genotypes with a
continuum of different levels of predisposition to sociopathy.
Second, in section 2.4.2, Mealey lists multiple environmental
risk factors that can increase the chances that a person becomes
a sociopath. Later we learn that there are also multiple environmental protective factors - such as good parenting, exciting
prosocial jobs, and so on - that can steer high sensation-seeking
individuals away from antisocial behavior. Why not simply
create a model that integrates nature and nurture in a straightforward (and more parsimonious) manner, stating that the
higher a person's genetic load of predisposing genes is, the more
likely the person is to learn sociopathic behavior if exposed to
risky environmental factors without adequate protective factors.
If sociopathy emerges as a combination of various genetic
loadings and exposure to countless developmental risk and
protective factors, it is easy to postulate a continuum of sociopathy with individuals distributed mostly near the low-end of
the continuum. Since only a small number of individuals would
receive both a heavy genetic load and experience the maximal
number of risk factors needed to produce maximally sociopathic
behavior, I would expect to see a skewed bell-shaped curve that
peaks near the low-end of the sociopathy continuum. Thus,
extreme sociopathy (which Mealey calls "primary") would be
rare - and the most intractable. Numerous levels of intermediate sociopathy would exist, being more common and more
treatable than the few extreme cases. Seeing a continuum of
possibilities (based on both nature and nurture acting in concert)
allows for a finer grained and more sensitive analysis of sociopathy than does Mealey's dichotomous model. The logic Mealey
uses to create the dichotomy also tends to elevate genetic causes
to a special status based on her a priori assumption that primary
sociopaths can only be understood in terms of "a genetically
determined strategy" (sect. 3.1.1).
Mealey often writes with an "either-or" logic, saying, for
example, that some people are emotionless and others are not.
Perhaps the dichotomous model (of primary and secondary
sociopathy) has emerged as an artifact of either-or logic. A
multiple factor model - that recognizes both multiple polygenetic causes and countless environmental risk and protective
factors - helps one avoid either-or logic and think in terms of
complex nature-nurture interactions with a continuum of outcomes ranging from zero to maximal manifestations of any
behavior.
Nondichotomous models also sensitize us to look for the many
types of socialization that can lead to high Mach scores, calcuBEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
543
Commentary I'Mealey: The sociobiology of sociopathy
lated thinking, and deficits in empathy. Since each component
of sociopathy is only partially correlated with the others, many
other types of socializations can lead to variable levels of each
one. I believe we need to examine each individual separately, to
identify sensitively as many of the developmental risk and
protective factors as possible. If some individuals appear to be
very susceptible to becoming sociopathic - and do so in a
protective environment with little exposure to risk factors - we
can hypothesize that they have a strong genetic loading of
the predisposing genes. If physiological markers or measures
are available to identify the level of genetic load, so much the
better.
In section 3.1.1, and elsewhere, we see Mealeys tendency to
think of "primary" sociopaths as professional gamblers, emotionless cheaters, and incorrigible criminals. Let us not forget that
they might also become TV evangelists, lawyers, politicians,
driven scientists, and others who ruthlessly pursue their career
without being thought of as criminals - though those who know
them might note a calculatingness and social insensitivity in
their actions.
In section 3.2,1 see no treatment benefits from dichotomizing
sociopathy. At present, we cannot change a person's genetic load
for the behavior. All we can do for both of Mealey's "primary"
and "secondary" sociopaths is try to make proactive and reactive
environmental interventions: first, we can proactively try to
reduce the risk factors and increase the protective factors that
affect the development of the behavior; or, second, for those
who already exhibit sociopathic behavior, we can use punishment or the threat of punishment for antisocial behavior along
with rewards for socially acceptable behavior. Thinking of sociopathy in terms of a continuum (not just primary or secondary
types) alerts us to the multiple variables we need to attend to
when proacting or reacting to any given individual's sociopathic
possibilities.
Sociopathy, evolution, and the brain
Ernest S. Barratt and Russell Gardner, Jr.
Department of Psychiatry and Behavioral Sciences, University of Texas
Medical Branch, Galveston, TX 77555-0443. ernest.barratt@utmb.edu
Abstract: We propose that Mealey's model is limited in its description of
sociopathy because it does not provide an adequate role for the main
organ mediating genes and behavior, namely, the brain. Further, on the
basis of our research, we question the view of sociopaths as a homogeneous group.
Although Mealey deals at length with sociobiology and impulsive mechanisms of genes and their resultant behaviors, she
does not deal in an evolutionary sense with the organ through
which the genes and behavior must be mediated: the brain.
She discusses neurochemistry related to personality traits and
correctly describes psychophysiological factors "as significant
causes, not just correlates" of sociopathy. Yet she does not
discuss from an evolutionary viewpoint, relevant neuropsychological constructs and selected brain structures that relate to
sociopathy (or psychopathy or antisocial personality disorders).
Since impulsiveness and impulse control are key constructs in
the definition of behavioral control, and since these constructs
have been related to selected brain areas, a discussion of the
development of the brain and related behavioral control or
inhibitory mechanisms would seem to be an appropriate and
essential part of the development of her model. For example,
damage to the orbital frontal cortex almost invariably results in
impulse control problems. The personality trait of impulsiveness has also been related to frontal cortex functions (Barratt
1993). Damasio et al. (1990) have written on "acquired sociopathy" stemming from brain damage in the orbital frontal region.
544
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
Thus, cross-species comparisons of frontal cortex functions related to behaviors similar to those of psychopaths should provide
data to strengthen and extend her argument. This is not to argue
that sociopathy is a brain disease; but caution should be used in
relying on population statistics and conceptual models such as
the Prisoner's Dilemma as ultimate and sufficient explanations
of complex constructs like psychopathy. Some personality traits
such as impulsiveness may have had (and may still have) adaptive functions among persons with intact brains. Acting "automatically" or without reflection may be helpful in emergency
situations. For example, it was estimated in a recent air crash
that the pilot had less than five seconds to observe and respond
appropriately to a warning light. Over the span of animal
evolution, acting quickly to protect territory or other natural
goods has always been at a premium. Perhaps this was true long
before mammals were primates, or for that matter, before
vertebrates were mammals. The population biology of sociobiologists often overlooks the length of time that Darwinian
mechanisms may have been operating. In modern society, to
react quickly and consistently without thinking can be a handicap when coupled with other personality traits (e.g., the high
impulsiveness and low anxiety seen in psychopaths) or in situations that call for more reflection. Again, we suggest that
Mealey's model should include more of a role for the main organ
through which the genes and behavior must be mediated,
namely, the brain.
In a study of impulsive aggression in our laboratory (currently
being prepared for publication), we studied prison inmates, all
of whom satisfied the criteria for antisocial personality as defined
in DSM-III-R and measured by a standard interview (PDI-R).
These inmates were all young male recidivists. Based on formal
disciplinary reports of aggressive acts, we classified the subjects
into impulsive aggressive and premediated aggressive groups.
Again, each aggressive act was classified using a standard interview. The measures of these aggressive acts were normally
distributed along a continuum from completely premeditated to
completely impulsive. We had hypothesized that the inmates
who committed impulsive aggressive acts would have higher
levels of impulsiveness and anger/hostility personality traits
than those who committed the premeditated acts. Both groups
of inmates had significantly higher levels of impulsiveness and
anger/hostility than noninmate controls (matched for sex, age,
education level, and race). The two inmate groups did not differ
significantly in these personality traits. They did differ significantly, however, in cognitive psychophysiological measures of
information processing during the performance of a simple
choice task. The results implicated the frontal cortex and several
other cortical areas. A frequency analysis of the EEGs (electroencephalograms) suggested that anticonvulsant medication
might be helpful in controlling impulsive aggression among the
inmates. The data to date are consistent with this suggestion.
Within Mealey's model, these inmates would also satisfy the
criteria for sociopaths. Yet, there were two distinct groups
among them that expressed aggression in different ways and had
significant differences in brain functions. Mealey's model suggests primary sociopaths are a homogeneous group. The above
data raise questions about such homogeneity.
You can cheat people, but not nature!
John Barresi
Department of Psychology, Dalhousie University, Halifax, Nova Scotia B3H
4J1, Canada. |barresl@ac.dal.ca
Abstract: The psychological mechanisms implicated in psychopathy do
not limit their activity to those behaviors that support a cheater strategy
in social games. They result in a number of other clearly maladaptive
behaviors that do not directly involve other individuals. Thus, any gains
Commentaryi'Mealey: The sociobiology of sociopathy
that might arise from the use of a cheater strategy in social situations are
probably lost elsewhere.
The primary sociopath, or psychopath (Cleckley 1941; Hare
1986), exhibits behaviors easily interpreted as a cheater strategy
in a reciprocal altruistic game. As such, the psychopath seems to
have a marginally adaptive strategy in social interactions with
other humans. But can the psychological mechanisms implicated in psychopathy limit their activity just to those behaviors
that support the cheater strategy in social interactions? I think
not. As I will show shortly, the same psychological mechanisms
also cause the psychopath to engage in a number of behaviors
not directly involved in social interactions with others that are
clearly maladaptive. Thus, the real question is whether there is
a net gain for the psychopath when some of his behaviors that do
not involve other agents in social games are maladaptive as
compared to nonpsychopaths, while other behaviors that do
involve other agents are adaptive as a low-frequency cheating
strategy in reciprocal altruistic games. This is a more difficult
question to answer; however, I would guess that the psychopath
obtains at best a net gain of zero from the costs that go along with
the benefits of this strategy. Whatever the psychopath gains
from reciprocators in direct competition through the use of a
cheater strategy, he loses to these very same people through the
indirect competition with them in situations not involving social
games.
According to Mealey, the gains from cheating that the primary
sociopath obtains occur because he takes a short- rather than
long-term view of social interaction, choosing immediate gain
over long-term gain through reciprocal altruism. She suggests
that this occurs because the primary sociopath lacks the social
emotions that bind individuals to each other, producing cooperation through time. It is the social emotions such as shame,
guilt, sympathy, and love that are an essential part of the
psychology that maintains the human social adaptation of reciprocal altruism, or of a cooperative strategy over multiple Prisoner's Dilemma-type social situations. The psychopath, lacking
these emotions, is inclined not to cooperate over the long term
with others, but prefers to cheat by taking the dominant defect
strategy in Prisoners Dilemma-like social situations.
1 do not wish to debate with Mealey over whether the primary sociopath, or psychopath, lacks these social emotions or
whether the lack of these emotions might provide an advantage
to the psychopath through leading him to defect in Prisoner's
Dilemma-type social situations. Indeed, the psychopath exhibits other related behaviors that seem to optimize the success
of this strategy, such as his tendency to wander from one
population to another so as to reduce multiple interactions with
the same individuals (Harpending & Sobus 1987). However,
there is every reason to think that the psychopath lacks not only
the relevant social emotions but also other emotions that inhibit
self-interested behavior even when other individuals are not
involved. For example, it has long been known that psychopaths
do not exhibit the usual physiological correlates of anxiety or fear
when they know that they are about to be shocked (e.g., Hare
1986) and that this apparent emotional deficit reduces their
capacity to learn responses that avoid both shocks and certain
social punishments (Lykken 1957; Schmauk 1970). Such a general deficit in passive avoidance learning relative not only to
other humans but to many other organisms (e.g., Mineka &
Zinbarg 1991) can hardly be viewed as an adaptive consequence
of psychopathy. It is a cost that must be balanced by the success
of cheating in social situations.
But there are further difficulties associated with the lack of
emotions in psychopaths that must also be considered, in
particular, their lack of prudent behavior concerning future
consequences of current activity. Even when other individuals
are not involved, the psychopath fails to show concern about his
own future. Especially in approach-avoidance conflict situations, the psychopath is unwilling to delay immediate gratifica-
tion for a better outcome later (e.g., Newman et al. 1992).
Although he is quite capable of calculating what will happen in
the future, he fails to take a sufficient interest in it to be
motivated by the consequences of present actions for his future
self. William Hazlitt pointed out long ago that it is the same
sympathetic imagination that "must carry me out of myself into
the feeling of others . . . by which I am thrown forward as it
were into my future being and interested in it. I could not love
myself, if I were not capable of loving others" (Hazlitt
1805/1969, pp. 2 - 3 ; Martin & Barresi 1995). In the case of the
psychopath, just as he fails to show sympathy for other people's
interests, he also fails to show sympathy for the interests of his
future self. And again we can ask whether the direct payoffs from
the cheater strategy offset these losses due to this lack of
sympathy by the psychopath for his own future self. [See also
Logue 1988; Rachlin 1995.]
My suspicion is that if the psychology of the psychopath is at
all successful as a low-frequency strategy for human social
interaction, it has developed through a form of genetic drift,
where gains that are made through cheating are offset by the
losses in noncheating situations. Turned around, what this
means is that the success of the cheating strategy allows an
intrinsically maladaptive behavioral disorder, psychopathy, to
exist in higher frequency than it normally would. This possibility makes one wonder whether there are other psychopathologies that are maintained at fairly high frequencies
through such "secondary gains" of the pathology. Conversion
hysteria might be a prime example of just such a psychopathology.
Secondary sociopathy and opportunistic
reproductive strategy
Jay Belsky
Department of Human Development and Family Studies, College of Health
and Human Development, Penn State University, University Park, PA
16802. jxb@psuvm.psu.edu
Abstract: Mealey s analysis of secondary sociopathy has much in common with Belsky, Steinberg, and Draper's (1991) evolutionary theory of
socialization. Both draw attention to the potential influence of early
rearing in the promotion of a cold, detached, manipulative, and opportunistic style of relating to others and, in so doing, raise the question of
whether secondary sociopathy represents a facultative reproductive
strategy.
The assertion that a cold, detached, manipulative, opportunistic
style of relating to others is, in some cases, the result of
contextual stresses and rearing practices during childhood has
much in common with an evolutionary theory of socialization
advanced by Belsky et al. (1991). Building upon many of the
same sociobiological foundations as Mealey, these theorists
argued that humans have evolved to be responsive to their
rearing circumstances in the service of reproductive goals (i.e.,
fitness). When parental care is harsh, rejecting, insensitive, and
inconsistent, as it is likely to be when economic resources are
limited and family stress is heightened, children are predisposed to develop insecure attachments to their parents, perceive others as untrustworthy and relationships as unenduring;
in consequence, they develop an opportunistic (including aggressive, especially in males) advantage-taking style of relating
to others (a sociopathic style?). This developmental trajectory,
Belsky et al. argued, was part of a reproductive strategy designed to favor growth and mating over parenting, and thus was
hypothesized to be associated with earlier timing of puberty,
earlier onset of sexual activity, unstable pair bonds, and limited
parental investment (see Fig. 1).
Like Mealey, Belsky et al. (1991, p. 650) highlighted
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
545
Commentaryi'Mealey: The sociobiology of sociopathy
TYPE I I
TYPE I
A. FAMILY CONTEXT
Spousal harmony
Adequate $ resources
B. CHILDREARING
Infancy / Early Childhood
Sensitive, supportive,
responsive
Positively aflectionate
Marital discord
High stress
Inadequate $ resources
Harsh, rejecting
insensitive
Inconsistent
Insecure attachment
Mistrustful internal
working model
Opportunistic interpersonal
orientation
<t
9
Aggressive
Noncompliant
Anxious
Depressed
Early maturation / puberty
Earlier sexual activity
Short-term, unstable
pair bonds
Limited parental investment
C. PSYCHOLOGICAL /
BEHAVIORAL
DEVELOPMENT
D. SOMATIC
DEVELOPMENT
E. REPRODUCTIVE
STRATEGY
Secure attachment
Trusting internal working
model
Reciprocally rewarding
interpersonal orientation
Later maturation / puberty
Later sexual activity
Long-term, enduring
pair bonds
Greater parental investment
Figure 1 (Belsky). Developmental pathways of divergent reproductive strategies
(Type I: Opportunistic; Type II: Mutually beneficial; Belsky et al. 1991, p. 651).
genotype-environment interactions, specifically underscoring
"differential susceptibility to environmental experience" in an
attempt to explain why an association between contextual stress
and an opportunistic reproductive strategy might not always
obtain (for a related argument, see Wilson 1994). In fact, in
discussing environmentally induced reproductive strategies,
Belsky et al. further pointed out that some genotypes might be
more reactive to specific environmental influences than others:
Whereas some individuals may be genetically predisposed to respond to contextual stress and insensitive rearing by maturing early
and engaging in relatively indiscriminate sexual behavior (and other
manifestations of sociopathy), others may be genetically predisposed
to respond to sensitive rearing by deferring sexual behavior and
establishing pair bonds in adulthood. (1991, p. 650)
When applied to Mealey's analysis, this observation raises the
question of whether a sociopathy-inducing environment would
foster (secondary) sociopathic behavior among all genotypes. To
the extent that the answer to this question is no, it suggests that
genotype-environment interaction is probably more complex
than Mealey's analysis of sociopathy implies.
