Physiol Rev
84: 1051–1095, 2004; 10.1152/physrev.00042.2003.
Molecular Structure and Function
of the Glycine Receptor Chloride Channel
JOSEPH W. LYNCH
School of Biomedical Sciences, University of Queensland, Brisbane, Australia
I. Introduction
A. Scope of this review
B. Glycine as an inhibitory (and excitatory) neurotransmitter
II. Diversity, Distribution, and Function of Glycine Receptor Subunits
A. Molecular diversity
B. Distribution and function in the rat nervous system
C. Distribution and function in other tissues
III. Structure and Assembly
A. General structural features
B. Transmembrane domains
C. NH2-terminal ligand-binding domain
D. Large intracellular domain
E. Receptor assembly
IV. Structure and Function of the Pore
A. Functional properties of the pore
B. Molecular determinants of ion selectivity and conductance
C. The channel activation gate
D. Molecular mechanisms of desensitization
V. Agonist Binding and Receptor Activation
A. Introduction
B. Kinetic models of glycine receptor gating
C. Agonist binding
D. Structural changes accompanying activation
VI. Glycine Receptor Modulation
A. Phosphorylation
B. Modulators of possible physiological relevance
C. Molecular pharmacology
VII. Glycine Receptor Channelopathies
A. Human startle disease
B. Murine startle syndromes
C. Bovine myoclonus
VIII. Outlook
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Lynch, Joseph W. Molecular Structure and Function of the Glycine Receptor Chloride Channel. Physiol Rev 84:
1051–1095, 2004; 10.1152/physrev.00042.2003.—The glycine receptor chloride channel (GlyR) is a member of the
nicotinic acetylcholine receptor family of ligand-gated ion channels. Functional receptors of this family comprise five
subunits and are important targets for neuroactive drugs. The GlyR is best known for mediating inhibitory
neurotransmission in the spinal cord and brain stem, although recent evidence suggests it may also have other
physiological roles, including excitatory neurotransmission in embryonic neurons. To date, four ␣-subunits (␣1 to
␣4) and one ␤-subunit have been identified. The differential expression of subunits underlies a diversity in GlyR
pharmacology. A developmental switch from ␣2 to ␣1␤ is completed by around postnatal day 20 in the rat. The
␤-subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin.
The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information has
recently emerged concerning GlyR molecular structure and function. Most of the information has been obtained
from homomeric ␣1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritable
mutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar
syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent
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0031-9333/04 $15.00 Copyright © 2004 the American Physiological Society
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JOSEPH W. LYNCH
modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide
inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of
muscle relaxant and peripheral analgesic drugs.
nAChR, particularly in areas where knowledge of the
GlyR is deficient.
I. INTRODUCTION
A. Scope of This Review
Ligand-gated ion channels permit cells to respond
rapidly to changes in their external environment. They are
particularly well known for mediating fast neurotransmission in the nervous system. The glycine receptor (GlyR) is
a membrane-embedded protein that contains an integral
Cl⫺-selective pore. When glycine binds to its site on the
external receptor surface, the pore opens allowing Cl⫺ to
passively diffuse across the membrane. The GlyR is a
member of the pentameric ligand-gated ion channel
(LGIC) family, of which the nicotinic acetylcholine receptor cation channel (nAChR) is the prototypical member.
Other members of this family include the cation-permeable serotonin type 3 receptor (5-HT3R), anion-permeable
GABA type A and C receptors (GABAAR and GABACR),
recently identified cation-permeable zinc and GABA receptors (34, 86), as well as invertebrate anion-permeable
glutamate and histidine receptors (130). Note that glycine
also directly activates a cation-selective ion channel of the
excitatory glutamate receptor family (62). The structural
and functional properties of this receptor class have recently been reviewed (94) and are not considered here.
Glycine was first proposed as an inhibitory neurotransmitter on the basis of a detailed analysis of its distribution in the spinal cord (16). Subsequent electrophysiological studies demonstrated a strychnine-sensitive hyperpolarizing action of glycine on spinal neurons (80,
395). This hyperpolarization was soon discovered to be
mediated by an increase in Cl⫺ conductance (81, 82, 396).
The receptors responsible for these actions were subsequently purified by strychnine affinity chromatography
(291, 292), and the first GlyR subunit was cloned in 1987
(138).
Current research into the GlyR can be divided into
two major strands. The first involves the investigation of
the molecular mechanisms of GlyR trafficking and clustering at synapses. This area is currently the subject of
intense investigation, and recent progress has been covered in several authoritative reviews (189, 190, 219). The
second research strand is concerned with understanding
the molecular structure and function of the GlyR. Research has intensified in this area over the past few years,
and the purpose of this review is to present a coherent
view of recent findings. Much of our understanding of
GlyR structure-function has been gained by comparison
with the structurally homologous nAChR. Hence, this review makes frequent references to research on the
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B. Glycine as an Inhibitory
(and Excitatory) Neurotransmitter
When the GlyR is activated, the resulting Cl⫺ flux
moves the membrane potential rapidly toward the Cl⫺
equilibrium potential. Depending on the value of the equilibrium potential relative to the cell resting potential, the
Cl⫺ flux may cause either a depolarization or a hyperpolarization. The GlyR is generally known as an inhibitory
receptor because the Cl⫺ equilibrium potential is usually
close to or more negative than the cell resting potential.
Subthreshold depolarizations can inhibit neuronal firing if
they are accompanied by an increase in membrane conductance that shorts out excitatory responses, a phenomenon termed “shunting inhibition.” However, in embryonic neurons, the intracellular Cl⫺ concentration is raised
substantially, with the effect that GlyR activation causes a
strong, suprathreshold depolarization. These large glycine-induced depolarizations gate a calcium influx that is
necessary for the development of numerous specializations, including glycinergic synapses (190). The switch to
the mature neuron phenotype is mediated by the expression of a K⫹-Cl⫺ cotransporter, KCC2, which lowers the
internal Cl⫺ concentration, thereby shifting the Cl⫺ equilibrium potential to more negative values and converting
the actions of the GlyR from excitatory to inhibitory
(355).
II. DIVERSITY, DISTRIBUTION, AND FUNCTION
OF GLYCINE RECEPTOR SUBUNITS
A. Molecular Diversity
Betz and colleagues (291, 292) originally purified the
rat spinal cord GlyR by affinity chromatography on aminostrychnine-agarose columns. Oligonucleotides designed
from peptide sequences of purified receptors were then
used to probe a rat spinal cord cDNA library, resulting in
isolation of cDNA clones corresponding to the 48 kDa
(␣1) and 58 kDa (␤) subunits (137, 138). Subsequently,
cDNAs of the rat ␣2- and ␣3-subunits were cloned by
homology screening (9, 199, 201). The ␣4-subunit, which
does not appear to exist in the rat or human, was first
identified in the mouse (254) and has subsequently been
found in the chick (157) and zebrafish (91). While the
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
␣-subunit genes are highly homologous, with primary
structures displaying 80 –90% amino acid sequence identity, the ␤-subunit has a sequence similarity of ⬃47% with
the ␣1-subunit (137).
The rat ␣1-subunit has a splice variant, termed ␣1ins,
which contains an eight-amino acid insert in the large
intracellular loop (244) that contains a possible phosphorylation site (see sect. VIA). Alternative splicing of the rat
␣2-subunit generates two splice variants, ␣2A and ␣2B
(199, 201). The ␣2B-variant differs from ␣2A by the V58I
and T59A amino acid substitutions. Another version of the
␣2-subunit, termed ␣2*, incorporates a single amino acid
substitution (G167E) that confers strychnine insensitivity
(202). Two transcripts of the human ␣3-gene, termed ␣3L
and ␣3K, have been characterized (280). The ␣3K-variant
lacks a 15-amino acid segment in the large intracellular
loop that exists in both the rat ␣3- and the human ␣3Lsubunits. A splice variant of the zebrafish ␣4-subunit contains a 15-amino acid insert in the ligand-binding domain
(91). Although a human ␤-subunit gene polymorphism has
been described (264), it does not appear to result in a
coding mutation.
All GlyR genes cloned to date share a similar exonintron organization with the coding region spread over
nine exons (254, 264, 346). This common organization
suggests a phylogenetic gene duplication (345).
B. Distribution and Function in the Rat
Nervous System
1. Distribution of functional GlyRs
A) DISTRIBUTION OF STRYCHNINE AND GLYCINE BINDING SITES.
Autoradiographic localization of [3H]strychnine binding
sites was first studied by Zarbin et al. (431) in the rat.
Strychnine binding sites were shown to exist at high
levels in the spinal cord and medulla and at lower levels
in the pons, thalamus, and hypothalamus, while being
virtually absent in higher brain regions. In the spinal cord,
their distribution is relatively diffuse throughout the gray
matter. In contrast, GlyRs in the brain stem are highly
localized to discrete nuclei, notably the trigeminal nuclei,
the cuneate nucleus, the gracile nucleus, the hypoglossal
motor nucleus, the reticular nuclei, and cochlear nuclei
(301). The retina, which also has a high concentration of
strychnine sites (297), is considered as a separate case,
below. Together, these areas comprise a subset of the
distribution as determined by glycine autoradiography
(270) or glycine immunoreactivity (310), a mismatch that
is understandable given that glycine is also associated
with glutamatergic synapses (270). An advantage of
strychnine autoradiography is that it reveals the presence
of surface-expressed receptors, but a disadvantage is that
its limited resolution does not permit the ultrastructural
localization of strychnine binding sites. Thus strychnine
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autoradiography does not necessarily define the distribution of glycinergic synapses.
B) DISTRIBUTION OF GLYR IMMUNOREACTIVITY. Many studies
have examined the immunocytochemical localization of
GlyRs at the light and electron microscopic levels using
generic GlyR ␣-subunit monoclonal antibodies. At the
light microscopic level, there is a strong correlation between the distribution patterns revealed by GlyR immunoreactivity and strychnine autoradiography (17, 370).
However, some significant differences have been observed. First, immunolabeling reveals the existence of
GlyRs in the cerebellum (17, 361) and olfactory bulb
(383), whereas none was seen using strychnine binding
(431). Second, the substantia gelatinosa in the spinal cord
is strongly labeled by strychnine (431), but not by GlyR
antibodies (23). The reasons for the differential labeling
are yet to be clarified.
Electron microscopic immunoreactivity reveals that
GlyRs at central synapses are concentrated into regions
closely apposed to presynaptic terminals (13, 23, 370, 371,
383), strongly suggesting a functional role. It is of interest
to note that this approach has demonstrated the colocalization of GlyRs and GABAARs at individual postsynaptic
densities in the spinal cord (43, 127, 369) and cerebellum
(100).
C) DISTRIBUTION OF FUNCTIONAL GLYCINERGIC SYNAPSES. Most
central nervous system neurons are inhibited by glycine
(279). Of course, the mere presence of functional GlyRs,
especially on dissociated or cultured neurons, does not
imply a physiological role. However, it has recently been
proposed that the activation of nonsynaptic GlyRs in embryonic cortical neurons may be important for development, and that taurine released from local glial cells may
be the endogenous ligand (114). Similarly, nonsynaptic
GlyRs on hippocampal CA3 neurons are proposed to be
held in a tonically active state by locally released taurine
or ␤-alanine (273). The idea that glycinergic ligands may
act on nonsynaptic GlyRs to mediate processes of physiological importance certainly warrants further attention.