Once it is acknowledged that (secondary) sociopathy as detailed by Mealey or the (highly correlated?) opportunistic reproductive strategy outlined by Belsky et al. may be the result of
contextual conditions and rearing practices and that individuals
may be differentially susceptible to these environmental influences, a number of interesting questions arise. We can ask first
whether secondary sociopaths mature earlier than individuals
with similar genotypes who are exposed to dramatically different rearing conditions. An affirmative answer to this query
would be consistent with the notion that the secondary sociopaths of Mealey's analysis are likely to be the same individuals
who are central to Belsky et al.'s opportunistic reproductive
strategy.
The explicit linking of these two sets of ideas highlights a
second important issue, one that Belsky et al. raised but could
not resolve. As applied to the topic of secondary sociopathy, it
takes the following form: Is secondary sociopathy best conceptualized as a continuous or a categorical construct? To the extent
that varying exposure to harsh, insensitive, and inconsistent
rearing systematically fosters varying degrees of sociopathic
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BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
behavior, one must wonder whether Mealey is describing a
specific form of psychopathology or a far more general behavioral and even developmental phenomenon.
A third important issue with regard to secondary sociopathy
concerns developmental timing and plasticity. Will secondary
sociopaths, as a result of early rearing experiences, be resistant,
like primary sociopaths, to many environmental efforts to modify their behavioral proclivities? In fact, might there be a point in
development after which deflection from a sociopathic developmental trajectory would be less, rather than more, likely?
The answer to these queries may themselves depend on the
reply to the last question raised in this commentary about
secondary sociopaths: Do the early experiences that promote
secondary sociopathy induce in the victims of such rearing the
same neurochemistry - high dopamine activity, low serotonin
activity, and low norepinephrine activity - that characterizes
sociopaths in general? That is, are primary and secondary
sociopaths as similar in terms of brain chemistry as they might
be in their overt behavior? To the extent that brain chemistry is
influenced by early developmental experiences, the prospect
arises that processes set in motion early in life may become selfsustaining for neurochemical reasons as much as for socialexperiential ones. And to the extent that neuronal pruning
further characterizes processes of brain development pertinent
to the understanding of secondary sociopathy, reservations
might have to be raised about the prospect of ameliorating this
psychopathological syndrome.
Group differences ^ individual differences
C. S. Bergeman and A. D. Seroczynski
Department of Psychology, University of Notre Dame, Notre Dame, IN
46556. cbergema@lrlshmvs.cc.nd.edu
Abstract: Mealey's etiological distinction between primary and secondary sociopathy blurs the delineation between individual and group
differences. She uses physiological evidence to support her claim of
genetic influences, neglecting variability within social classes, fre-
Commentary/Mea\ey: The sociobiology of sociopathy
quency of delinquent behavior in upper and middle classes (measured
by self-report), and discontinuity of criminal behavior across the life
span. Finally, Mealey's proposals for differential intervention fall short
of a future agenda, which should tailor to individual needs, not social
classes.
Mealey presents a provocative model of the development of
sociopathy, which blends research from a wide variety of disciplines. Unfortunately, the assumptions upon which the theory is
based are often unsubstantiated; we will attempt to outline a few
of the discrepancies.
First, Mealey defines two types of sociopathy - primary and
secondary. She contends that primary sociopathy is due to a
strong genetic endowment and accounts for most of the sociopathic individuals found in the upper and middle socioeconomic
classes. Secondary sociopathy, on the other hand, is "not as
clearly tied to genotype" (sect. 3.3, para. 4) and can be explained
by environmental risk factors such as high population density, competition for limited resources, poor parenting skills,
and other factors often associated with low social class. Mealey contends that secondary sociopaths will almost always
come from lower-class backgrounds, whereas primary sociopaths are found almost exclusively in the middle and upper
classes.
Unfortunately, to support this distinction Mealey has drawn
the mistaken conclusion that physiological responses or differences are necessarily genetic. In other words, to support her
contention that primary and secondary sociopathy have different underlying etiologies, Mealey has assumed that if physiological characteristics (which have been shown to have a genetic
component) relate to behavioral differences between social
classes (stronger relationship in upper-class individuals), then
those differences must be due to genetic influences in upperand middle-class individuals and environmental factors in the
lower class. For example, she cites research (Raine & Venables
1981; 1984; Satterfeld 1987) that indicates that physiological
response differences between social classes have been associated with differences in antisocial behavior in children. Because
these physiological variables (heart rate, skin conductance,
EEGs) have been shown to have a strong genetic component, it
is assumed that this is the "genetic link." These physiological
differences found between social classes might not necessarily
be genetically determined. Environmental variables such as
familial stressors or systematic desensitization to violence might
account for the observed physiological differences. A later work
by Raine et al. (1990) found no relationship between socioeconomic status and psychophysiological measures, even
though there was a relationship between central and autonomic
measures of arousal at age 15 and criminality at age 24. Raine
et al. conclude that "social and academic factors do not appear
to be important mediators of the crime-arousal relationship"
(p. 1006).
A second problem is Mealey's lack of consideration of variability within social classes. A multitude of studies across the
social sciences have focused on average group differences and
have paid little attention to the relative amount of variability
within the groups. This neglect of variability can inhibit both
conceptual and empirical development in the study of behavioral phenomena. The problem is that average group differences
are not necessarily related to individual differences, thus we will
learn little about the description or explanation for individual
differences in the development of sociopathy by studying group
differences. In addition, research has indicated that the differences between individuals within a group are far greater than
average differences between groups (Plomin 1990). If all we
know about individuals is whether they come from the upper,
middle, or lower socioeconomic class, we will know very little
about their sociopathic tendencies. For example, the causes of
individual differences in the development of sociopathological
behavior could be entirely influenced by genetic factors; however, the average differences between social classes could be
environmental in origin.
Mealey violates this premise by assuming that delinquents
from high-SES (socioeconomic status) families don't come from
"bad homes.' Even if Mealey's theory is "right on the mark," and
genetic factors are responsible for the development of primary
sociopathy in middle- and upper-class families, namely, the
extent to which the trait runs in families for genetic reasons, we
would expect it to relate to parenting behavior and family life.
That is, high-SES families of sociopathic kids should exhibit
similar deviant tendencies. To date, no research has specifically
tested the differences in genetic and environmental influences
between high- and Iow-SES groups.
A third concern with this model is that Mealey views unsocialized and socialized juvenile delinquents as precursors of
primary and secondary sociopathy, respectively. One problem
with this line of reasoning is that it relies on research that defines
delinquent behavior based on police records and public criminal
files. It is not surprising that these differences could be associated with social class, considering that there is an increased
likelihood that lower-class individuals will be caught and convicted of crimes due to an uneven distribution of police or biased
police handling (Sampson 1986; Staples 1987). Over 15 years
ago, Tittle et al. (1978) refuted the argument that social class is
tied to criminality. More recent work by Rowe (1983) has
indicated that the use of self-report measures of delinquency
show no social class differences.
A fourth issue is that much of the work focuses on children
and adolescents. What is the expectation for the other three
quarters of the life-span? Research has indicated that less than
half of juvenile delinquents become repeat offenders and the
majority do not continue their criminal behavior in young
adulthood (Farrington 1979; West & Farrington 1977). Moreover, some people do not commit their first crime until adulthood (Guttridge et al. 1983). In addition, research has indicated
that criminal behavior in adulthood shows a stronger genetic
relationship than does juvenile delinquency in adolescence
(Plomin 1991). The Mealey model does not go far enough to
explain the complexity of this behavior across the entire life
span.
Finally, Mealey states that "since the genetics and life histories of primary and secondary sociopaths are so different, successful intervention will require differential treatment of different cases" (sect. 3.3, para. 6). She has failed, however, to show
that the genetic and environmental influences relating to these
two types of sociopathy are different. She also makes the
assumption that if trait is genetic, then it is immutable and
nothing can be done to change the behavior, or that the best we
can do is provide socially acceptable outlets for primary sociopaths. The result is her suggestion that interventions should be
directed toward secondary sociopathy and not primary
sociopathy.
The one issue on which we do agree with Mealey is th'at a
single intervention is unlikely to work for all individuals. But,
rather than to direct differential interventions toward members
of different social classes, we suggest the need to identify the
individual maladaptive behaviors and to tailor intervention
strategies accordingly. That is, if poor parenting strategies
are identified, then the intervention should be directed toward
parental education. Likewise, if social skill deficits or drug
abuse are problems, then let us direct intervention at these
issues.
In conclusion, what the target article points out is the woeful
lack of research in this area. Future research should be directed
toward assessing genetic and environmental influences on primary and secondary sociopathy and the extent to which physiological indices associated with sociopathic behavior share the
same underlying genetic and environmental influences. It
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
547
Commentaryl Mealey: The sociobiology of sociopathy
should attempt to determine what intervention strategies are
effective with each type of sociopathy, regardless of social class.
Putting cognition into sociopathy
R. J. R. Blair and John Morton
MRC Cognitive Development Unit, London WC1H OBT, and Department of
Psychology, University College, London WC1E6BT, England.
ucjtrjb@ucl.ac.uk; John@cdu.ucl.ac.uk
Abstract: We make three suggestions with regard to Mealey s work.
First, her lack of a cognitive analysis of the sociopath results in underspecified mappings between sociobiology and behavior. Second, the
developmental literature indicates that Mealey's implicit assumption,
that moral socialisation is achieved through punishment, is invalid.
Third, we advance the use ofcausal modelling to map the developmental
relationships between biology, cognition, and behaviour.
The target article is an interesting attempt to relate sociobiological theory, the physiological data on various antisocial populations, and criminal behaviour. During the attempt, Mealey joins
the growing band of authors (e.g., Fagan & Lira 1980; Moffitt
1993) who distinguish between primary and secondary sociopaths. The groups display similar behaviour, which we might
expect to be controlled by roughly equivalent cognitive structures. Finally, we are in total agreement with at least one of
Mealey's descriptions of sociopaths, that they will acquire a
theory of mind without access to empathic responding. Indeed,
we have shown that psychopaths perform well on complex
theory-of-mind tasks in the absence of physiological arousal
responses to distress cues (Blair 1994).
Our major problem with Mealey's work is the lack of a
cognitive analysis; there is little reference to the kinds of
cognitive structures that might mediate sociopathic behaviour.
Indeed, some of the core features of her model, for example
cheating strategies, are not considered at a cognitive level at all.
We believe that if such an analysis had been carried out, some of
the difficulties in Mealey's argument might have become clear.
For example, Mealey claims that rape and spouse abuse are
"genetically influenced, developmentally and environmentally
contingent cheating strategies" (sect. 2.2.1, para. 4) that have
been selected for. It seems unlikely that these behaviours per se
are part of the phenotype; that motor programs for antisocial
behaviours, such as spouse abuse, are laid down in the genotype. Thus, Mealey's position requires that some more general cheating mechanism has been selected for - what she calls
the cheating strategy - which appears, from her argument, to
correspond to some form of inborn personality trait. Presumably, given Mealey's position on cost-benefit analysis in the
sociopath, this corresponds at the cognitive level to some form of
value-specifying mechanism that undervalues prosocial actions
and overvalues antisocial actions. Of course, even if this reading
of her theory is correct, an account of how the cheating strategy
leads to the development of particular cheating strategies such
as spouse abuse still needs to be formulated.
Our second problem concerns the claim that sociopaths suffer
from a hypoaroused nervous system. First, it should be noted
that the data concerning this claim are, at best, equivocal (e.g.,
Patrick et al. 1993). Second, and far more important, the theoretical importance of this claim is unclear. Mealey makes the
assumption, as others in the sociopath/psychopath literature
have done before her (e.g., Eysenck 1964; Patterson & Newman
1993), that moral socialisation is achieved through the use of
punishment. The difficulty with this assumption is that there is
no evidence for it. Indeed, the evidence that exists indicates
that moral socialisation is best achieved through exposure to and
focus on the victims of moral transgressions (see, for review,
Hoffman 1977). Moreover, much of the exposure and focus on
548
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
victims appears to be independent of primary caregivers and
occurs in interactions with teachers and peers (Nucci & Nucci
1982).
Finally, it should be noted that we applaud the fact that
Mealey is attempting to model a disorder developmentally and
at several levels simultaneously. Too often, models of sociopathy
have ignored development (e.g., Gorenstein & Newman 1980).
However, we suggest that this developmental multiple level
modelling must be formalised to increase clarity. One way to
increase clarity is to use causal models; formal models of developmental interlevel relationships - genetics, physiology, social,
cognitive, and behaviour (Morton & Frith, in press). Causal
modelling has been used to model the development of several
disorders, including autism (Morton & Frith, in press), dyslexia
(Morton & Frith 1993), and psychopathy (Blair, in press). These
models require the clear representation of the cognitive underpinning of behaviour and are a particularly powerful tool for
representing the fact that behaviours could arise from different
developmental histories. With specific reference to criminal
behaviour, high levels of violence are associated with a variety of
different groups. With delinquents, this violence has been
linked to disinhibition caused apparently by frontal lobe deficits
and/or maldevelopment (e.g., Moffitt 1993). Although the
high level of aggression in psychopaths has also been attributed
to a particular frontal lobe deficit (Gorenstein & Newman 1980),
psychopaths do not seem to be any more deficient in general
frontal lobe functioning than other criminal groups (see, for a
review, Kandel & Freed 1989). The second crucial developmental relationship that causal models clearly express is that the
behavioural consequences of particular structures are mediated
by the rest of the cognitive system. This phenomenon is hinted
at in the target article; Mealey suggests that the genotype for
sociopathy that is expressed in males as sociopathy may be
expressed in females as Briquet's Syndrome. However, she
offers no consideration of what differences in the cognitive
structures of males and females might mediate this distinction.
In conclusion, we appreciate that other researchers are now
using a multiple level, developmental approach to sociopathy.
We only suggest that without cognition sociopathy cannot be
fully understood.
Sociopathy or hyper-masculinity?
Anne Campbell
Psychology Department, Durham University, Science Laboratories, Durham
DH1 3LE, England, a.campbell@durham.ac.uk
Abstract: Definitional slippage threatens to equate secondary sociopathy with mere criminality and leaves the status of noncriminal sociopaths ambiguous. Primary sociopathy appears to show more environmental contingency than would be implied by a strong genetic trait
approach. A reinterpretation in terms of hypermasculinity and hypofemininity is compatible with the data.
Mealey's criteria for distinguishing primary and secondary sociopaths are that the former show a greater genetic predisposition, less environmental contingency of their behaviour, an
absence of secondary emotions, and more marked hypoarousal.
But when we turn to the data, this distinction, critical to policy
and program development, starts to dissolve. Primary sociopaths become equated with chronic criminal offenders (sect.
2.2.2, para. 2), despite Mealey's initial statement that sociopaths
may constitute as few as 33% of this population (Introduction,
para. 1). In accounting for secondary sociopaths, who are more
canalised by early developmental experiences, she asserts that
studies of juvenile delinquents can be used as a reliable guide
to their childhood environments. If secondary sociopaths are
simply criminal offenders who begin their careers as juvenile
Commentary/ Mealey: The sociobiology of sociopathy
delinquents (and most do), then sociopathy becomes coterminous with criminality. Although the DSM-III requires criminality as a defining feature of sociopathy, Mealey herself criticises it on these very grounds. If the term sociopath is instead
used (as Mealey prefers) as descriptive of a particular constellation of behavioural and affective traits that have adaptive advantage, we should expect sociopaths to thrive wherever there is
competition and individual disadvantage, be it on Wall Street or
in the Bronx. Mealey recognises this but asserts confusingly
both that noncriminal sociopaths are "subclinical" (sect. 2.5.1,
para. 2), implying a rather weak behavioural manifestation of the
trait, and that "almost all sociopaths from the upper-classes will
be primary sociopaths" (sect. 3.1.1, para. 1), implying a rather
strong manifestation.
The distinction between simple criminality and sociopathic
criminality is an important one for Mealey's argument, because
it leads to different predictions about the age-crime curve.
Gottfredson and Hirschi (1990) have argued that high crime
rates during the teen years and early twenties transcend nation,
offence, sex, and race - in line with Daly and Wilson's (1988)
evolutionary account. However, Blumstein et al. (1988) point
out that a small subset of chronic offenders show a constant rate
of offending throughout their criminal careers with no evidence
of a decrease with age. Mealey suggests that these chronic
offenders are likely to be sociopaths who "consistently use the
same strategy in every situation" (sect. 1.2, para. 2) and hence
we should expect to see a similar invariance in their offending
rates, but this is not found. Hare et al. (1992) computed mean
number of convictions per year free for sociopaths and nonsociopaths. Both groups show a similar increase and decrease in
convictions over the age span 16 to 46. This suggests that, unlike
chronic offenders, sociopaths' offending follows the same age
trajectory as that of other criminals. The age-relatedness of
criminal behaviour by both sociopaths and other criminals
suggests that their behaviour may be more environmentally
contingent than Mealey suggests.
I offer the following proposal. Cheating, as discussed by
Mealey, implies a strategy in which an individual exploits others'
cooperation selfishly by a failure to respond in kind. As she
notes, most of us do not cheat because it is a risky strategy, both
in the short term (immediate detection and retaliation) and the
long (social shunning). Yet whereas cheating is only one form of
risk taking, the converse is not true, making riskiness the
superordinate construct. Daly and Wilson (1988) have amply
documented the evolutionary relevance of risky behaviour to
males in their early reproductive years, arguing that the greater
preference for risk by males is part of our evolved psychology.
Mealey rightly observes that women show less cheating (sociopathy), but they also show less risk taking overall, as measured
by, for example, driver deaths and interpersonal violence. This
suggests that there might be a continuum of psychological
readiness to take risks (including cheating), with the male mean
"shifted up" relative to that of females.
Two psychological dimensions powerfully discriminate between males and females. Initially called masculinity and femininity, they are now more widely referred to as instrumentality
and expressivity (Spence 1985). Instrumental traits include
being individualistic, aggressive, and willing to take risks, embodying a competitive view of relationships and a readiness to
assert oneself at the expense of others. Expressive traits include
being gullible, sensitive to the needs of others, and compassionate, embodying an interdependent view of relationships and a
willingness to sacrifice oneself for others. While Mealey posits
sociopathy as the underlying continuum behind cheating,
I suggest that instrumentality and expressivity may be twin
continua lying behind riskiness, and that sociopaths may be
viewed as a case of simultaneous hyper-masculinity and hypofemininity. Like sociopathy, instrumentality and expressivity
have a substantial genetic component (Bouchard & McGue
1990; Mitchell et al. 1989; Rowe 1982). Just as sociopaths exceed
normals, men exceed women on both the psychoticism and the
sensation-seeking scale. Neurochemically, male rats and primates have lower serotonin levels than females, and estrogen
reverses the inhibitory effect of dopamine on the pituitary. The
direct excitatory effect of estrogen is even greater than that of
norepinephrine, and estrogen also inhibits cortical responsivity to the inhibitory transmitter adenosine (see Hoyenga &
Hoyenga 1993, for references and their proposal of relating
masculinity to aggression). In evolutionary terms, men possessing masculine qualities of independence and aggression probably had a fitness advantage whereas the simultaneous presence
of feminine characteristics of nurturance and empathy may have
mediated social bonding and cooperation. Although sociopaths
show no change over the life course in their lack of concern for
others (Hare et al. 1992), their willingness to engage in risky
behaviour diminishes after the peak reproductive years when,
in evolutionary terms, it is of most value. In primary sociopaths,
we may be seeing the far end of the male instrumental continuum completely untempered by the softening effects of
expressivity.
Cheaters never prosper, sometimes
H. Lome Carmichael
Department of Economics, Queen's University, Kingston, Ontario, Canada
K7L 3N6. carmykle@qucdn.queensu.ca
Abstract: In the Frank (1988) model, a small increase in the number of
cheaters will soon be reversed. It is not clear that this prediction holds
for sociopathy. There are also many attractive evolutionary models that
do not admit a small, stable proportion of cheaters. Hence, without
definitive evidence about the character of early human society, we
cannot conclude that sociopathy has an evolutionary origin.
In reviewing earlier issues of BBS for examples of fine commentary, I was struck by the virulence of the debates surrounding
sociobiological papers like this one. To some proponents, the
fact that human life evolved on this planet is sufficient to relegate
all opposition to the realm of divine myth (e.g., Thornhill &
Thornhill 1992, especially the Authors' Response). Of the opponents' position, sometimes the phrase "society is to blame"
seems altogether too weak to be an effective parody. Indeed,
suggestions to the contrary are sometimes branded harmful and
worthy of suppression (e.g., the comment by Dupre (1992) in
the above collection).
Sociobiology has the attraction that it is at least a well-defined
theory. It has assumptions that can be expressed clearly, that are
amenable to logical manipulation, and that can generate clear
predictions. This, in an area as complex as human behavior,
makes it an easy target. No such model will ever be completely
true. Rather, one hopes that a simple model will capture aspects
of reality that are particularly useful and relevant to the questions at hand. Unfortunately, the target article fails to meet even
this weaker standard.
Linda Mealey has convinced me that primary sociopathy is
present in individuals at a very early age and develops regardless of parenting, and that secondary sociopathy requires a
combination of inherent vulnerability and bad luck. I am willing
to accept that these traits are heritable, although I do not think it
proven. The rings on this target article center elsewhere - on its
main hypothesis that sociopathy exists for a particular reason
illuminated by simple evolutionary theory.
Even if we accept that evolutionary processes are determinate, I wager that most of us could create a plausible society
that, had it existed for 100,000 years or so, would have selected
for just about any trait purported to exist in the species today. In
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
549
Commentary/Mealey. The sociobiology of sociopathy
the absence of a definitive historical record, therefore, the
empirical strength of the current paper rests entirely on the
charm, as a metaphor for human interactions, of the random
matching one-shot Prisoner's Dilemma model with imperfect
identification of types. But this is not the only charming model
available, and some of the others (including the same one with
different parameter values) do not admit a stable proportion of
cheaters, or do not admit cheaters at all. Also, if taken seriously,
the model chosen makes predictions about sociopathy that do
not seem to be true.
I will address these criticisms in reverse order. Recall that if a
mixed strategy profile is evolutionarily stable, then if a particular
strategy A increases (or decreases) in frequency, the payoffs to all
the strategies must change so that A is relatively disadvantaged
(or advantaged). Natural selection will then ensure that strategy
A goes back to its proper place. If we are to take the current
application of Frank's (1988) model seriously, then the proportion of sociopaths in a society should follow a similar dynamic.
But does it? Would primary sociopaths in an ancient society
with "too many" of their own kind have had difficulty gaining
access to resources and mating partners? We just do not know,
and the target article is not helpful. Perhaps cooperators would
become more diligent, but if cheaters ganged up under a
charismatic Attila one suspects large numbers would be an
advantage.
The dynamic for secondary sociopathy is discussed in the
paper, but things seem to go the wrong way. One has to be
careful here - if I move from Kingston to New York City and as a
result my kids are more likely to become sociopathic, this could
be because environmental differences lead New York to have a
higher proportion of cheaters. Rather, suppose that a small
group of young sociopaths move to Kingston, all else the same.
According to the Frank/Mealey model, Kingston children will
now be less likely to become sociopathic. I have no evidence; but
like most parents, I think not.
Frank's (1988) model is a variant of the round robin, infinitely
repeated Prisoner's Dilemma introduced by Axelrod (1984) in
his celebrated competition. But there are other models that
have equal "charm' and are therefore equally (un)likely to
capture the essence of the conditions facing primitive homo
sapiens. Here are two examples:
Consider a model where individuals match up randomly, play
a one-shot Prisoner's Dilemma, and then have the choice of
continuing or terminating the match (Carmichael & MacLeod
1994; Stanley 1993). If the match ends, both parties go back,
anonymously, to the matching market. If not, the partners may
stay matched until death, continuing to play a Prisoner's Dilemma each period. For a modern image, think of the matching
market as a large, dimly lit singles' bar.
Even though cooperators play a repeated Prisoner's Dilemma, the strategy "tit for tat" does very poorly. It is quickly
invaded by cheaters who defect at the first opportunity and then
move on to a new match. An interesting evolutionarily stable
strategy is for cooperators to offer (and demand) an exchange of
gifts at the beginning of any new match (Carmichael & MacLeod
1993). Cheaters in this society would have to buy a succession of
gifts, and this effectively screens them out. This model makes
quite a few predictions about the form of the gifts that must be
used. l
bargainers will be vulnerable to the "terrible twos" strategy of
demanding almost everything, backed up with the emotional
threat to ensure that otherwise the hyena gets everything.
Faced with such an opponent, a rational bargainer cuts his
losses, takes what is offered, and moves on. Of course an entire
society of two-year-olds does very poorly, and can be invaded by
a group whose members fight if they do not receive at least half
the spoils. This strategy is evolutionarily stable - it quickly
reaches agreement with itself and can do no worse than any
invader it meets. 2 Again, in this simple model, there is no room
for cheaters.
Readers will recognize the "bourgeois" strategy of Maynard
Smith (1982), but there are some new twists. In particular, if
people are of two types, there are many equilibria where one
type does better than the other. If men fight whenever they get
less than one-third and women fight whenever they get less than
two-thirds, for example, this is evolutionarily stable. Equilibria
like these require that one's notion of territory be socially
determined. There must be a way for early experience and
teaching to establish and coordinate internal notions of what one
deserves out of life. Sociobiology can therefore account for the
existence of systemic discrimination, and society may indeed, at
least partly, be to blame.
The point, of course, is not that these are better models of
reality than the one used in the target article, but they do seem
equally plausible, and they have implications that are at least as
attractive and intriguing. Perhaps they each capture relevant
but separate aspects of reality. If so, Mealey's conclusion - that
evolution is unable to rid society of a small proportion of cheaters
- is not robust. (The rule of emotions in these models, by
contrast, is robust.) Cheaters do prosper, no doubt. But until we
have excellent evidence about the exact nature of early human
society, or until we can show that in any sensible evolutionary
model there will survive a small proportion of cheaters, sociobiology will not be able to tell us why.
Here is another one (Carmichael 1994). Suppose we retain the
one-shot matching framework of Frank but change the game
from a Prisoner's Dilemma to a bargain. People meet and have to
decide how to divide the spoils of some joint venture (the carcass
of some animal, perhaps). If they can agree quickly on a division,
then all is well. If they cannot, the spoils disappear, dragged
away by a hyena.
Strategies that do well in these bargains will proliferate into
the future. An intraspecies arms race might develop, where
"bargaining ability" grows over time. Rational and unemotional
In a target article of exceptional scholarship and originality,
Mealey has put forward an interesting new interpretation of
sociopathy. Given the vast range of material covered by the
article and the limited space available for my commentary, I
shall confine my comments to the specific game theoretic model
that underpins Mealey's interpretation. I shall argue that it
cannot do what is required of it, and I shall suggest an
alternative.
Like most earlier theorists who have used game theory to
explain the evolution of social behavior, starting with Maynard
550
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
NOTES
1. Among other things, gifts should have low use value, be overpriced, and should be hard to recycle as gifts in a subsequent match. Cut
flowers and chocolates work - house plants and money do not.
2. Unless, of course, the invader has a weapon. The arms race in this
model is real.
Prisoner's Dilemma, Chicken, and mixedstrategy evolutionary equilibria
Andrew M. Colman
Department of Psychology, University of Leicester, Leicester LE1 7RH,
England, amc@lelcester.ac.uk
Abstract: Mealey's interesting interpretation of sociopathy is based on
an inappropriate two-person game model. A multiperson, compound
game version of Chicken would be more suitable, because a population
engaging in random pairwise interactions with that structure would
evolve to an equilibrium in which afixedproportion of strategic choices
was exploitative, antisocial, and risky, as required by Mealey's
interpretation.
Commentary/Mealey:
(a)
Smith (1974; Maynard Smith & Price 1973), Mealey relied on a
two-person game, specifically the Prisoner's Dilemma game. A
generalized payoff matrix for any two-person, two-choice game
can be represented as follows:
CD
C RS
DTP
The row and column players each choose between two pure
strategies, C and D, the payoffs shown in the matrix are those to
the row player. Thus the payoff to the row player following a C
choice is ft or S, depending on whether the column player
chooses C or D, respectively, and following a D choice it is Tor P,
depending on whether the column player chooses C or D,
respectively. In the Prisoner's Dilemma game, C represents
cooperation and D defection, and by definition T > R > P > S,
so that the row player receives the best payoff by choosing D
(defect) while the column player chooses C (cooperate), the
second-best payoff by choosing C while the column player
chooses C, and so on. Although it is customary to show only the
row player's payoffs, the game is the same from the column
player's point of view, so that the column player also gets the
best payoff by choosing D while the row player chooses C, and
so on.
The standard two-person model is of limited value in deterining evolutionary processes. We need to establish what will
ippen in an entire population in which individuals interact
ith one another in pairwise games with this strategic structure,
:suming that the payoffs represent units of Darwinian fitness
he lifetime reproductive success of the individual players) and
lat the propensity to choose C or D is heritable. For this
uqjose, we need to construct a multiperson compound game
dolman 1982, pp. 163-66, 243-49), in which it is assumed that
very player plays the same average number of two-person
;ames either with each of the others or with a random sample of
he others.
Considering the situation from a single player's viewpoint,
suppose that the number of other players is n and the number of
Dther players choosing C is c. The total payoff to a player
choosing C, denoted by P(C), and the total payoff to a player
choosing D, denoted by P(D), are then defined by the following
payoff functions:
P(C) = Re + S(n - c),
P(D) =
P(n - c).
The total payoff to a player adopting a mixed strategy is just a
weighted average of P(C) and P(D).
The values of the P(C) and P(D) payoff functions at their endpoints are found by setting c = 0 and c= n. Thus, if none of the
other players chooses C (i.e., c = 0), the payoff to a solitary C
chooser is Sn and the payoff to a D chooser is Pn. If all of the
other players choose C (i.e., c = n), then a C chooser gets Rn and
a solitary D chooser is Tn. It is clear that in the case of the
Prisoner's Dilemma game Tn can be interpreted as the temptation to be the sole D chooser, fin the reward for collective
cooperation, Pn the punishment for collective defection, and Sn
the sucker's payoff for being the sole C chooser.
Figure l(a) shows clearly that, in the case of the Prisoner's
Dilemma game (with T > R > P > S), the P(D) payoff function
strictly dominates the P(C) payoff function, which means that a
D choice pays better than a C choice irrespective of the number
of others choosing C. The evolutionary optimal strategy is
therefore not frequency-dependent, and the population will
(regrettably) evolve to a stable equilibrium in which every
player chooses D in every two-person encounter. This means
that the Prisoner's Dilemma game cannot provide a basis for
I&
The sociobiology of sociopathy
P(D)
(b)
P(Q
P(D)
P(C)
Figure 1 (Colman). Multiperson compound games based on 2 X
2 matrices. Panel (a) on the left is multiperson Prisoner's Dilemma; (b) on the right is multiperson Chicken. The P(C) and
P(D) functions indicate the payoffs to a player choosing C or D
when c of the other players choose C. Dashed circles indicate
stable equilibria.
Mealey's interpretation of sociopathy, in which the "cheater
strategy" (the D choice) corresponds to various criminal, delinquent, and generally antisocial or predatory forms of behavior
that she claims exist at a low frequency, in every society and
are maintained through frequency-dependent Darwinian selection.