Traditionally, however, a functional role for GlyRs in
neurons has required the demonstration of strychninesensitive synaptic currents.
There is abundant evidence for the existence of functional glycinergic synapses in the retina (see below) in
spinal cord motor reflex pathways (219) and in spinal
cord pain sensory pathways (4). Glycinergic neurotransmission has also been demonstrated in various brain stem
nuclei. For example, it has been well characterized in
several brain stem nuclei of the central auditory pathways. In the medullary cochlear nucleus, which receive
inputs directly from the auditory nerve, glycinergic synapses occur onto stellate cells (108) and bushy cells (230).
In the trapezoid body, a subsequent major relay station in
the auditory pathway, GlyRs are located presynaptically
at calyceal synapses onto principal cells (373). The prom-
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JOSEPH W. LYNCH
inent output from this nuclei extends to the superior
olivary complex of the pons, where glycinergic synapses
are also found (194, 351). Functional glycinergic synapses
also exist on neurons in the medullary trigeminal (421),
abducens (325), and hypoglossal motor nuclei (248, 347,
375). In the cerebellum, glycinergic synapses mediate inhibitory neurotransmission between Lugaro cells and
Golgi cells in the cerebellar cortex (93), and between
interneurons and principal cells in the deep cerebellar
nuclei (182). This list may expand as other brain stem
nuclei are characterized in detail.
It is relevant to note that glycine may not be the sole
inhibitory neurotransmitter at many of these synapses.
Mixed GABA-glycine synapses may mediate neurotransmission at individual synapses in the spinal cord (174),
brain stem (194, 283, 325), and cerebellum (100). Interestingly, GlyR activation appears to be able to inhibit
GABAARs via a phosphorylation-dependent mechanism
(229). This process may be important for regulating inhibitory synaptic current magnitude at mixed GABA-glycine
synapses. There is evidence that the GABAergic component of inhibitory neurotransmission at mixed synapses
may be upregulated in individuals suffering from heritable
disorders of glycinergic neurotransmission (see sect. VII).
Finally, presynaptic GlyRs have been functionally
characterized at calyceal synapses (373) and on terminals
synapsing onto rat spinal sensory neurons (172). Surprisingly, in both cases GlyR activation is excitatory, leading
to increased neurotransmitter release.
2. Distribution of GlyR subunits
In situ hybridization was the first approach employed
to localize the distribution of individual GlyR subunits in
the rat. An advantage of this approach is its subtype
specificity, but a disadvantage is that transcript expression does not necessarily imply the surface expression of
functional receptors. Expression of ␣1-subunit mRNA in
adult rats was highest in the brain stem nuclei and spinal
cord, but it was also found in the superior and inferior
colliculi and in regions of the thalamus and hypothalamus
(245, 331). It was notably absent from cortical regions.
Thus, with few exceptions, its distribution is similar to
that of functional GlyRs as described above. In the rat,
expression is detectable at embryonic day 15 and increases to a maximum at around postnatal day 15, without substantial changes in its distribution (245). Northern
blot analysis reveals that ␣1ins shares a similar distribution (244).
Prenatally, transcripts of the ␣2-subunit gene are
found throughout most of the central nervous system.
However, postnatally they decline sharply with little label
remaining by postnatal day 20 (9, 245, 392). Detectable
amounts of ␣2-transcripts do persist into adulthood, however, notably in the retina (see below), auditory brain
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stem nuclei (293), and some higher brain regions (245).
The ␣2A-isoform is expressed more abundantly than ␣2B
during development, although the ␣2B-isoform is present
at higher levels in the adult (199).
The distribution and developmental changes in ␣3transcripts generally resemble those of ␣1-transcripts,
with the exception that ␣3-expression is much less intense at all developmental stages (245). As with the ␣1subunit, its expression intensity increases postnatally to
reach a maximum at around 3 wk (245). The ␣3L- and
␣3K-variants share similar distribution patterns (280).
GlyR ␣4-subunit transcripts are expressed at very low
levels (if at all) in the adult rat (293), although they are
strongly expressed in the spinal cord, dorsal root ganglia,
sympathetic ganglia, and the male genital ridge of the
chick (157).
GlyR ␤-subunit transcripts are distributed widely
throughout the embryonic and adult central nervous system (125, 245). Although present at low levels prenatally,
expression increases dramatically after birth and persists
into adulthood (245). The reason for this broad expression profile is somewhat puzzling given that these subunits do not form functional receptors in the absence of
␣-subunits.
3. Developmental switch from ␣2 to ␣1␤
Becker et al. (27) showed using protein expression
that fetal GlyRs are predominantly ␣2-homomers,
whereas adult receptors are predominantly ␣1␤-heteromers. Such a switch is also supported by the mRNA
expression patterns described above. In the neonatal rat,
the ␣1-, ␣2-, and ␤-subunits exist in abundance, implying
a mixture of receptor isoforms, but the switch towards
the adult isoform is complete by around postnatal day 20
(27, 121, 392). The sparse expression of the ␣3- and ␣4subunits suggests they may also be included in a minority
of adult GlyRs. Recent evidence suggests this switch may
not be as complete as originally thought and that ␣2subunit expression may remain at significant levels
throughout adulthood in the retina (see below) and auditory brain stem (293). Although the mechanism responsible for triggering the developmental switch is not known,
it does not seem to require the activation of the GlyRs
themselves (247).
Given that ␣2-subunits alone are expressed in embryonic neurons, is it possible that homomeric ␣2-GlyRs may
mediate synaptic transmission? Takahashi et al. (361)
showed that the single-channel conductance and kinetic
properties of recombinant homomeric ␣2- and ␣1-GlyRs
were similar to those of native GlyRs in rat spinal neurons
at embryonic day 20 and postnatal day 22, respectively.