A more appropriate game theoretic model might be a compound version of the game of Chicken, which Maynard Smith
(1976; 1978) and Maynard Smith and Price (1973) call the HawkDove game. This game is defined by the inequalities T> R> S
> P, and the P(C) and P(D) payoff functions are shown graphically in Figure l(b). In this case, the population will evolve to a
mixed-strategy equilibrium point, where the two payoff functions intersect. To the left of the intersection, when relatively
few of the others choose C (c is small), the C function lies above
the D function, which means that the fitness payoff from a C
choice is higher than from a D choice, so the number of C
choosers will increase relative to D choosers and the outcome
will move to the right as c increases. To the right of the
intersection, exactly the reverse holds: D choosers will increase
relative to C choosers and the outcome will move to the left as c
decreases. At the intersection, and only there, the strategies are
best against each another and are in equilibrium, and any deviation
from the mixture at that point will tend to be self-correcting. By
setting the parameters (values of the payoffs T, R, S, and P)
appropriately, the intersection point, and thus the proportion of
"predatory" D-choices, can be made as small as required.
It appears, therefore, that the Prisoner's Dilemma game
cannot underpin an evolutionary explanation of sociopathic
behavior, but that a multiperson compound game version of
Chicken, in which cheating is at least frequency-dependent,,
might be more promising. Chicken is the archetypal dangerous
game, because a player can outdo a co-player only by cheating
(choosing D) while the co-player behaves cautiously (by choosing C), and any such attempt to get the best payoff (T in the
payoff matrix above) involves a necessary risk of the worst
possible payoff (P). The interpretation of criminal, delinquent,
and generally antisocial behavior in terms of strategic choices
seems more natural in the game of Chicken. (For a more
detailed discussion of the strategic properties of Chicken and
some observations on its application to antisocial and criminal
behavior, see Colman 1982, pp. 98-104; 1995, sect. 9.6.)
ACKNOWLEDGMENT
Preparation of this commentary was facilitated by Grant No. L122251002
from the Economic and Social Research Council as part of the Framing,
Salience and Product Images project.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
551
Commentaryi'Mealey: The sociobiology of sociopathy
The sociopathy of sociobiology
Wim E. Crusio
G6n6tique, Neurog6n6tique et Comportement, URA 1294 CNRS. UFR
Biom&dicale, University Paris V Ren6 Descartes, 75270 Paris Cedex 06,
France, crusio@clti2.fr
Abstract: Mealey's evolutionary reasoning is logically flawed. Furthermore, the evidence presented in favor of a genetic contribution to the
causation of sociopathy is overinterpreted. Given the potentially large
societal impact of sociobiological speculation on the roots of criminality,
more-than-usual caution in interpreting data is called for.
Mealey's target article provides an enormous compilation of
data on vastly different subjects, varying from social psychology to evolutionary theory. Admirable as the amount of work
involved with gathering, digesting, and discussing this large
body of literature may be, the result is unfortunately not very
enlightening. The line of reasoning has become buried under an avalanche of uncritically enumerated research findings,
so that after some time the reader loses sight of exactly what
the author is trying to say, based on what evidence, and what
the assumptions are on which the author's interpretations are
based.
In consequence, I am not quite sure what Mealey's main
thesis is. As far as I see, she is trying to make two, not necessarily
related, points. First, that sociopaths can be divided into two
subtypes: primary and secondary sociopaths. Second, that this
subdivision has come into existence because of selective forces,
exerted during evolution on a genetic substrate underlying
sociopathy. I trust that other commentators who are more
competent on this subject than I will comment on the first point.
I will direct my scrutiny mainly at the second one, but not
without noting that neither evolution nor genetics appear to be
very relevant for Mealey's arguments regarding the first point,
and they clearly are irrelevant to the exclusively environmental
recommendations presented as following from the model (sects.
3.2 and 3.3).
To start with the evolutionary part, Mealey asserts that the
two forms of sociopathy came into existence as a result of two
different evolutionarily stable strategies (ESSs; sect. 1.2). Although it does not become clear what role natural selection is
playing here, the description of these ESSs clearly shows the
logical impossibility of having two different ESSs for one single
phenotype within one single population. If two different mechanisms are at work, then this implies that primary and secondary
sociopathy are two different, independent phenotypes. Yet, in
the last paragraph of section 2.3.1 it becomes clear that this is
not what the author has in mind. I would like to see some
clarification of this serious contradiction and an explanation of
how natural selection may have acted in order to result in two
different ESSs for one single phenotypical continuum. In addition, might it not be necessary to consider whether and how the
increasingly dramatic changes in living conditions over the last
few thousands of years (and even more so in the last few
centuries) may have influenced the nature of the natural selection involved, in the process perhaps rendering all this evolutionary speculation rather pointless?
The last paragraph of section 2.3.1 poses a number of other
problems, too. It is argued that some persons become primary
sociopaths because of their genotype. Others become secondary
sociopaths because of a smaller genetic load combined with
certain environmental causes (see also n. 14). Perhaps this is so.
Unfortunately, the evidence provided to support these ideas is
highly inadequate. Let me give a few examples. A number of
physiological factors are discussed, for which primary sociopaths
apparently differ from other people; this supposedly suggests a
genetic basis for primary sociopathy. But physiological variables
are phenotypes, too; there is nothing inherently different about
them that would suggest they are somehow more "heritable"
552
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
than other types of characters. Still, this appears to be implicitly
assumed. Take the finding that upper class, but not lower class,
boys who subsequently became delinquent showed physical
signs of hypoarousal (sect. 2.3.3, last para.), which is presented
as evidence for the idea that the upper-class individuals became
delinquent as the result of a particularly strong genetic predisposition. Only when hypoarousal would be a phenotype with a
heritability equal to unity and perfectly correlated with sociopathy would this conclusion be warranted. The finding that
almost all upper-class subjects that had been arrested came from
a biological high-risk group (sect. 2.3.3, last para.) is not conclusive either. As "biological high-risk group" probably means that
subjects descended from parents with an antisocial background,
the familial environment of these subjects is very likely to have
differed from that of control subjects too. Mealey herself seems
to recognize the importance of environmental influences, even
for primary sociopaths, given the recommendations at the end of
her article.
The only reasonably solid evidence provided for any genetic
basis for either form of sociopathy is heritability estimates,
derived from twin and adoption studies, 1 of around 0.60 (but
see BBS commentary on Wahlsten, 1990, for a discussion of
the scientific value of such estimates). However, even when
rather high, this value still tells us that genotype and environment explain about equal parts of the interindividual
variation observed. Summarizing, the genetic component of
Mealey's model is nothing more than just some elaborate speculation.
If the target article were dealing, for example, with the
mating behavior of black widow spiders or some other subject
without direct societal impact, the author might have indulged
in some speculation without much possible harm. But in the
present case we are dealing with people, not spiders. Scientific
speculation, however interesting in itself, may easily be interpreted by politicians, journalists, or others as stated scientific
fact - the more so if the speculative nature of an author's writings
is not clearly indicated as such. Sociobiologists appear to be
rather prone to this kind of oversight, hence the title of my
comment.
ACKNOWLEDGMENTS
The preparation of this commentary benefitted from support by the
CNRS (URA 1294), UFR Biomedicale (University Paris V Rene Descartes), DRED, and the Fondation pour la Recherche M6dicale.
NOTE
1. It should be noted that in all these studies at least the early part of
childhood environment is confounded with possible genetic effects.
Together with possible prenatal influences, this perhaps explains in part
why human behavior-genetic analyses so often render heritability estimates (in the broad sense, see Mealey's n. 6) that are much higher than
anything reported from animal research. Note also that experimental
animals are generally bred and reared in a rigorously standardized
environment in order to maximize genetic effects relative to environmentally induced variations (i.e., boosting heritability).
A neuropsychology of deception and
self-deception
Roger A. Drake
Department of Psychology, Western State College, Gunnison, CO 81231.
rdrake@wsc.colorado.edu
Abstract: As more criminals are imprisoned, other individuals change
their behavior to replace them, as predicted by the "floating niche"
theory of strategic behavior. The physiological correlates of sociopathy
suggest that research in cognitive neuroscience can lead toward a
solution. Promising pathways include building upon current knowledge
of self-deceit, the independence of positive and negative emotions, the
Commentary/Mealey:
lateralization of risk and caution, and the conditions promoting prosocial
behavior.
Mealey provides a useful summary of research from a variety of
disciplines on sociopathology. One of her best insights is the
differentiation between primary and secondary sociopaths and,
therefore, differences in the way they must be treated, to
protect ourselves from their deception and predation.
Of particular interest is her idea of a floating niche for deceit
and exploitation. This fits with the reality that although the
United States has a million individuals in prison, crime has not
decreased. Trivers (1985) predicted this in his discussion of
deception, when he said that, "In species with multiple mimics
[of honesty], the frequency of mimics is controlled by the
relative frequency of their models" (p. 420).
This statement implies that as deceivers - who are mimics of
honesty - become too common, there is greater detection of
their dishonesty. But if some deceivers are removed, for example by imprisonment, other individuals will shift to a strategy of
deception, because it will prove to be a more effective strategy
than honesty. Mealey argues that this produces a massive
practical problem, because of the great harm that sociopaths and
other deceivers do to others.
Trivers (1985) further argues that the most effective deceivers
will be those who can deceive themselves. This reduces the
possibility of cues of anxiety giving them away. Research by
Drake and Sobrero (1987) indicates that relative activation of the
right hemisphere of the brain produces behavior that is independent of a person's previously measured traits and attitudes.
This recommends a fruitful direction for research into a neurological basis for self-deception.
Mealey offers solutions to the predation of primary sociopaths
on a trusting population that are based principally on game
theory (introduced in sect. 1.2). She proposes that society can
reduce antisocial behaviors in primary sociopaths by establishing a reputation for detection, identification, and retaliation
(sect. 3.2.1). In other words, in an analogy with the Prisoner's
Dilemma game, she advocates that the outcomes for deception
must be harsh and must be publicized.
The problem with this approach is that primary sociopaths are
rarely deterred by fear or threat (Patrick 1994), but they do
approach pleasure. This implies that one direction for research
should be toward a greater knowledge of the separation of
positive from negative emotions. This is an established concept
within psychology (Cacioppo & Berntson 1994; Diener & Emnions 1985; Warr et al. 1983). Such research can profitably
gain from integration with the neuropsychological evidence
for distinctive regions of the brain associated with positive
versus negative emotions (Ahem & Schwartz 1985; Gainotti
et al. 1993; Merckclbach & van Oppen 1989; Schiff& Lamon
1989; Van Strien & Morpurgo 1992; Wittling & Rosehmann
1993).
Mealey describes physiological correlates and perhaps causes
of sociopathology (in sect. 2.3.3), yet physiological solutions to
predatory deception arc never mentioned. Her proposals are
mostly extensions of current government efforts, such as parental education, reduction of social stratification, and exciting
alternative careers. These approaches are not novel. Perhaps
the field of cognitive neuroscience could contribute more
through both basic and applied research on the differences in
brain structure, hormones, and neurotransmitters that differentiate the sociopath. These may eventually lead to treatments for
the lack of fear and other negative emotions that typify the
predatory sociopath.
The biobehavioral scientific community can further contribute with basic and applied research on the neuropsychology
of caution and risk (Drake & Ulrich 1992; Miller & Milner
1985) and on the conditions for the promotion of prosocial
behaviors (Levine et al. 1994). Mealey has provided us with a
strong outline of the extent of the problem, and has helped to
The sociobiology of sociopathy
point in the directions research should take us toward possible
solutions.
The role of emotion in sociopathy:
Contradictions and unanswered questions
Nancy Eisenberg
Psychology Department, Arizona State University, Tempe, AZ 85287-1104.
atnhe(« asuvm.inre.asu.edu
Abstract: Emotion is critical in Mealey s conceptual arguments. However, several of her assertions about the role of emotion in sociopathy are
problematic. Questions are raised regarding the link between lack of
anxiety and low levels of secondary emotions such as love and sympathy,
the argument that sociopaths are low in anxiety but high in neuroticism,
and the designation of anxiety as a secondary emotion.
In Mealey's arguments, emotion plays a crucial role; Mealey
linked the unique characteristics of primary psychopaths to
genetically based emotional mechanisms. A critical point in the
target article is that "guilt, anxiety, and sympathy are social
emotions that primary sociopaths rarely, if ever experience,"
(sect. 2.5.2, para. 8) although fluctuations in mood (e.g., anger,
depression, and optimism) are experienced by primary (and
secondary) sociopaths. A distinction is made between primary
emotions, such as fear, anger, and disgust, and secondary
emotions, which are viewed as more dependent upon learning
and socialization (note 1). Primary sociopaths are viewed as
experiencing primary emotions but as being very low in the
experience of secondary, social emotions such as shame, guilt,
sympathy, and love. In contrast, secondary sociopaths apparently are viewed as capable of experiencing normal amounts of
both types of emotional reactions.
Although Mealey's assertions are often supported by some
empirical literature, several conceptual issues can be raised.
One question concerns why primary sociopaths seem to have a
diminished capacity to experience secondary social emotions. In
note 1, secondary emotions are described as involving a critical
element of learning. Given that primary sociopaths are not
described as lacking in cognitive capacities or in the capacity for
primary emotions, it is unclear why they seldom "learn" to
experience such emotions. Is Mealey hypothesizing a biological
mechanism in sociopathy that affects proneness to learn secondary emotions? Lack of anxiety is used as an explanation for low
levels of guilt, but lack of anxiety does not explain low levels of
love, sympathy, and some other emotions.
A related point is Mealey's designation of anxiety as a secondary social emotion (sect. 2.5.2, para. 8). This is a critical point,
given that proneness to anxiety is viewed as a crucial element in
conditionability and in the development of conscience (i.e.,
negative emotional arousal) when one "cheats" or otherwise
deviates. However, anxiety is an emotion closely tied to the
primary emotion of fear (Plutchik 1984, p. 215) and is quite
different from secondary social emotions such as love, empathy,
sympathy, and true guilt. If primary sociopaths are described as
experiencing primary emotions, such as fear, at normal levels, it
is not obvious why they would not be as prone to anxiety as other
people.
On a related issue, Mealey noted that sociopaths score high
on neuroticism as assessed by Eysenck (1976). Yet the neuroticism scale contains items pertaining to anxiety (worry) and
guilt. Thus, the link between neuroticism and sociopathy would
argue against sociopaths being immune to anxiety and hence
being difficult to socialize. Mealey contradicts herself by saying
that primary sociopaths are low in anxiety and then implying
(sect. 2.3.2, para. 4) a link between neuroticism and primary
sociopathy.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
553
Commentary I Mea\ey. The sociobiology of sociopathy
In actuality, the link between neuroticism and primary
sociopathy may not be consistent or strong. Eysenck (1976)
argued that primary sociopaths are high in extroversion (E) and
psychoticism (P), with average scores on neuroticism (N),
whereas typical secondary psychopaths are high in E and N,
with average P scores. In other words, rather than being high on
anxiety, worry, and other negative emotions linked to neuroticism, primary sociopaths appear to be high on hostility and to
lack social feelings (elements of P). Perhaps primary sociopaths
are actually low on some aspects of neuroticism (e.g., guilt,
anxiety) and higher on others (e.g., proneness to experience
irritability and emotional lability). But if primary psychopaths
are not especially low in anxiety, the argument that they are
difficult to socialize due to lack of the inability to condition
would not seem plausible.
In brief, several points regarding anxiety are inconsistent in
the target article. My resolution of the inconsistencies is as
follows. It is likely that anxiety is not comparable to the secondary social emotions of guilt, shame, empathy, and love. If this is
true, then Mealey's assertions that primary sociopaths tend not
to experience secondary social emotions but do experience
primary emotions seem reasonable. In addition, primary psychopaths are probably average in general proneness to anxiety
and not particularly high in neuroticism. The reason that primary sociopaths are difficult to socialize is not that they are low
in anxiety per se; it is because they are low in the tendency to
experience social emotions, such as love, shame, and empathy,
that are likely to result in anxiety when people are in social
situations in which needs linked to social emotions are in
jeopardy (e.g., in socialization situations in which children
might elicit disapproval or rejection from their parents). If
primary sociopaths also do not seem to condition well in situations that do not involve social emotions and motivations, it may
be due to other factors, such as their hypoaroused nervous
system (which, as noted by Mealey, may impair learning), or
their tendency to approach rather than inhibit in approach/
avoidance situations.
This brings me to one final issue concerning the apparent low
baseline physiological arousal of sociopaths and their tendency
to be sensation seekers. Larsen and Diener (1987) proposed that
people high in affective intensity use emotion for stimulation to
compensate for a chronically low level of baseline arousal. If this
were true, one would expect people high in primary sociopathy
to be high on scores of affect intensity. Yet, at least for adults
(results for children are more complex), we have found that
emotional intensity is associated with sympathy (a secondary
social emotion; Eisenberg et al. 1994). Thus, interesting questions for future consideration concern the role of baseline
arousal in the tendency to experience primary and secondary
emotions intensely and whether intensity of emotional experience is linked to sociopathy.
Extending arousal theory and reflecting on
biosocial approaches to social science
Lee Ellis
Department of Sociology, Minot State University, Minot, ND 55701.
ellls@warp6.cs.misu.nodak.edu
Abstract: This commentary extends arousal theory to suggest an explanation for the well-established inverse correlation between church
attendance and involvement in crime. In addition, the results of two
surveys of social scientists are reviewed to reveal just how little impact
the biosocial/sociobiological perspective has had thus far on "mainstream" social science.
Mealey's target article provides a useful summary of how neurological and hormonal factors help to orchestrate human varia-
554
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
tions in criminal and antisocial behavior within an evolutionary
context. The evidence that has been amassed is consistent with
my work on possible neurohormonal (Ellis 1987a; 1990a) as well
as evolutionary (Ellis 1988; 1990b) underpinnings of crime and
delinquency.
Here two issues will be addressed briefly. First, I propose that
information reviewed in the target article can be extended to
explain a controversial observation, namely, that religious
people are less criminal than nonreligious people. Second, I
allude to two recent surveys that remind us of how little the
research reviewed by Mealey has thus far influenced the way
most social scientists think.
Mealey appropriately notes that arousal theory has garnered
substantial empirical support in recent years (sect. 2.3.2). According to arousal theory, for neurological reasons, persons who
are most prone toward criminal or antisocial behavior tend to be
under-aroused (or what I call suboptimally aroused; Ellis 1987a).
Thus, from the standpoint of arousal theory, criminal and antisocial behavior is an expression of sensation seeking and boredom
avoidance rooted in a nervous system that prefers stimulus
intensity and novelty that are greater than the environment
normally provides. [See also Zuckerman "Sensation Seeking: A
Comparative Approach to a Human Trait" BBS 7(3) 1984.]
In addition to predicting that people who are suboptimally
aroused will be more likely to commit crimes, arousal theory
suggests that criminally prone persons will attend religious
services infrequently. This follows from noting that most church
services are anything but exciting. As a result of infrequent
church attendance, criminally prone persons should also come
to hold relatively unorthodox religious views (Ellis 1987b; in
press). These deductions are supported by numerous studies
showing a significant inverse correlation between criminality
and religiosity (reviewed in Ellis 1985; in press).
If the hypothesized inverse association between religiosity
and criminal/antisocial behavior continues to be supported, one
could even postulate a new evolutionary explanation for religion. Perhaps religious institutions, rituals, and dogmas have
evolved in part to help individuals and their parents screen out
antisocial individuals from those being considered as prospective marriage partners. Females especially, who are prone
toward investing most heavily in offspring production, may use
religion as a filtering device in mate selection. In so doing, they
should minimize their risk of marrying someone who is extremely antisocial.
After reading Mealey's synthesis of the literature, it is hard
not to be impressed by how strong the evidence now is for the
involvement of biological factors in criminal and antisocial behavior. Nonetheless, a perusal of most criminology texts will
show that the sympathetic attention of most criminologists to
such factors is still minimal. Also, two surveys have indicated
how social scientists continue to resist the evidence that Mealey
reviews.
The first survey asked 182 criminologists in 1986 what theories they thought had the most merit in explaining criminal
behavior (Ellis & Hoffman 1990). Arousal theory was not mentioned by a single respondent, and less than 20% of the criminologists were sympathetic toward viewing either genetic or
neurological factors as significant causes of criminal behavior.
More recently, 164 of my fellow sociologists were asked to
estimate how much of the variance in criminality could be
attributed to biological, as opposed to social environmental,
factors (Sanderson & Ellis 1992). The mean percentage attribution was 13% for serious crime and 9% for minor crime and
delinquency. The median percents were even lower.
Overall, Mealey's article shows that major progress has been
made over the past two decades in identifying biological contributors to criminal and antisocial behavior. Nevertheless, much
remains to be accomplished in the way of disseminating this
information to the social science community at large.
Commentary/Mealey:
Sociopathy and sociobiology: Biological
units and behavioral units
Carl J. Erickson
Department of Psychology - Experimental, Duke University, Durham, NC
27708. carl@psych.duke.edu
Abstract: Behavioral biologists have long sought to link behavioral units
(e.g., aggression, depression, sociopathy) with biological units (e.g.,
genes, neurotransmitters, hormones, neuroanatomical loci). These units,
originally contrived for descriptive purposes, often lead to misunderstandings when they are reified for purposes of causal analysis. This
genetic and biochemical explanation for sociopathy reflects such problems.
The ill-defined field of sociobiology has been a mixed blessing.
At its best it has been a heuristic force, shaping new questions
and providing new insights into sex ratios, altruism, and a
variety of behavioral phenomena. At its worst it has fostered a
simplistic set of inferences in which utility means adaptation
cans evolution means natural selection means heritability
means genes. Mealey's analysis reflects both the strengths and
the weaknesses of sociobiology s contribution.
The research on infanticide provides a useful model from
which to examine Mealey's views on sociopathic behavior.
Infanticide, in both animals and humans, has often been dismissed as maladaptive, pathological behavior; however, sociobiologists, encouraged by a renewed interest in sexual selection,
have argued that infanticide might serve biological fitness.
Twenty years of research has strengthened that supposition.
Mealey suggests that sociopathy, like infanticide, has utility for
those individuals who exhibit it, and she notes that it is important for society to recognize this fact in coming to grips with the
behavior. Such perspectives are welcome and deserve encouragement, but the comparison to infanticide also illuminates
some of the limitations of her argument. Sociobiological interpretations work best when they relate to very specific behavior
patterns having a direct bearing on reproductive success. Even
then they can be dismissed as "just so" stories unless they carry
enough detail and eliminate enough alternatives to make the
adaptationist argument inescapable. For example, Vom Saal
(1985) finds that the frequency of infanticide among male mice
increases dramatically following ejaculation, and it remains high
until just before their own young are born. It then drops to a low
level until their young disperse, at which time it rises again.
These patterns are so complex, so challenging to alternative
explanations, and yet so consistent with an adaptationist viewpoint that it is difficult to escape the sociobiological conclusion.
Moreover, when males kill the infants of other males, there is an
obvious impact on inclusive fitness. An evolutionary explanation
is therefore tenable, if not compelling. On the other hand, with
the possible exception of rape, sociopathic behavior has no
obvious connection to inclusive fitness. To be sure, a competitive edge in the most general sense is implied when it occurs at
low frequency in the larger population, but how this advantage
translates into a gain in inclusive fitness (a necessity for the
evolutionary argument) remains unclear.
Mealey suggests (sect. 3.1.1) that sociopathic behavior actually takes two forms: (1) a primary, "inborn" pattern and (2) a
secondary, "environmentally contingent" form. Although only
the first of these is explicitly presented as a "genetically determined strategy," both are presented as "genetically based." Such
preformationistic positions have drawn a barrage of criticism
over the past twenty years (cf. Lewontin et al. 1984), and even
the most traditional ethologists and sociobiologists now concede
that although genes make a difference, they do not govern any
behavior in such deterministic fashion. Increasingly, developmental biologists and psychologists recognize that the developing organism is in a sense "self-organizing," and that there is no
"blueprint" for behavior in the genome. Even if one accepts
Mealey's categories of primary and secondary sociopathy (which
The sociobiology of sociopathy
I am reluctant to do), there still would be no logical reason to
assume that genes are any more involved in one than the other.
Moreover, even if the patterns vary in the frequency-dependent
way Mealey describes (and there appears to be little evidence
for this), it would be virtually impossible to distinguish the
secondary sociopathy of an evolutionary adaptation from the
coping behavior of a remarkably flexible human cognitive process. Brain mechanisms for the latter have evolved, to be sure,
but the specificity of their role is very different from those that
sociobiologists envision.
Speaking more generally I might add that hormones, neurotransmitters, brain nuclei, and so on also make a difference in
behavior, but again, they do not control behavior in the strong
sense implied by Mealey's characterization. Testosterone's action is a case in point. Mealey's exposition would lead one to
believe that higher levels of this hormone make male adolescents bigger, stronger, and more aggressive. The research
literature remains unclear on testosterone's relationship to aggression, but it is quite clear on its relationship to body size.
Testosterone terminates the growth of the long bones. Young
boys who take anabolic steroids are short, and the castrati of
18th- and 19th-century Italy were excessively tall. Mealey
suggests that aggression and testosterone are mutually stimulating and generate a positive feedback loop, but the relationship is
far more complex than she suggests. True, subnormal levels of
testosterone may indeed correlate with subnormal levels of
behavior, but once serum testosterone reaches normal levels or
above, the relationship breaks down. A panoply of internal and
external contextual conditions modulate testosterone's influence. Liver activity (which may reflect drug and alcohol use),
carrier proteins, receptor numbers, and past experience are
among the many factors that complicate the relationship between blood testosterone level and aggressive or sexual behavior. Moreover, many of these factors have rate-limiting effects,
preventing excessive testosterone from having excessive effects
on behavior. In short, neither testosterone nor any other behaviorally relevant chemical plays a determining role in any behavior. Generally speaking, biological units (genes, hormones,
neurotransmitters, or chunks of brain) do not have a one-to-one
causal relationship with specific behavior patterns.
Mealey musters an impressive volume of literature in her
analysis of sociopathy. Much of it contributes to a better understanding of this intriguing behavior, but the sociobiological
perspectives are largely untestable and add little. Moreover, by
suggesting that the behavioral variants are genetically determined, this formulation encourages the unfortunate conclusion
that developmental studies are irrelevant when, in fact, they are
critical.
Psychopathology: Type or trait?
H. J. Eysenck
Professor Emeritus of Psychology, University of London, London SE5 8AF,
England
Abstract: Mealey proposes two categorical classes of sociopath, primary
and secondary. I criticize this distinction on the basis that "type"
constructs of this kind have proved unrealistic in personality taxonomy
and that dimensional systems capture reality much more successfully. I
suggest how such a system could work in this particular context.
Mealey's discussion of the sociobiology of sociopathy is remarkably complete and succinct; it covers much ground and establishes its primary contentions admirably. I have little to criticize,
other than the main point of the paper, namely, establishing the
two categorical classes of sociopaths - primary and secondary
(with criminals who are not sociopathic as a third type,
presumably).
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
555
Commentary/Mealey: The sociobiology of sociopathy
I have always argued that the psychiatric commitment to a
categorical sphere of diagnosis is fundamentally wrong and has
to be replaced by a dimensional system, if we are to be governed
by factual evidence (Eysenck 1960; 1970). The low reliability of
categorical diagnoses, resulting from overlapping classifications,
is notorious, and the claim that the intersituation has been
remedied by successive editions of the APA Diagnostic and
Statistical Manual has not been found to be justified (Kirk &
Kutchins 1992). Even here, sanity is breaking in; DSM-IV
agrees that a dimensional approach is scientifically preferable,
although refusing to use it in actuality because of old, established habits of medical diagnosis.
Mealey suggests an absolute contrast between primary and
secondary sociopaths but reports no evidence that these could
be separated diagnostically with any acceptable reliability.
From experience, I would be surprised if agreement between
professional observers would be greater than 0.3 or thereabouts;
clearly insufficient for scientific or practical purposes. I believe a
much better picture would be one in which individuals were
represented by points in a hollow, three-dimensional globe, the
three diameters of which would represent Extraversion, Neuroticism, and Psychoticism (Eysenck & Gudjonsson 1989); the
group of primary and secondary sociopaths would then appear as
clusters of points in the E + N + P + octant, but certainly not as
two quite separate and distinct clusters. The differences Mealey
notes would locate primary sociopaths further out toward the
periphery, and possibly closer to P than secondary sociopaths,
who might be closer to the centre, and nearer to £ and N. But all
differences would be dimensional, with no absolute demarcations. And, clearly, a system of diagnosis referring each point in
this globular universe to the three dimensions, as a three-digit
number, would be more reliable and more valid than a verbal
type of construct, as suggested by Mealey.
It would be possible, of course, to rotate these three reference
axes to accommodate the theories of Gray and Cloninger, as
Mealey suggests. I believe that both have made important
contributions to the psychophysiological interpretation and understanding of personality-related behaviour, but I am not
convinced that the evidence presented by them is adequate to
suggest the rotation of the three primary axes. The latest study
of the Cray system (Carver & White 1994) suggests to me that
the activity of the BAS system aligns it with extraversion, that of
the BIS system with neuroticism. But however that may be, and
even if future research should force some degree of rotation, the
principle of dimensional diagnosis would remain unaffected. If
nature has not in fact produced some 300 separate psychiatric
illnesses, as DSM suggests it has, then no human effort to force
diagnoses into this Procrustean bed is likely to be successful.
Nor will efforts to force behaviours into a two-type system be any
more successful. Primary and secondary psychopaths may occupy different positions in our globe, but there are innumerable
gradations observable in the points lying between pure examples of these points. If the notion of categorical disease entities
breaks down completely when such time-honoured groups as
schizophrenics and manic-depressives are concerned (Kendell
& Brockington 1980), what hope is there for primary and
secondary sociopaths?
Primary sociopaths are said to be mainly activated by genetic
factors, secondary ones by environmental ones, but surely no
one would argue that the ratio of these two factors is not
infinitely variable, and at present incapable of measurement. All
the genetic and environmental correlates and possible causes of
both conditions (and criminality as well) are continuously variable; they cannot conceivably give rise to two categorically
distinct groups. Indeed, it is not even clear just how Mealey
would diagnose her primary sociopaths phenotypically. She says
that "there will always be a small, cross-culturally similar and
unchanging baseline frequency of sociopaths and a certain
percentage of sociopaths . . . will always appear in every culture, no matter what the socio-cultural conditions" (sect. 3.1.1,
556
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
para. 1). But how would we ever test such an hypothesis, in the
absence of methods of diagnosing the genetic make-up of a given
individual? And is it really suggested that each and every one of
these primary sociopaths in fact had the identical genetic makeup? Surely there would always be a more-or-less and a gradual
fading into the genetic make-up of the secondary sociopath, with
no clear-cut boundary between them. And it is of course quite
doubtful whether the proportion of sociopaths would indeed be
identical from group to group. Rushton (1994) has reported
evidence of very marked differences in criminality between
racial groups; it is not unlikely that this portends similar differences in the number of primary sociopaths.
Mealey also seems to suggest that treatment, whether preventive or later in life, should be categorically different for
primary and secondary sociopaths. This again seems to go
counter to fact. Unless diagnosis was well above the 0.70 level,
any scheme of allocating treatment differentially to primary and
secondary sociopaths would be doomed to failure. Considering
that the great majority would fall between the two extreme pure
groups, what should we do with them? Again, a graded, dimensional system would seem much better able to accommodate the
facts of human diversity. Fortunately, it should not be difficult to
translate the numerous psychological, hormonal, and physiological characteristics of Mealey's two types into a dimensional
language, thus transforming an unnatural typological system
into a much more natural dimensional one.
The epigenesis of sociopathy
Aurelio Jose Figueredo
Department of Psychology, University of Arizona, Tucson, AZ 85721.
ajf@arizvms/ajf@ccit.arlzona.edu
Abstract: Mealey distinguishes two types of sociopathy: (1) "primary," or •
obligate, and (2) "secondary," or facultative. Either sociopathy evolved
twice, or one form is derived from the other, e.g., through: (1) genetic
assimilation generating polymorphism in the relative strength of biases
favoring the development of otherwise facultative strategies, or (2)
independently heritable but strategically relevant characteristics biasing the optimal selection of facultative strategies.
Mealey's target article represents an important contribution to
the study of sociopathy. She is to be congratulated on a theoretically insightful synthesis and creative reinterpretation of a wideranging assemblage of scientific findings on the topic. This kind
of work is a sure sign that evolutionary psychology is rapidly
maturing in its ability to incorporate and accommodate otherwise fragmented and disparate empirical content from the
traditional social sciences by providing a meaningful functional
context and a powerful interpretive framework. Mealey's major
claim is that sociopathy can be productively seen as an evolved
adaptive strategy for intraspecific social parasitism, or "cheating." Although this identity remains far from conclusively established, the case for functional equivalence is too compelling to
dismiss as mere coincidence. A less persuasive secondary claim,
however, is the proposed distinction between "primary" and
"secondary" sociopathy in both phenomenology and etiology. It
is about this second and more minor claim that I harbor certain
reservations.