They also demonstrated an increased decay rate of the
glycinergic inhibitory postsynaptic currents (IPSCs) over
the same period that was consistent with the change in
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
channel kinetic properties (361). Subsequent studies have
supported these findings (12, 347). However, it is unlikely
that homomeric ␣2-GlyRs mediate inhibitory neurotransmission for the following reasons. First, because ␤-subunits are required for GlyR postsynaptic clustering (188,
259), it is not certain how the ␣2-homomers would undergo the prerequisite aggregation at postsynaptic densities. Second, a recent study has found that ␣2-homomeric
GlyRs activate too slowly to effectively mediate synaptic
transmission (246). Given their wide distribution throughout the nervous system during development, and the fact
that Cl⫺ fluxes are excitatory in developing neurons (114,
355), it seems more likely that homomeric ␣2-GlyRs mediate nonsynaptic cell-to-cell communication that could
be important for neuronal differentiation and synaptogenesis (190). Glycinergic synapses in immature neurons
are probably comprised of ␣2␤-heteromeric GlyRs (219).
The single-channel conductance of synaptic GlyRs from
embryonic neurons is consistent with such a conclusion
(12, 347).
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The synaptic distribution of GlyR subunits is spatially
distinct from that of GABAAR subunits, although individual ganglion cells may possess both types of synapse (195,
329). Recent studies have begun to address the possibility
that glycinergic and GABAergic transmission may have
distinct physiological roles. Although functional differences between GABAergic and glycinergic IPSPs in retinal neurons have been demonstrated (119, 302), the physiological significance remains unknown. However, structural studies have provided evidence that the GABA and
glycine synaptic pathways participate in different functional circuits. In particular, glycinergic synapses are
thought to play a specific role in the transmission of
dark-adjustment signals through the off-channel of the
rod pathway from amacrine cells to off-bipolar cells and
hence to off-ganglion cells (146, 330, 391). This circuit
contributes to the switch from day to night vision.
C. Distribution and Function in Other Tissues
1. Spermatozoa
4. A special case: the retina
This is considered separately because the profile of
GlyR subunit distribution is atypical and has been mapped
in detail and because a specific role for glycinergic synapses has been proposed. GABA and glycine both function as inhibitory neurotransmitters in the retina (143,
176, 297, 390). In situ hybridization and immunohistochemistry both show that GlyR ␣1-, ␣2-, ␣3-, and ␤-subunits have different patterns of distribution in the adult
rat (136, 146, 330). The ␣1-subunit is distributed predominantly on bipolar cells and on some ganglion cells in the
inner plexiform layer (136, 144, 145, 231). The ␣2-subunit
is distributed on amacrine cells and on almost all ganglion
cells, whereas the ␣3- and ␤-subunits are distributed
widely throughout the inner plexiform layer (136). A detailed study in the mouse concluded that ␣1-subunits are
associated with synapses in the rod pathway between AII
amacrine cells and off-cone bipolar cells, whereas ␣3subunits are restricted to cone pathways (159). Together
these results indicate a spatial distribution of GlyR subunit composition throughout the adult retina. Consistent
with this picture, Enz and Bormann (105) detected mRNA
for all four GlyR subunits in RNA from whole retina, but
mRNA for only ␣1- and ␤-subunits in RNA isolated from
individual rod bipolar cells.
Electron microscopy has confirmed that the punctate
immunoreactivity seen with the light microscope is due to
clusters of GlyRs at the postsynaptic densities (146, 330).
Consistent with these anatomical studies, electrophysiological investigations in the rat have revealed the presence of glycinergic inhibitory postsynaptic potentials
(IPSPs) in identified amacrine cells (119), ganglion cells
(153, 302, 368), and rod bipolar cells (77, 99, 303).
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The front of the mammalian sperm head contains a
large secretory vesicle termed the acrosome. The process
of fertilization is initiated when the sperm head contacts
the outer coat, or zona pellucida, of the egg. A zona
pellucida-specific glycoprotein, ZP3, forms the sperm receptor. Its interaction with sperm initiates a complex
intracellular signaling mechanism inside the sperm that
culminates in a calcium elevation that is thought to be
mediated at least partly by an influx through voltage-gated
calcium channels (115). This event, termed the acrosome
reaction (AR), results in the release of acrosome hydrolytic enzymes by exocytosis. These enzymes induce various protein modifications to ensure that the sperm remains tightly bound to the zona pellucida while fusion
takes place between the sperm and egg plasma membranes.
The activation of GlyRs and GABAARs in the sperm
plasma membrane appears to be essential for the AR
(257). There is considerable evidence that GlyRs exist in
sperm plasma membranes. For example, immunochemical studies have demonstrated the existence of GlyR ␣and ␤-subunit protein in porcine, mouse, and human
sperm (49, 258, 332). An immunofluorescence study localized the ␣-subunits to cell membranes in the periacrosomal regions of live mouse sperm (332). In addition, strychnine binding studies have revealed the presence of GlyRs
in hamster sperm (232).
Functional evidence for GlyR involvement has also
been demonstrated. For example, glycine initiated the AR
in a manner that was inhibited by strychnine or a GlyR
␣-subunit antibody (49, 332). Furthermore, studies using
fura 2-loaded human sperm showed that 50 nM strychnine
was also able to inhibit the ZP3-mediated calcium influx
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JOSEPH W. LYNCH
(49). Finally, sperm from homozygous spasmodic and
spastic mice (which possess defective GlyR ␣1- and
␤-subunits, respectively) are deficient in their ability to
undergo the AR (333).
Thus the GlyR is likely to have a central role in the
AR. However, two questions remain about this process.
What is the concentration of Cl⫺ inside sperm? Presumably, it must be high enough to force an outward (i.e.,
depolarizing) Cl⫺ flux upon GlyR activation. Second, what
is the glycine concentration in the oviduct where fertilization takes place? Do the GlyRs remain tonically active
holding the sperm in a depolarized state preceding
the AR?