Although I have never worked explicitly on sociopathy, I have
recently been involved in various collaborative research projects dealing with the deviant social and sexual strategies of
competitively disadvantaged males. A major and recurring
problem in this line of research concerns whether the deviant
behavior patterns represent what behavioral biologists distinguish as "alternative" versus "conditional" adaptive strategies
(Alcock 1989; cf. Mayr 1974). An "alternative" strategy can be
defined as an adaptive strategy that is obligate for the individual,
Commentary/Mealey:
but for which the genetic predisposition is polymorphic within
the population. A "conditional" strategy can be defined as an
adaptive strategy that is facultative for the individual - contingent for its phenotypic expression upon environmental circumstances - but for which the genetic preparedness is monomorphic throughout the population.
In a study of domestic violence, Figueredo and McCloskey
(1993) found that a structural equations model, based on 21
manifest indicators that were theoretically related to the depressed mate quality of the perpetrator, predicted 60% of the
variance in spouse abuse and an associated 25% of the variance
in child abuse. In a clinical sample of juvenile sex offenders,
Russell et al. (in preparation) found that a series of moderated
multiple regressions predicted a sequential progression from
psychosocial deficits to sexual deviance to nonsexual criminality
and, finally, to sexual criminality, with psychosocial deficits
functioning as significant upregulators of that sequential progression. In both of these path models, the development of
deviant strategies was driven by individual failure at mainstream social and sexual strategies, seemingly favoring the
"conditional" strategy hypothesis.
In an explicitly behavior-genetic study, however, Rowe et al.
(in press) found that the single most important predictor of
juvenile delinquency, a measure reflecting the relative allocation of reproductive effort into mating effort (as opposed to
parental effort), was highly heritable. In this study, individual
failure at mainstream social and sexual strategies seemed more a
consequence of this behavioral deviance than its initial precondition, seemingly favoring the "alternative" strategy hypothesis.
Regrettably, we have never measured sociopathy per se in any of
these studies but it would be surprising if it were not among the
major contributors to these deviant behaviors. Nonetheless,
Mealey's proposal that sociopathy is discriminate into two
distinct types, based primarily on degree of genetic preparedness (cf. Garcia & Ervin 1968; Garcia et al. 1974; Seligman 1970;
Seligman & Hager 1972), would greatly mitigate the apparent
discrepancies between these various findings.
I am nevertheless reluctant to accept that formulation. For
one thing, conditional and alternative strategies are not necessarily mutually exclusive mechanisms. Certain microevolutionary processes, such as genetic assimilation (Waddington 1957)
may produce developmental biases favoring the development of
certain phenotypes over others in response to specific environmental contingencies. Thus, even within the context of conditional strategies, genetic polymorphism in the relative strength
of these developmental biases could be generated in a population of otherwise facultative strategists. In addition, it is possible
that the chance individual possession of independently heritable (but strategically relevant) characteristics may bias the selection of adaptive strategies (i.e., "reactive heritability'). For
example, an individual with a sexually attractive phenotype will
be more effective at pursuing certain mating strategies than
others and would, thus, be rationally expected to select them
preferentially.
In the absence of more conclusive evidence I am personally
biased toward the belief that it is more parsimonious to postulate
a continuous trait than a discontinuous typology. This is not to
say that such discrete typologies do not exist, as there are
numerous nonhuman animal examples (Alcock 1989). In this
case, however, it may be necessary to postulate that sociopathy
arose not once but twice in human behavioral evolution; otherwise it is necessary to derive one form secondarily from the
other, as the mechanisms proposed above purport to do. These
and other plausible alternative hypotheses should be more fully
explored, if not exhausted, before accepting the reality of a more
complex state of affairs. Nevertheless, I must acknowledge that
Mealey's theory is at least consistent with all of the data in this
line of research that I have had direct involvement with, and
holds the further promise of reconciling certain seemingly
discrepant results.
The sociobiology of sociopathy
"Just So" stories and sociopathy
Andrew Futtermana and Garland E. Allen"
"Department of Psychology, College of the Holy Cross, Worcester, MA
01610; bDepartment of Biology, Washington University, St. Louis, MO
63130. futterman@hcacad.holycross.edu; allen@biodept.vmstl.edu
Abstract: Sociobiological explanation requires both a reliable and a valid
definition of the sociopathy phenotype. Mealey assumes that such
reliable and valid definition of sociopathy exists in her "Integrated
Evolutionary Model.' A review of psychiatric literature on the diagnosis
of antisocial personality disorder clearly demonstrates that this assumption is faulty. There is substantial disagreement among diagnostic
systems (e.g., RDC, DSM-III) over what constitutes the antisocial
phenotype, different systems identify different individuals as sociopathic. Without a valid definition of sociopathy, sociobiological theories
like Mealey's should be viewed as entirely speculative.
Sociobiologists who study human behavior are confronted with a
daunting task. They seek to provide a compelling evolutionary
explanation for the emergence of behavioral phenotypes such as
altruism, sociopathy, alcoholism, homosexuality, or any other
trait that seems at first glance to be individually maladaptive. In
order to do so, they must demonstrate that the phenotype
advances the fitness of individuals, or relatives who demonstrate
it. Darwinian fitness is claimed first by demonstrating that the
behavioral phenotype in question is stable and consistent across
environments, and then, by demonstrating that this behavioral
phenotype in some manner confers an adaptive advantage over
rival traits. In circumstances in which they cannot demonstrate
consistency of phenotype or little is known about genetic and
developmental mechanisms, sociobiologists often introduce
evolutionary "models" to "explain" the trait's persistence. Another term for these models is "just so" stories, taken from the
Rudyard Kipling book in which animal curiosities are fancifully
and allegorically explained for the amusement of children (e.g.,
"How the Leopard Got His Spots"). However troubling or
important these behaviors may be, such behavior-genetic explanations amount to little more than speculations that may sound
reasonable but for which there is little or no evidence.
In the case of the "just so" story to explain the persistence of
sociopathy developed by sociologist Linda Mealey, not only are
the mechanisms and developmental processes underlying the
sociopathy trait not specified, but the stability and consistency
for the sociopathy phenotype itself has not even been demonstrated. And unless it can be demonstrated empirically that a
sociopathy phenotype remains stable over a range of environments, a study of its genetic base becomes impossible.
One reason for this is that the classification scheme for
sociopathy is not firmly fixed so that we should have any
confidence that descriptions of the trait are either reliable or
valid. Unlike the classification of Kettlewell's moths as speckled,
dark, or white in rural and industrial Britain - a classification
scheme that is broadly understood relative to selection effects
and remains largely unchanged and unquestioned after 90 years
- the validity of the classification scheme for sociopathy has
changed frequently, and the properties that comprise the syndrome have been and still are assessed in a multitude of different
ways. Moreover, unlike judgments of moth color, identification
of sociopathy requires judgments of properties such as "lack of
remorse," "impulsivity," "reckless disregard for others," and
other social behaviors (American Psychiatric Association 1994)
that are subjective and of highly variable description. Thus, that
Mealey treats sociopathy "inclusively" should not prompt us to
concede that she has identified sociopathy in any meaningful
way or that she or other independent observers can assess the
disorder reliably. As Kitcher (1985, p. 124) rightly points out:
"Those who engage in studies of evolution of behavior need to do
some work before they can reach the point from which Kettlewell began." [See also BBS, multiple book review of Kitcher's
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
557
Commentary/Mealey:
The sociobiology of sociopathy
Vaulting Ambition BBS 10(1) 1987.] Unless sociobiologists like
Mealey can demonstrate that sociopathy is defined consistently
and in meaningful ways, namely, that her definition meets the
standards of an evolutionary, stable strategy, she has not reached
"the point from which Kettlewell began." Her whole theory is
built like a house of cards - if the bottom card falls, the whole
edifice collapses.
Recent research on the validity of psychiatric diagnosis clearly
suggests that sociopathy - or antisocial personality disorder
(ASP) as the disorder is known - is not a demonstrably reliable
or valid diagnosis. For one thing, ASP has received four different "standard" definitions in the past 15 years - the Research
Diagnostic Criteria (RDC, Spitzer et al. 1978), the Diagnostic
and Statistical Manual, Third edition (DSM-III, American Psychiatric Association 1980), Third edition-Revised DSM-III-R,
APA, 1987), and Fourth edition (DSM-IV, APA 1994). Over twodozen changes have been made in the definition of the ASP
syndrome from the RDC to DSM-IV. Although some of these
changes are apparently minor — for example, the inclusion of an
additional 12th symptom "initiation of fights" prior to age 15
(DSM-III) opposed to the 11th symptom listed in the RDC),
others are more theoretically significant and suggest a different
conception of ASP (e.g., the inclusion of the adult symptom
"lack of remorse" in DSM-III-R). It is important to note that the
different classification schemes do not typically identify the
same people as antisocial. Prevalence rates (the percent of those
identified as ASP) can differ by as much as 800% using different
definitions in the same sample. Moreover, agreement rates for
different methods of classification of ASP are generally poor
(e.g., interview-based versus self-report versus family history
methods; Kosten & Rounsaville 1992; Zimmerman & Coryell
1990). Thus, it is not true that one researcher's RDC ASP
will necessarily be like another researcher's DSM-IV ASP, or
that sociopathy assessed with one method interviewer will be
like sociopathy assessed by another. The upshot here is that
when we focus on sociopathy, we are literally seeing a moving
target.
Difficulties also exist in demonstrating the reliability and
validity of particular symptom ratings, for example, "lack of
remorse" or "impulsivity," that comprise ASP diagnoses. Existing data clearly show that items assessing core traits of sociopathy have very low inter-rater agreement and are poorly
correlated with other criteria for the ASP syndrome (cf. Carroll
et al. 1993). Moreover, researchers have different views of what
constitutes necessary features for ASP (and for that matter, most
personality disorders; Livesley et al. 1987). Thus, even at the
level of prototypic symptoms, sociopathy remains a muddy and
an ill-conceived notion.
So where are we? Without a clear idea of what the sociopathy
is, sociobiological theories of sociopathy such as Mealey's develop a mental terrain that is only tangentially connected to
available data. That rarely stops the press from uncritically
accepting and presenting such theories as "state of the art."
What is worse, such representations present a hopelessly simplistic and naive picture of genetics and human behavior that
serves as an explanation for mounting economic and social
problems in modern American society. If increase .in violence,
crime, and other so-called sociopathic behaviors can be attributed to innate biological defects, then current social practices
and policies are not to blame. The victims themselves are to
blame; no matter that more jobs and benefits have been cut in
the past three years than in the preceding decade, that pay and
vacation time have been reduced, and that working hours and
workplace stress have both increased. The evolutionary model of
sociopathy focuses the problem back on the individual, and the
status quo remains unchanged. That more powerful forces in our
society - those elites on Wall Street and in Washington who
manage our economic and political system - solicit and give
wide publicity to such theories is, of course, Machiavellian. That
some sociobiologists and psychologists flock to provide such
558
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
explanations based on minimal or hypothetical data, borders on
irresponsibility.
The primary/secondary distinction of
psychopathy: A clinical perspective
Gisli H. Gudjonsson
Department of Psychology, Institute of Psychiatry, Denmark Hill, London
SE5 8AF, England.
Abstract: In this brief commentary the author concentrates on the
treatment perspectives of Mealey's model. The main weakness of the
model is that it does not provide a satisfactory theoretical connection
between treatment and different types of target behavior. Even within
the primary-secondary distinction, there are large individual differences
that should not be overlooked in the planning of treatment.
Mealey has made a formidable attempt to produce an "integrated evolutionary model' of psychopathy. Her main conclusion is that there are two fundamentally different etiologies or
pathways to psychopathy - "primary" and "secondary" - which
have distinct prevention and treatment implications. The primary/secondary distinction is, of course, not a new idea, and
Mealey's main contribution is to integrate these concepts into a
comprehensive evolutionary model.
In this brief commentary I shall focus on the clinical perspectives of Mealey's model, particularly in relation to treatment
issues. I agree with Mealey that a proper theoretical framework
is essential for the classification, diagnosis, prevention, and
treatment of psychopathy.
Mealey's model postulates that only "secondary psychopaths"
are likely to be responsive to treatment interventions. In contrast, "primary psychopaths" require a firm response from the
criminal justice system while simultaneously providing them
with constructive alternative activities to crime. How this might
be achieved in practice is not specified.
The main weakness of the model is that it fails to provide a
clear theoretical connection between treatment techniques and
different types of target behavior. The goals identified and the
techniques recommended for modifying the undesirable behavior, including the psychopathy itself, seem rather vague. The
most important message is that because the background histories of psychopaths are so variable, the programs used have to
suit the needs of the individual psychopath.
From a clinical perspective, the extensive work of Blackburn
(1994) and Copas et al. (1984) on psychopaths in British hospital
settings is important, but there is no reference to it in Mealey's
target article. I believe this is a serious omission, since some of
the ideas in Mealey's article overlap with those of British authors
and do not represent distinct "new" knowledge.
The British work of Blackburn and Copas and his colleagues
indicates that psychopaths seen in clinical settings most typically resemble secondary psychopaths. However, even within
the classification of secondary psychopathy, not all subjects so
diagnosed are equally treatable. For example, the study of
Copas et al. (1984) showed that as far as therapeutic community
treatment is concerned, the anxious and intropunitive psychopaths responded most favourably to treatment programs,
whereas treatment was least effective with the anxious psychopaths with extrapunitive personality. These findings suggest
that even within the secondary psychopath category there are
likely to be individual differences in responsiveness to treatment which are related to personality. Indeed, it may be
unwise, for the purposes of treatment programs, to view secondary psychopaths as a homogeneous group.
In their 1984 paper, Copas and his colleagues also expressed
important treatment implications from their findings for "secondary" and "primary" psychopaths, along very similar lines to those
Commentaryi'Mealey: The sociobiology of sociopathy
recommended by Mealey. That is, the secondary psychopaths
were as a group more responsive to treatment that assisted them
to overcome their high anxiety and acting-out behavior, whereas
the primary psychopaths, in contrast, needed an approach that
confronted them with the consequences of their behavior.
Primary psychopaths, according to Mealey's model and previous empirical research cited in her paper, are deficient in
social emotions such as shame, guilt, and empathy, making them
susceptible to continued irresponsible and criminal behavior. In
addition, according to Mealey's model, this lack of propensity
for social emotion in the primary psychopath is crucial in
differentiating them from secondary psychopaths, and it makes
them relatively unresponsive to environmental influences, such
as those found in most treatment programs.
Unlike primary psychopaths, secondary psychopaths do experience some feelings of guilt and anxiety. Indeed, in our own
work (Cudjonsson & Roberts 1983; 1985) we found that both
male and female secondary psychopaths obtained significantly
higher guilt and anxiety scores on the Eysenck Personality
Inventory and the Mosher True-False Guilt Inventory, respectively. Therefore, not only do secondary psychopaths report
feelings of anxiety and guilt, unlike primary psychopaths, but
their scores significantly exceed those typically reported by
normal subjects. In addition, we found that, unlike normal
subjects, the secondary psychopaths reported constantly experiencing strong feelings of shame and guilt that were unrelated
to specific situational transgression. That is, these feelings
seemed to be a reflection of their generalized poor self-concept
and negative preoccupation rather than a consequence of specific situational transgression. This may explain why feelings of
guilt and shame in secondary psychopaths appear ineffective in
preventing future transgressions (i.e., feelings of guilt do not
show the typical increase in subsequent prosocial behavior as
predicted in Mealey's model). The implication is that treatment
with anxious and guilt-ridden secondary psychopaths should
focus on improving their self-concept as well as on helping them
cope with their high levels of anxiety and acting-out behavior.
Implications of an evolutionary
biopsychosocial model
Harmon R. Holcomb III
Department of Philosophy, University of Kentucky, Lexington, KY
40506-O027. holcomb.ukcc.vky.ech
Abstract: Mealey's work has several interesting implications: It refutes
the charge that sociobiology paints a cynical portrait of human nature
and adopts a one-sided reductionisin; it exemplifies a general theoretical
scheme for constructing evolutionary biopsychosocial models of human
behavior; and it has the practical effect of promoting and informing early
intervention in children at risk for psychopathic disorder.