Harvey et al. (157) found that the ␣4-subunit gene is
expressed on the developing male genital ridge of the
chick and proposed that GlyRs containing this subunit
may contribute to the development of immature spermatogonia.
2. Endocrine pancreas
A pancreatic cell line, GK-P3, expresses functional
GlyRs. When activated, these receptors cause a depolarization that increases the intracellular calcium concentration (393). A glycine receptor antibody displayed immunoreactivity with GK-P3 cells and with isolated rat pancreatic islet cells (393) prompting the authors to surmise
that GlyRs may also be expressed in islet cells in vivo.
However, there is as yet no electrophysiological evidence
for GlyRs in pancreatic islet cells.
3. Adrenomedullary chromaffin cells
High-affinity [3H]strychnine binding sites have been
shown to exist in catecholamine-secreting chromaffin
cells of the adrenal medulla (415, 416). The same group
subsequently demonstrated that glycine can stimulate significant catecholamine secretion from chromaffin cells in
both in vitro and in vivo assays (414, 417). The presence
of GlyR ␣3-subunit mRNA (but not ␣1 or ␣2) was also
demonstrated by RT-PCR from RNA extracted from rat
adrenal glands. However, direct electrophysiological evidence for glycine-activated currents in chromaffin cells is
conspicuously absent to date.
4. Kupffer cells and other macrophages
A variety of pharmacological evidence, summarized
in Reference 184, suggests that GlyRs may at least partially mediate the anti-inflammatory effects of glycine in
macrophages and leukocytes. Research on GlyR involvement in these processes has focused on Kupffer cells,
which are specialized macrophages found in the liver.
Glycine has been shown to reduce the magnitude of lipopolysaccharide-induced calcium transients in these
cells in a strychnine-dependent manner (122, 167). Similar
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observations have also been made in neutrophils (398)
and hepatic parenchymal cells (304). Recent evidence
from Western blots, RT-PCR, and RNAse protection assays indeed suggest the presence of GlyR ␣1-, ␣2-, ␣4-,
and ␤-subunits in Kupffer cells (122).
5. Neural stem progenitor cells
Strychnine-sensitive glycine-gated currents are present in postnatal, nestin-positive neural stem progenitor
cells (278). Consistent with this observation, RT-PCR and
immunocytochemical methods revealed the presence of
␣1-, ␣2-, and ␤-subunit RNA transcripts and ␣-subunit
protein, respectively.
III. STRUCTURE AND ASSEMBLY
A. General Structural Features
The nAChR is the most intensively investigated member of the LGIC family. Consequently, most of the structural features of the GlyR have been deduced from its
homology with this receptor. LGIC receptors contain five
subunits arranged pseudo-symmetrically around a central
ion-conducting pore. The membrane topologies of all
LGIC subunits are similar. This topology includes a large
NH2-terminal extracellular domain that contains the agonist binding sites. A defining feature of LGIC subunits is
the conserved cysteine loop in this domain. All GlyR
subunits also harbor a second cysteine loop (309) that
incorporates a principal glycine-binding domain. As discussed in detail below, the crystal structure of acetylcholine-binding protein (AChBP) provides an excellent model
for understanding the structure of this domain (52).
Hydropathy analysis originally predicted an arrangement of four ␣-helical transmembrane domains (TM1–
TM4) per subunit. Although evidence exists for the inclusion of ␤-sheet in the TM regions (134), the recent elucidation of the crystal structure of the Torpedo nAChR TM
domains provides an overwhelming argument in favor of
the original four ␣-helical model (267). This structure,
determined by cryoelectron microscopy to a resolution of
4 Å by Miyazawa et al. (267), is a major advance in the
field. Finally, TM3 and TM4 are linked by a large, poorly
conserved, intracellular domain that contains phosphorylation sites and other sites for mediating interactions with
cytoplasmic factors. The structure and function of each of
these regions is now considered in detail.
B. Transmembrane Domains
1. Spatial organization
The principal role of the TM domains is to provide a
sealed barrier to separate the ion permeation pathway
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from the apolar region of the lipid bilayer. In most ion
channels of known structure, this is achieved by a close
packing of amphipathic ␣-helices at angles close, but not
quite perpendicular, to the plane of the membrane (353).
This arrangement also applies to LGIC receptors, with
each subunit contributing an ␣-helical TM2 domain to the
lining of a single central water-filled pore. The TM1, TM3,
and TM4 domains surround TM2 and provide the interface
with the lipid bilayer, thereby isolating TM2 from direct
contact with the surrounding hydrophobic environment.
Viewed from the synapse, TM1–TM4 are arranged consecutively in a clockwise manner, with TM1 and TM3 located
closest to TM2 (267). In the nAChR, the TM domains splay
outwards towards the extracellular membrane surface
and extend about two helical rotations (⬃10 Å) beyond
the hydrophobic membrane core. As noted by Miyazawa
et al. (267), the extracellular spaces between the splayed
helices appear to afford a lateral pathway (in addition to
the large central outer vestibule pathway) for ions to
access the pore.
The remainder of this section attempts to relate the
Miyazawa TM domain structure with an abundance of
earlier information that also bears upon TM domain structure and function. However, before doing so, it is worth
briefly considering three functionally based techniques
that have been of particular value in defining the secondary structure of ion channel pore-lining domains.
2. Methods for probing TM domain
secondary structure
A) SUBSTITUTED CYSTEINE ACCESSIBILITY METHOD. The substituted cysteine accessibility method (SCAM) was initially applied as a means of identifying the secondary
structure of ion channel pore-lining domains (5, 8). The
method entails introducing cysteine residues one at a time
into the protein domain of interest. Cysteine reactivity is
then assayed by exposure to highly soluble, sulfhydrylspecific reagents, generally methanethiosulfonate (MTS)
derivatives (180). If a functional property of the channel is
irreversibly modified upon exposure to such a reagent,
the cysteine is assumed to be exposed at the wateraccessible protein surface. If every second residue is
reactive, then the secondary structure is interpreted as
␤-sheet (8), whereas if every third or fourth residue is
exposed, the structure is interpreted as ␣-helical (7). This
approach is now applied more widely to probe structural
changes in extramembranous domains (e.g., Ref. 234).
However, a drawback of applying this approach outside
the pore is that a lack of functional modification does not
necessarily mean that the residue has not reacted. In
other words, negative results cannot be interpreted. However, this limitation is less likely to apply in the spatially
restricted environment of an ion channel pore, where
attachment of a large side chain is more likely to affect
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1057
current flow, thus providing a generally more reliable
measure of cysteine reactivity. Various extensions to this
technique have also proven useful. For example, by determining changes in cysteine reactivity in various functional states (e.g., closed, open, and desensitized), it may
be possible to draw conclusions about state-dependent
structural changes. Similarly, by comparing state-dependent reaction rates of positively and negatively charged
reagents, it may be possible to estimate the local electrostatic potential (289, 405). The originators of SCAM have
provided an excellent review of its capabilities and limitations (180).
B) TRYPTOPHAN SCANNING MUTAGENESIS. This approach involves introducing tryptophan residues one at a time into
the domain of interest. Because tryptophan side chains
are bulky, it is assumed that if they protrude into the
relatively fluid lipid bilayer they should be less likely to
disrupt receptor structure and function than if they protrude towards the protein interior (71). Experimentally,
one or more basic functional properties (e.g., agonist
EC50) of each mutant receptor is measured, and then a
correlation is drawn between the position of the introduced tryptophan and the severity of the functional consequence. As with SCAM, any resulting periodicity is interpreted as ␤-sheet or ␣-helix.
C) HYDROPHOBIC REAGENT REACTIVITY. In this approach,
employed extensively by Blanton and colleagues in the
nAChR (22, 40, 41), labeled hydrophobic reagents are
incubated with the receptor. The identity of the residues
that are covalently modified by these compounds is then
determined using biochemical assays. Residues thus identified are assumed to be exposed to the lipid bilayer.
Again, any resulting periodicity is interpreted in terms of
secondary structure.
3. TM1
By connecting directly with the NH2-terminal domain, TM1 is ideally located to act as a linkage between
the ligand-binding site and the channel activation gate.
Hence, an unequivocal understanding of its structure and
relationship with TM2 is essential. The Torpedo nAChR
crystal structure identifies TM1 as an ␣-helix that is initiated at the residue corresponding to Y222 of the ␣1-GlyR
and enters the membrane at around M227. As stated
above, it is likely that water-filled space surrounds the
extracellular portion of this helix. In support of this, an
aqueous tyrosine-specific reagent labeled two tyrosines
(Y223, Y228) in TM1 of the ␣1-GlyR (222). Furthermore,
SCAM analysis on the nAChR revealed several extramembranous TM1 residues that are accessible to modification
by hydrophilic reagents (432). Several lines of evidence
implicate the extramembranous TM1 residues in LGIC
gating (e.g., Refs. 6, 38, 102, 363, 432). Throughout the
membrane-embedded portion of the nAChR TM1 there
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appears to be a distinct absence of van der Waals contacts
with TM2, implying that a water-filled pocket separates
the respective domains (267). However, by homology
with nAChR, there may be a hydrophobic bond linking
I234 (or L237) and M12⬘ in TM2 of the ␣1-GlyR. At its
intracellular end, the TM1 ␣-helix is probably terminated
by the aspartic acid at position 247.
4. TM2
Affinity labeling experiments employing pore-blocking substances first suggested an ␣-helical open state
structure of the nAChR TM2 (reviewed in Ref. 18). The
Torpedo nAChR structure of Miyazawa et al. (267) confirms the long-held view that this domain forms an ␣-helix
throughout its entire length (267). As summarized in Figure 1A, an ␣-helical structure is also strongly supported
by SCAM analysis. Indeed, the luminal exposure patterns
as determined by SCAM and the Miyazawa structure are
entirely in agreement. SCAM also reveals a highly conserved pattern of residue exposure in the nAChR,
GABAAR, and 5HT3R (Fig. 1A), suggesting that a similar
pattern applies to all LGIC members.
To facilitate comparison between different LGIC
members, a common TM2 numbering system is used
(265). This system assigns position 1⬘ to the putative
cytoplasmic end of TM2 and 19⬘ to the outermost residue
(Fig. 1A). (Note that these assignments are confirmed by
the Miyazawa TM domain structure.) A complete SCAM
analysis of the GlyR TM2 is yet to be published. However,
experiments conducted to date indicate the following
GlyR ␣1-subunit residues line the pore: G2⬘, T6⬘, R19⬘, and
A20⬘ (234, 341). By homology with other LGICs (7, 234,
341, 409), the following residues are also likely to line the
pore: T7⬘, L9⬘, T10⬘, T13⬘, S16⬘ and G17⬘ (Fig. 1A). When
viewed on an ␣-helical net, these residues form a hydrophilic “strip” along one side of an otherwise hydrophobic
␣-helix (Fig. 1B). A predicted cross-section through the
␣1-GlyR pore is shown in Figure 1C. The pore exposure
pattern of residues intracellular to G2⬘ cannot currently
be predicted for anionic LGICs because, as discussed in
detail in section IVB, they contain an additional proline at
position ⫺2⬘ that is likely to induce structural disruptions
around the internal pore boundary. Because the GlyR
␤-subunit TM2 has an unusually low sequence homology
with all other LGIC TM2 domains (Fig. 1A), it will be of
interest to determine whether it also shares the consensus
residue exposure pattern.