Mealey's evolutionary biopsychosocial model of psychopathy
refutes the standard charge that sociobiology paints a cynical
view of human nature. As products of natural selection, humans
are motivated to do whatever it takes to perpetuate their genes.
This is commonly interpreted to mean that we are egoistic
manipulators who make cold-hearted calculations (or act as if we
do) toward the end of reproductive success. By inferring psychological states (egoistic manipulators) directly from evolutionary
processes (individual selection), the standard charge fails to
distinguish proximate and ultimate levels of description.
Mealey's lesson is this: first distinguish, then integrate these
levels of description. If the standard charge were correct, we
would all be psychopaths and the sociobiology of psychopathy
would apply to all humans. On the contrary, Mealey's sociobiological study reveals that psychopaths arise from one of two
very specific, developmentally distinct etiologies that emerge
from two specific evolutionary mechanisms, showing how and
why psychopaths differ from the rest of us.
Whereas critics of sociobiology often contrast "genetic," "biological," and "evolutionary" with "psychological" and "social,"
evolutionary theory applies to human behavior only when
species-typical and local biological, psychological, and social
(cultural) circumstances are taken into account. Instead of a onesided reductionisin, sociobiology adopts an integrated causal
model of human behavior. Mealey's integration of biological,
psychological, and social factors within evolutionary theory
helps us see how we can change human behaviors by changing
factors in any of these three categories; sociobiology need not
"provide a biological excuse for the status quo."
Although the specific mix of biopsychosocial factors Mealey
exhibits for psychopathy is unique, it illustrates a general
scheme for constructing evolutionary biopsychosocial models of
human behavior. Can she tell us more, in general, about how
such models apply the evolutionary conception of human actors
as fitness strategy (or adaptation) executors, and how such
models interrelate biological, psychological, and social factors as
inputs to development?
By.going beyond heritability studies to focus on development, we can better discern how genetic factors are pertinent to
a practical understanding of human behavior. Mealey cites
research into psychopathy using quantitative genetics, which
analyzes correlations gained from studying variations among
individuals. But heritability studies have nothing to say about
the causes of traits in particular individuals or about whether the
same individuals treated differently or raised in different environments would develop the same or different traits. For a
causal analysis pertinent to social policy directed at changing
psychopathic behavior, developmental analysis is necessary.
Given the two developmentally distinct types of psychopaths,
Mealey's reasonable suggestion is that deterrence will work for
primary psychopaths and that reduction of risk factors will work
for secondary psychopaths. However, it would be a mistake to
assume that what is special to psychopaths is the only route to
effective intervention. Children who turn into killers often report
that they acted out of fear, hatred, or self-defense; both psychopathic and nonpsychopathic killers are responding to perceived
threats to their fitness. They feel trapped and have learned that
violence is their only way to remove these threats. Teaching
people to identify other ways to solve problems - how to deal with
problems without crime, manipulation, and violence - is just as
applicable to psychopaths as to everyone else. Deterrence and
reduction of risk factors may very well work better when supplemented with problem-solving guidance than when used alone.
There are limits to what environmental intervention will
achieve. We cannot teach primary psychopaths how not to manipulate people; they do not have a "moral conscience" that could get
the message. Suppose we tried to teach them how to manipulate
people without breaking the law. A limit is imposed by the fact
that many laws are enacted to prevent people from manipulating
others, which is why psychopaths are so often criminals.
Indeed, standard diagnostic evidence for antisocial behavior
concerns behaviors often regulated by law. The Diagnostic and
Statistical Manual of the American Psychiatric Association (1987)
used at least four of the following criteria to indicate antisocial
behavior after the age of 15: (1) inability to sustain consistent work
behavior, (2) failure to conform to social norms with respect to
lawful behavior, (3) irritability and aggressivity, as indicated by
repeated physical fights or assaults, (4) repeated failure to honor
financial obligations, (5) failure to plan ahead, or impulsivity,
(6) no regard for truth, as indicated by repeated lying, use of
aliases, or "conning" others, (7) recklessness regarding one's own
or others' personal safety, as indicated by driving while intoxicated or recurrent speeding, (8) inability to function as a responsible parent, (9) failure to sustain a monogamous relationship
for more than one year, and, (10) lacking remorse (feeling justified in having hurt, mistreated, or stolen from another). To re-
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
559
Commentary/Mealey:
The sociobiology of sociopathy
duce drastically the criminal behavior of psychopaths, we would
have to change the laws to make extreme forms of these behaviors
legal, something we will never do. The law is biased against
psychopaths by design; laws are made for and by nonpsychopaths.
Health care professionals already use combinations of biological, psychological, and social traits to diagnose health conditions. An easy way to make evolutionary biopsychosocial models
practically useful is to use them to inform such diagnostic
criteria. Mealey's thesis provides an example. First, the manual
cited above reserves a diagnosis of "antisocial personality disorder" for adults (18 or over) and uses its typical childhood signs to
diagnose "conduct disorder." A developmental approach suggests that the diagnosis of antisocial personality disorder should
be expanded in scope to cover both children and adults. Second,
the diagnostic criteria should be augmented to include the many
symptoms of psychopathy cited in the variety of studies Mealey
reviews. Third, the diagnostic criteria should distinguish diagnoses of primary and secondary sociopathy.
Genes, hormones, and gender in sociopathy
Katharine Hoyenga
Department of Psychology, Western Illinois University, Macomb, IL 61455.
hoyengak@ccmall.wlu.bgu.edu
Abstract: Although serotonin, testosterone, and genes contribute to
sociopathy, the relationships are probably indirect and subject to modifiers (e.g., present only under certain conditions of rearing and temperament). Age at menarche may be a marker variable as well as a causal
factor. Since the genders differ in all four areas, sex differences in
sociopathy represent a very complex interaction of these factors.
Overall, I find Mealey's arguments persuasive and her theory
provocative. I do have a few quibbles; those of us who look for
biosocial covariates must take into account not only the consistent but also the discrepant results.
First, the genetic evidence is not quite as clear or as consistent
as implied. As Mealey pointed out, adoption studies find evidence for the heritability of property crimes only, not violent
crimes. In addition, although heritability estimates of twin studies
are often higher because of nonadditive genetic influences,
identical twins also have stronger tendencies to imitate each
other's violence (Carey 1992; Rowe 1990), which would spuriously inflate the heritability estimates derived from twin studies.
Second, the relationship between testosterone (T) and violence is also not very consistent (Hoyenga 1993; Hoyenga &
Hoyenga 1993a). I think that some of the variability in results,
and weakness of the effects, can be attributed to genetic and
developmental variability. People differ in their sensitivity to T,
and before T can be related to aggression, certain other traits
such as personality traits (with genetic and developmental
components) might also have to be present. Overall, aggression
may be more sensitive to postpubertal than to perinatal T, but
failures to replicate are more numerous than positive results.
Age at menarche may be a marker variable, as well as serving
as a causal factor as described by Mealey. Age at menarche has
genetic components and is also sensitive to disruptions in family
life (Hoyenga & Hoyenga 1993b). Thus, early menarche might
be a marker for mechanism 1 (primary), as well as being a causal
component for mechanism 5 (secondary).
Although Mealey covered some aspects of the role of temperament in the development of sociopathy, most of the work
summarized in that section concerned psychopathological traits,
and sociopathy can also be related to extreme forms of normal
personality traits. For example, in a factor analytic study done
with the Psychopathy Checklist, Revised, two factors were found
(Harpur et al. 1989). The first corresponded almost perfectly to
the interpersonal personality trait of hostile dominance, which is
a blend of dominance and lack of nurturance. The second factor
560
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
found in the Harpur study - the one most closely related to acts of
violence - did not seem to be a personality trait. Similarly,
Foreman (as described by Hare et al. 1991) found that PCL-R
total scores were also positively correlated with staff ratings of
dominance and negatively correlated with nurturance.
Although the relationship between impulsive violence and
low serotonin is strong and often replicated, there are inconsistencies here as well. Some people tend to respond to an increase
in brain serotonin with an increase rather than with the expected decrease in hostility and anger. This somewhat atypical
reaction may be found more often in anxiety neurotics (Germine
et al. 1992; Kahn et al. 1988). Also, in both humans (the Madsen
[1985] reference cited by Mealey) and many nonhuman primates, dominance is positively correlated with brain serotonin
levels (e.g., Brammer et al. 1991; Raleigh & McGuire 1986;
Steklis et al. 1986). It seems that in some primate males,
becoming dominant depends on successfully affiliating with
several females; males with high aggression cannot affiliate, but
males with high serotonin show more afRliative behaviors and so
can increase their dominance status in the group.
Sex differences in sociopathy may reflect something more
complicated than implied by a two-threshold model. For example, females of a variety of species reliably have more brain
serotonin or more active brain serotonergic systems than do
males (Hoyenga & Hoyenga 1993a; 1993b). In addition, because
of socialization and sex limitation, the sexes differ in the development of the relevant temperamental traits, including the
traits of dominance and nurturance mentioned above (Hoyenga
1993). Thus, gender-related differences in sociopathy may reflect the simultaneous actions of multiple interacting factors.
Al Capone, discrete morphs, and complex
dynamic systems
Douglas T. Kenrick and Stephanie Brown
Department of Psychology, Arizona State University, Tempe, AZ
85287-1104. atdtk@asuacad.bitnet; assrb@asuacad.bltnet
Abstract: We consider four mechanisms by which apparent discontinuities in the distribution of antisociality could arise: (1) executive
genes or hormonal systems, (2) multiplicative interactions of predisposing factors, (3) environmental tracking into a limited number of social
roles, and (4) cross-generational gene—environment interactions. A
more explicit consideration of complex self-organizing dynamic systems
may help us understand the maintenance of antisocial subpopulations.
Even normal people occasionally consider antisocial acts such as
homicide (Kenrick & Sheets 1994). Most of us keep such inclinations under restraint, though, and cannot empathize with characters like Al Capone, who invited three men to dinner, then
had them tied to their chairs and beat them with a baseball bat.
Capone showed the signs of primary sociopathy: personal
charm, an early career of violence, and little remorse, fear, or
sympathy. Though such cases fit Mealey's distinction, much of
the evidence she reviews suggests a continuous distribution
from well-socialized individuals through "secondary sociopaths"
and "primary sociopaths." Is a discontinuity between primary
sociopaths and the rest of the population plausible?
What could produce a discontinuous distribution of antisocial
behavior? We consider four mechanisms that could contribute to
discontinuity in antisociality.
1. Executive genes or hormonal systems. If people vary continuously in levels of testosterone, adrenaline, serotonin, and
dopamine, and if one hormonal system is independent of another, one expects a continuous rather than discontinuous
distribution of antisociality. However, internal organization
could yield a smaller number of discrete settings. Mealey
mentions testosterone's developmental organizing role. In a
related vein, gender differences result in two discrete morphs,
Commentary/Mealey: The sociobiology of sociopathy
not continuous variation in size of reproductive glands. Selflimiting discrete organizations may occur within each sex, as in
certain fish species, when some individuals of the same sex grow
into completely different adult morphs (Gross 1984).
2. Multiplicative interactions of predisposing factors. Apparent
typologies or quasi-typologies could arise from multiplicative
interactions of underlying physiological dimensions (see Table 1).
Imagine several individuals with settings of either 2 or 4 on three
underlying dimensions. Assuming additive combination, an individual with a 4 on each dimension would be twice as antisocial
as one with all 2s (12 vs. 6). Assuming multiplicative combination,
however, the highest setting would be eight times as antisocial as
the lowest (64 vs. 8), yielding a distribution appearing more like a
typology. Even assuming continuously distributed characteristics, multiplicative interactions enhance dissimilarity between
highs and lows (see Fig. 1), and allow for antisociality even with a
high environmental threshold (as found for the upper classes).
3. Environmental tracking into a limited number of social roles.
Small and continuous differences between children amplify over
the lifespan. A child with a small initial edge in basketball,
music, or chess gets training and grows into a professional many
times better than grade-school cronies. Mealey discusses how
children having common troubles with authorities band together and amplify one another's antisociality.
Environmental tracking would capitalize on initial genetic
proclivities. Just as animals with tools such as talons have
different cost/benefit weightings for aggression; an outlaw strategy could yield a more favorable cost/benefit ratio for attractive,
lowfemotion people.
4. Cross-generational gene-environmental interactions. Societal
forces may artificially select extremes of exploitativeness. Over
time, laws punishing cheaters could increase differentiations between cheaters and cooperators, since half-hearted or inept exploitation will be punished, and the middle levels become less populous.
Antisocial behaviors may also be maintained by reward structures. If physically attractive high-risk individuals choose one
another as mates, the package of Cleckley characteristics is
increasingly maintained by speciation-Iike processes. Note that
ruthless promiscuous individuals who disregard social convention include spies and military heroes as well as criminals. Al
Capone became incredibly wealthy and politically powerful,
and in Sicily, mafiosi received esteem from the local populace as
well as advantageous marital and extramarital mating opportunities (Serviado 1976). Payoffs for "antisocial" behavior come
not only from cheating and backstabbing: people respect and are
attracted to "fearlessness."
thing but your success. Conventional individuals sometimes
band together to lynch or tar and feather those who take more
than their share of reproductive and material resources. On this
view, it is fairer to envision dynamic conflict between different
mating strategies and jettison the term "antisocial" for those
playing one strategy.
Complex dynamic systems from simple underlying properties.
Mealey uses the term "dynamic equilibrium" but does not
discuss emerging notions about complex self-organizing dynamic systems. Interesting things emerge when the Prisoner's
Dilemma is expanded to larger matrices. We modelled primitive 6 x 6 networks and found that, when all individuals in an
imaginary "neighborhood" were set to a similar strategy (act
aggressive if 50% of neighbors were aggressive on the previous
iteration), self-maintaining pockets of mutual aggression and
mutual cooperation emerge, with occasional pockets of peripheral instability. Individuals set to more aggressive thresholds
remain peaceful in unaggressive neighborhoods but catalyze the
spread of pockets of aggressiveness.
Such models are being used to map complex interactions
between genes, between neurons in cognitive systems, and
between species in ecosystems. How do cognitive dynamics
map onto internal physiological systems and inputs from the
larger social environment? Perhaps underneath the seemingly
continuous variation of the social world are only a (relatively) few
interlocking mechanisms. Though the proliferation of scientific
findings sometimes moves us toward information overload, the
insights of evolutionary science, dynamic systems theory, and
cognitive neuroscience could be aiming us toward a metasynthesis of Copernican magnitude.
At our present level of understanding of these systems,
though, interventions such as Mealey suggests may be presumptuous and even dangerous. More certain punishments for
antisociality, for instance, could further widen the gap between
conventional and unconventional individuals. Draconian measures exact a high cost on marginally conventional and generally
nondangerous individuals, while selecting the ranks of the
Capones for even more deviousness and ruthlessness.
(A) ADDITIVE MODEL
Oscar Wilde noted, however, that people forgive you anyLOW
Table 1 (Kenrick & Brown). A depiction of eight
HWH
Physiological Risk Factors
hypothetical individuals varying on three
(B) MULTIPLICATIVE MODEL
dimensions relevant to antisocial behavior
Resulting antisociality
Traits
Anxiety
Sensation
seeking
Behavioral
inhibition
Additive
model
Multiplicative
model
2
2
2
4
2
4
4
4
2
2
4
2
4
2
4
4
2
4
2
2
4
4
2
4
6
8
8
8
10
10
10
12
8
16
16
16
32
32
32
64
Physiological Risk Factors
Note: Higher scores indicate more proclivity toward antisocial
behavior.
Figure 1 (Kenrick & Brown). How multiplicative interactions
of underlying factors can produce an apparent typology. If
several underlying factors contributed to antisocial behavior,
and combined additively, the results would be a fairly continuous distribution, with many individuals at or near the threshold, as in (A). However, multiplicative interaction can enhance
differences, so that some individuals would clearly be above
most environmental thresholds, and others clearly below, as in
(B). The threshold for engaging in antisocial behavior could vary
as a function of environmental inputs, such as population size,
availability of resources, and normative pressures.