Structural analysis shows TM2 to be kinked radially
inwards, attaining a minimum pore diameter at its midpoint (267). Miyazawa et al. (267) propose that this constriction facilitates a tight hydrophobic coupling between
TM2 residues of neighboring subunits at two levels in the
central region of the pore. The first contact is thought to
occur between L9⬘ of one subunit and the 10⬘ residue of the
adjacent subunit. In all GlyR ␣-subunits the 10⬘ residue is a
threonine, but in the ␤-subunit it is a serine. The second
intersubunit contact occurs between residues homologous
to Q14⬘ and T13⬘ in neighboring TM2s of the GlyR ␣1-subunit
(or E14⬘ and S13⬘ in the ␤-subunit).
Although the 19⬘ position defines the external border
of membrane-embedded portion TM2, the ␣-helical structure extends into extracellular space for another 2.5 turns
before terminating at the residue corresponding to V280
in the ␣1-GlyR (267). SCAM analyses on the GlyR and
FIG. 1. Pore-lining residues in the ␣1 glycine receptor
(GlyR). A: sequence alignments of the TM2 domains of
indicated LGIC subunits. Positively charged residues are
shaded in blue, and negatively charged residues are
shaded in yellow. Note that only cationic LGICs have a
negatively charged residue at ⫺1⬘. Circles denote porelining residues as identified by SCAM analysis (7, 234, 341,
409) or by cryoelectron microscopic analysis in the nicotinic acetylcholine receptor (nAChR) (267). In the case of
the serotonin (5-HT3) receptor, dark circles denote those
residues identified as pore-lining by both Refs. 288 and
316, whereas light circles denote residues identified as
pore-lining by Ref. 316 only. Additional GlyR ␣1-subunit
residues that are assumed by structural homology with
other LGICs to line the pore are identified by squares. B:
helical net representation of GlyR ␣1-residues with the
putative pore-lining residues denoted by a white background. C: hypothetical cross-sectional view through the
␣1 GlyR pore. Pore-lining residues are indicated by the
white backgrounds. The exposure pattern of residues
deeper than G2⬘ cannot currently be modeled.
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GABAAR confirm that most extramembranous TM2 residues have extensive contact with water (37, 234). In fact,
the SCAM analysis on the GABAAR even predicted an
␣-helical structure for these residues (37). The TM2-TM3
linker is formed by the ␣1-subunit residues, V280 to D284.
The roles of TM2 in forming the channel gate, in
controlling ionic selectivity, and in forming the binding
sites for agents of physiological and pharmacological importance are considered in sections IV and VI.
1059
By structural homology with the nAChR, the ␣1-GlyR TM4
is likely to be initiated at K385 and terminated at V418,
with the intramembranous portion extending from K389
to I408 (267). Thus the ␣-helix extends about 2.5 turns
beyond the external membrane boundary. Although TM4
is closely apposed to TM3 throughout its length, its contact TM1 appears confined to its intracellular half (267).
C. NH2-Terminal Ligand-Binding Domain
5. TM3
According to the Miyazawa TM domain structure, the
␣1-GlyR TM3 ␣-helical domain starts at I285, with the
membrane-embedded portion extending from A288 to
H311 (267). There is strong support from functionally
based techniques that at least the external (NH2-terminal)
half of this domain forms an amphipathic ␣-helix. For
example, evidence from the nAChR using lipophilic
probes identified an ␣-helical like periodicity in the lipidfacing residues (40). A tryptophan scanning analysis identified an identical periodicity in the same set of residues
(76). In addition, SCAM analysis of the GABAAR using
water-soluble reagents also supported an ␣-helical periodicity in the outer half of TM3 (400, 401). A satisfying
aspect of these studies was that the water-facing residues
were generally displaced by one from the lipid-facing
residues (see Ref. 223 for review). Consistent with structural predictions (267), the SCAM results strongly suggest
that this portion of the domain contributes to the lining of
a water-filled pocket distinct from the channel pore. Recent SCAM studies on the GABAAR in the absence and
presence of pharmacological agents suggest that this
pocket changes conformation as the channel gates (400 –
402). An abundance of evidence from both the GABAAR
and GlyR, reviewed in section VI, provide a strong case
that residues in this pocket form binding sites for alcohols
and volatile anesthetics. The Miyazawa TM domain structure reveals that residues from TM3 are closely apposed
to residues from both TM2 and TM4 at several points
throughout their lengths (267). However, TM3 appears to
contact TM1 only towards the intracellular membrane
surface.
6. TM4
In addition to direct structural evidence (267), several lines of functional evidence imply that this domain
forms an ␣-helix throughout its entire length. First, the
pattern of lipid-exposed residues is consistent with an
␣-helical periodicity as determined by both hydrophobic
probes in the nAChR (40, 41) and tryptophan scanning
mutagenesis in the GABAAR (171). Second, proteolytic
studies on GlyR ␣1-homomers did not identify cleavages
in membrane-associated fragments of this domain (221), a
result that is also consistent with an ␣-helical structure.
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1. Structural homology with AChBP
The fresh water snail, Lymnaea stagnalis, produces
and stores a soluble AChBP in glial cells located near to
cholinergic synapses. When released by acetylcholine
stimulation, AChBP buffers the acetylcholine in the synaptic cleft (350). This protein forms a stable homopentamer and binds acetylcholine, ␦-tubocurarine, and ␣-bungarotoxin with much the same affinity as does the ␣7homomeric nAChR (350). AChBP comprises 210 amino
acids and, although it lacks the TM domains, it provides a
full-length model of the NH2-terminal ligand-binding domain of LGICs. It also incorporates the signature cysteine
loop that is a unique feature of the LGIC family. It shares
a 20 –24% amino acid sequence homology with nAChR
subunits and a 17% homology with the GlyR ␣1 subunit
(Fig. 2). The crystal structure of this protein (52) represents a major breakthrough in our understanding of LGIC
structure and function. Due to both its significant sequence homology and to its functional similarity with the
␣7-homomeric nAChR, its structure is considered an accurate template of the NH2-terminal ligand-binding domain of the nAChR and, by inference, of other LGIC
members.