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
561
Commentary/Mealey:
The sociobiology of sociopathy
An evaluation of Mealey's hypotheses based
on psychopathy checklist: Identified groups
David S. Kossona and Joseph P. Newmanb
"Department of Psychology, Finch University of Health Sciences /Chicago
Medical School, North Chicago, IL 60064; "Department of Psychology,
University of Wisconsin - Madison, Madison, Wl 53706.
jpnevvman@facstaff.wisc.edu
Abstract: Although Mealey's account provides several interesting hypotheses, her integration across disparate samples renders the value of
her explanation for psychopathy ambiguous. Recent evidence on Psychopathy Checklist-identified samples (Hare, 1991) suggests primary
emotional and cognitive deficits inconsistent with her model. Whereas
high-anxious psychopaths display interpersonal deficits consistent with
Mealey's hypotheses, low-anxious psychopaths' deficits appear more
sensitive to situational parameters than predicted.
We applaud Mealey's attempt to integrate diverse literatures
and to elucidate the significance of the historical distinction
between primary and secondary psychopathy. However, although several of her ideas are quite interesting, much is lacking
in the empirical basis for her proposals regarding primary and
secondary psychopathy.
Mealey acknowledges the problems associated with failures to
distinguish psychopathy from criminality and antisocial personality disorder, but she proceeds to integrate data without regard
to this distinction. There is now more than a decade of research
on the Psychopathy Checklist (PCL; Hare 1980; 1991), a measure that assesses psychopathy based on individuals' personality
as well as antisocial conduct. The PCL has impressive reliability
and validity (Hare 1991; Kosson et al. 1990) and greater specificity than the American Psychiatric Association (1987) diagnosis of
Antisocial Personality Disorder (see Hare et al. 1991). Moreover, applying taxometric methods to the PCL, Harris et al.
(1994) have presented evidence that PCL ratings identify a
discrete category or taxon. At least two partially correlated
dimensions of psychopathy have been identified (Harpur et al.
1989). Chronic, impulsive antisocial behavior defines one dimension (viz., Factor 2); a second (viz., Factor 1) addresses the
shallow, callous exploitation of others, regarded by many as the
personality core of the disorder.
To the extent that Factor 1 is essential for distinguishing
psychopaths from antisocial personality and criminality, reviews
like Mealey's that rely heavily on studies of criminals to bolster
propositions about psychopaths are apt to be misleading. Case
studies distinguish psychopaths from ordinary criminals (e.g.,
Cleckley 1976) and most research on psychopaths includes
nonpsychopathic inmates as a control group. Moreover, psychopaths constitute only about 20% to 33% of male prison inmates
(Hare 1991), so that differences between psychopaths and other
criminal groups are obscured by studies on criminals as a whole.
Indeed, most of the evidence Mealey uses to characterize
primary psychopaths is probably more relevant to Antisocial
Personality Disorder (i.e., Factor 2) than to psychopathy. Below,
we examine Mealey's assertions about the psychological characteristics of primary and secondary psychopaths using, except as
noted, PCL-defined psychopaths. Due to space constraints, we
limit our review to the domains of emotional and cognitive
function.
1. Emotional function In psychopaths. Emotional deficits play
a central role in Mealey's account of primary psychopathy:
psychopaths are deficient in social but not in primary emotions
or moods. Preliminary studies of emotional processing in PCLdefined psychopaths provide some support for this position.
Unlike nonpsychopaths, psychopaths' lexical decisions are not
faster for emotional words (Williamson et al. 1991), and viewing
unpleasant (vs. pleasant) slides does not increase their startle
responses (Patrick 1994; Patrick et al. 1993). Moreover, these
deficits are particularly related to Factor 1.
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With regard to emotional responsiveness, findings are inconclusive. Psychopaths display less autonomic arousal than nonpsychopaths during fear imagery (Patrick et al. 1994). Initial
studies using film inductions provide evidence that psychopaths
also show less disgust (Forth 1992) and report less affect to
negative emotion film segments (Patterson 1990) but are not
hyporesponsive on other measures. Thus, these studies suggest
limited deficits for primary emotions.
Few studies bear on Mealey's hypotheses regarding social
emotions with PCL-identified psychopaths. However, extant
studies provide little evidence that emotional deficits lead
primary psychopaths "to continually play for the short-term
benefit." (sect. 3.1.1, para. 2) Studies using the Prisoner's
Dilemma game indicate that neither psychopaths (meeting
Cleckley's 1976 criteria) nor low-anxious psychopaths (meeting
the PCL or MM PI criteria) play more selfishly than nonpsychopaths, although high-anxious psychopaths do (Hare &
Craigen 1974; Whitehill 1987; Widom 1976a). Sutker (1970)
found psychopaths (defined by agency diagnoses and MMPI
scores) as likely as controls to forfeit reward to prevent shocks to
a confederate. In addition, studies of social sensitivity suggest
that anxious or neurotic but not low-anxious psychopaths are
deficient (e.g., Rime et al. 1978; Widom 1976b; Patterson 1990).
Thus, although anomalies in psychopaths' emotional processing
and responsiveness have been observed, there is little evidence
that these underlie problems in interpersonal cooperation, and
interpersonal deficits are largely limited to high-anxious
psychopaths.
2. Integrating emotion and self-regulation. On the other
hand, studies evaluating psychopaths' responsiveness to feareliciting stimuli while pursuing immediate rewards show, as
Mealey notes, performance deficits in psychopaths. But in
contrast to Mealey's view that such findings suggest emotional
deficits per se, their situation-specificity is more consistent with
deficits in information-processing integration (Newman & Wallace 1993). In comparison to low-anxious controls, low-anxious
psychopaths display inhibitory problems in situations involving
both reward and punishment incentives. Moreover, their "insensitivity" to punishment stimuli appears to reflect difficulty in
shifting attention and interrupting ongoing behavior, which
impairs their ability to profit from environmental cues (Kosson
& Harpur, in press; Patterson & Newman 1993).
Moreover, high-anxious psychopaths do not display deficits
under these circumstances. They appear more able to interrupt
goal seeking in response to punishment, whereas low-anxious
psychopaths' responsiveness to punishment contingencies may
depend on situational factors that make punishment salient
prior to their initiating goal-directed behavior (e.g., Newman et
al. 1990; Newman et al. 1992). This distinction resembles
Mealey's assertion that secondary psychopaths respond to environmental contingencies, whereas a priori expectations dictate
primary psychopaths' behavior.
3. Cognitive function In psychopaths. The proposal that psychopaths often adopt a cognitive (vs. affective) style of processing information was also stated by Newman and Wallace (1993).
However, Mealey's proposal for accurate cost-benefit analyses
implies intact cognition. Although psychopaths often perform as
well as nonpsychopaths, subtle cognitive deficits are evident in
specific situations.
Becent evidence suggests specific deficits in divided attention, including reduced breadth of attention and deficient shifts
of attention (Howland et al. 1993; Kosson & Harpur, in press).
Moreover, these occur while subjects are actively engaged in
goal-directed behavior and are related to both dimensions of
psychopathy (Kosson 1995). Biases in appraisal have also been
reported. Psychopaths display both hostile, attributional biases
(Sterling & Edelmann 1988; Serin 1991; cf, Millon 1981) and
optimistic biases, with the latter linked to their excessive goal
seeking (Patterson 1990; Siegel 1978).
Commentary/Mealey: The sociobiology of sociopathy
4. Toward a classification of secondary psychopaths. Compared with previous proposals for secondary psychopaths,
Mealey's characterization of this group is unusual. Most proposals for "secondary" psychopathy explain antisocial behavior
as secondary to some internal causal factor: neurotic conflict or
anxiety (e.g., Karpman 1961; Lewis 1991), underlying schizotypy or schizoid tendencies (Hodgins 1994; Raine & Venables
1984), or inadequacy or passivity (e.g., Craft 1965). By contrast,
Mealey's emphasis on competitive disadvantage, like Quay's
(1986) category of "socialized delinquent," implicates individuals who are not emotionally or interpersonally impaired but
develop antisocial values via social learning.
Basic relations between competitive disadvantages, primary
psychopathy, and other candidates for secondary psychopathy
are also unclear. If anxiety and schizotypy are competitively
disadvantageous, should antisocial behavior attributable to such
factors be considered secondary psychopathy? Do low- and
high-anxious psychopathy reflect separate genetic diatheses or a
common (psychopathy) diathesis whose expression is moderated by level of anxiety? Answers to such questions appear
fundamental to Mealey's prescriptions for change.
In contrast to Mealey's general review, our more specific
analysis of PCL-defined psychopaths suggests that they are
characterized by deficits in both emotional and cognitive functioning that are more sensitive to situational parameters than
Mealey predicts. Whereas some differences between low- and
high-anxious psychopaths appear consistent with Mealey's distinction between primary and secondary psychopaths (e.g.,
integration of emotion with self-regulation), others do not (e.g.,
interpersonal sensitivity). In addition, correlations between
self-reported anxiety and Factor 2 (Hare 1991) suggest that highanxious psychopaths should be well represented in the genetic
studies of criminality that Mealey reviews. Given the apparent
social deficits also displayed by those individuals, we imagine
she would regard them as primary psychopaths. Indeed the
possibility that Mealey s account could explain the behavior of
high-anxious psychopaths remains an intriguing one which
appears worthy of study with well-defined groups.
Fatherless rearing leads to sociopathy
David T. Lykken
Department of Psychology, University of Minnesota, Minneapolis, MN
55455-0344. lykkeOOI@staff.tc.umn.edu
Abstract: Endorsing Mealey s analysis, it is pointed out that increasing
rates of crime and violence are due to increasing proportions of children
being reared in circumstances radically different from the extendedfamily environment to which we are evolntionarily adapted, that is, they
are reared without fathers.
Mealey's valuable paper was especially interesting to me because, reasoning in terms of evolutionarily stable strategies, she
arrives at a taxonomy of the family of antisocial personalities that
is very similar to that which I derived from quite a different
model (Lykken 1995).
We can suppose that our species is adapted to live reasonably
amicably in extended-family bands or tribal groups as they
probably did in the Pleistocene. One of our species-specific
characteristics is the capacity to become socialized, which
means: (1) to develop a conscience that leads us to avoid
antisocial behavior; (2) to develop prosocial inclinations such as
empathy, altruism, respect for authority, and cooperativeness;
and (3) to accept adult responsibilities, including those of parenting and pulling our own weight in the communal effort.
However, like our capacity for language, this disposition toward
socialization needs to be elicited and practiced during early
childhood, and this training, in ancestral as in present-day
traditional societies, was a responsibility shared by the parents
and the extended family.
Because we are adapted to grow up and live in such groups,
extended-family child rearing is very effective in socializing
youngsters and therefore intramural crime in traditional societies today is infrequent. The only chronic offenders are those
individuals whose innate temperaments make them very difficult to socialize - the people Mealey calls "primary sociopaths"
but whom I refer to as "psychopaths" in the diagnostic tradition
that goes back to Cleckley (1941) and earlier.
In her important study of mental illness in non-Western,
primitive societies, the anthropologist Jane Murphy found that
the Yupic-speaking Eskimos in northwest Alaska have a different
name, kunlangeta, for this type of individual. The
man who, for example, repeatedly lies and cheats and steals things
and does not go hunting and, when the other men are out of the
village, takes sexual advantage of many women — someone who does
not pay attention to reprimands and who is always being brought to
the elders for punishment. One Eskimo among the 499 on their island
was called kunlangeta. When asked what would have happened to
such a person traditionally, an Eskimo said that probably "somebody
would have pushed him off the ice when nobody else was looking. "
(Murphy 1976, p. 1026)
Because traditional methods of socialization are so effective in
tribal societies, where the extended family rather than just a
particular parent-pair participate in the process, the kunlangeta
probably possesses inherent peculiarities of temperament that
make him unusually intractable to socialization.
When a modern man and woman, often without any prior
experience in dealing with young children (experience they
would have had growing up in a traditional society), undertake
the demanding task of parenting on their own, the failure rate
(i.e., the crime rate) will inevitably be higher. I use "sociopath"
to designate antisocials whose lack of socialization is primarily
due to incompetent parenting. Because parenting is difficult,
the failure rate is higher still for single parents. About 70% (!) of
institutionalized delinquents grew up in homes without a father
(Lykken 1995). My "sociopaths' are roughly equivalent to those
whom Mealey calls "secondary sociopaths. "
It is important to realize that sociopath and psychopath
(Mealey's secondary and primary sociopaths) are endpoints on a
continuum. With the best parents and home environments, the
only antisocial offspring will be those who are the most fearless,
aggressive, impulsive, and so on - psychopaths with truly hardto-socialize temperaments. In the worst home environments, a
large fraction of all offspring will remain unsocialized. Over the
broad middle range of parental competence and environmental
risk factors, the incidence of antisocial offspring will be a
product-function of parental incompetence (or indifference or
parental sociopathy) and the child's innate proclivities. A complicating factor is that the worst parents are likely to contribute
hard-to-socialize genetic tendencies as well.
This line of thinking explains why the rate of juvenile violent
crime has increased 100% since 1972 and is six to eight times
higher among blacks than whites in the United States. The
proportion of births to unwed mothers in the U.S. increased
from about 5% in 1960 to about 30% today. In 1925, 85% of black
families in Harlem were headed by fathers. By 1965, when
Daniel Patrick Moynihan wrote his famous memorandum on the
break-up of the black family, the rate of black illegitimacy had
risen to 25%. Among urban blacks today, it is a rare youngster
who is being reared by both biological parents. Since 1965, the
increasing rates of arrest of juveniles in the U.S. for crimes of
violence, plotted separately for blacks and whites, very closely
parallel the increasing proportions of the two 12-17-year-age
groups that were born out of wedlock (Lykken 1995).
It is also important to realize that penal rehabilitation, psychotherapy, and other efforts at remediation do not work; the
only solution to the increase in numbers ofjuvenile sociopaths is
BEHAVIORAL AND BRAIN SCIENCES (1995) 18:3
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CommentarylMealey:
The sociobiology of sociopathy
prevention. Parental training, as Mealey proposes, is a good
investment for those few high-risk parents who can or want to be
trained. But, for the majority of high-risk children, programs of
professionalized foster care, of all-day care on the successful
kibbutzim model, of single-sex boarding schools, and the like
will be required. If each sociopath costs society at least $3
million in his lifetime (Westman 1994), such expensive programs
may be justified. But, until the source of nascent sociopaths children born to incompetent parents - is substantially reduced
(my proposal is parental licensure), this large, expensive, and
dangerous problem will stay with us.
Sociobiology, sociopathy, and social policy
Richard Machalek
Department of Sociology, University of Wyoming, Laramie, WY 82071.
machalek@uwyo.edu
Abstract: Evolutionary analysis suggests that policies based on deterrence may cope effectively with primary sociopathy if the threat
of punishment fits the crime in the cost/benefit calculus of the sociopath, not that of the public. On the other hand, policies designed to
offset serious disadvantage in social competition may help inhibit the
development of secondary sociopathy, rather than deter its expres-
Many social scientists in policy-oriented fields, especially criminology and sociology, have long feared that sociobiology necessarily leads to fatalism and hopelessness about antisocial behaviors. They reason, incorrectly, that adopting an evolutionary
approach to human behavior means resigning oneself to the
view that "biology is destiny." Mealey's analysis of sociopathy
demonstrates clearly that this is not true. Instead, sociobiology
can provide unique insights and help us clarify our options as we
form policy responses to social problems such as those created
by sociopathy.
Consider what it means to replace the more conventional
medical view of sociopathy with one informed by evolutionary
reasoning. As the term itself suggests, the medical model
attributes sociopathy to a "pathogen," in this case an emotional
deficit that may be genetically rooted and physiologically expressed. This results in behavior that is described as "antisocial" (and thus "pathological"), because it is emotionless and
uncompromisingly selfish. Although few among us would quarrel with characterizing sociopathy as pathological (except, perhaps, those who celebrate the rise of a Nietzschean, postmodern culture), evolutionary theory takes us beyond mere
diagnostic descriptors and prompts us to ask whether such
antisocial behaviors may, in some fundamental sense, be advantageous to those who express them. This is what Mealey
does by exploring a conceptualization of sociopathy as a "life
strategy" (Introduction, para. 6). In this view, the sociopath is
often a highly rational but morally bankrupt strategist motivated uncompromisingly by self-interest. Framing sociopathy
in evo