In three dimensions, AChBP forms a hollow cylinder
with an external diameter of 80 Å, a height of 62 Å, and an
inside diameter of 18 Å. Its size and general shape are in
good agreement with that previously determined from
electron diffraction images of Torpedo nAChRs (266). A
model of the GlyR ␣1-subunit ligand-binding domain
based on the AChBP structure is shown in Figure 3. Each
of the five subunits is positioned in a radially symmetrical
manner around the central pore. When viewed from
above (i.e., from the synapse, looking towards the membrane), the protein is said to resemble “a 5-bladed windmill toy” (52). Individual subunits contain an ␣-helix near
the NH2-terminal extremity and then a series of 10
␤-sheets with short 310 helices following the second and
third ␤-sheets. The ␤-sheets 1–7 form a “twisted ␤-sandwich” with ␤-sheets 8 –10, resulting in 2 separate hydrophobic cores. Together the ␤-sheets form a modified immunoglobulin fold. Pockets are present at the subunit
interfaces, approximately midway between the top and
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JOSEPH W. LYNCH
FIG. 2. Amino acid sequence alignment of AChBP and the human ␣1 and ␤
GlyR subunits. Secondary structural elements of AChBP and Torpedo nAChR are
shown in gray, with ␤-strands represented by arrows and helices represented
by cylinders (52, 267). Membrane-spanning sections of the TM ␣-helices are
shaded in dark gray. The NH2-terminal
␣-helix is labeled by ␣, and two short
polyproline helices are identified by 310.
Cross-linked cysteines are indicated by
the black brackets. The approximate locations of the AChBP binding domains
are identified by red lines (and labeled
A–F, as appropriate) with known glycine
and strychnine binding residues boxed in
black. The numbered assembly boxes are
outlined in green. Residues on the plus
and minus sides of the interface are
shaded yellow and blue, respectively.
Zinc-coordinating histidines are shown in
pink. See Ref. 276 for justification of the
sequence alignment used in the zinc-coordinating region. Putative glycosylation
sites are shaded in green, and putative
phosphorylation sites are shaded in red
and labeled accordingly. A region of the
TM3-TM4 domain involved in determining single-channel conductance in
5-HT3Rs is shaded in gray. The TM3 insertion domain, the gephyrin binding domain, and SH3 homology domains are
boxed and labeled in light blue, dark
blue, and orange, respectively.
bottom of the protein, and abundant evidence (reviewed
in Refs. 74, 179) identifies these as ligand binding sites.
This pocket is lined by three loops from one subunit that
form the “principal” (or ⫹) side of the ligand-binding
pocket, whereas three ␤-sheets from the adjacent subunit
form the “complementary” (or ⫺) side of the pocket.
Viewed from the top of the complex, the complementary
side of each AChBP binding site is situated anticlockwise
relative to the principal side (52). The AChBP binding site
Physiol Rev • VOL
opens to the outside of the complex and, unlike the
Torpedo nAChR electron diffraction images (266), there is
no entry to the binding site from the central pore side of
the protein.
The AChBP structure reconciles many years of biochemical and electrophysiological investigations into the
structure and function of the nAChR. As discussed later in
sections V and VI, it also reconciles an accumulation of
structure-function data from the GlyR. In particular, it
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
1061
FIG. 3. Homology model of the ␣1 GlyR
ligand-binding domain. A: backbone ribbon
representation of the ␣1 GlyR viewed along
the 5-fold axis of symmetry from the synaptic
cleft. The sequence alignment used to generate this model is shown in Fig. 2. Different
colors indicate different subunits. Dotted lines
mark the subunit interfaces with ⫹/⫺ signs
indicating the subunit faces that contribute to
the interface. The putative inhibitory zincbinding region is shown in blue with H107 and
H109 side-chains shown as bonds in pink.
[Model from Nevin et al. (276).] B: stereo view
of the inhibitory zinc-binding site (as proposed
in Ref. 276) viewed from within the vestibule
lumen along the direction of the arrow in A.
Only two subunits are shown for clarity. Sidechains of H107 and H109 from the ⫹ face
(right) and H107, H109, E110, and T112 from
the ⫺ face (left) are shown as bonds, with
standard coloring according to atom. The pink
sphere indicates the location of bound zinc.
(Image courtesy of Dr. Brett Cromer and Prof.
Michael Parker.)
provides an excellent basis for understanding the glycine
and strychnine binding sites and the zinc inhibitory site.
It was recently proposed that sections of the ␣1-GlyR
NH2-terminal domain between residues 158 –165 and 181–
191 may be associated with the plasma membrane (223).
Because the AChBP domain corresponding to 158 –165 is
located at a subunit interface well away from the membrane, a direct membrane interaction seems unlikely.
However, residues 181–191 indeed lie toward the lowest
Physiol Rev • VOL
point of the structure, between ␤-sheets 8 and 9, and thus
could conceivably dip into the membrane.
Apart from the direct polypeptide chain linkage between ␤-sheet 10 and TM1, structural and functional evidence suggest 2 likely points of contact between the
ligand-binding domain and the transmembrane domain.
These regions are the conserved cysteine loop and the
loop linking ␤-sheets 1 and 2 of AChBP. This later loop is
also known as “loop 2.” Both loops have been proposed to
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JOSEPH W. LYNCH
interact closely with the TM2-TM3 linker domain (181,
267), and the nature of these proposed interactions is
considered in more detail in section VD.
2. Glycosylation
As shown in Figure 2, the GlyR ␣1-subunit contains a
glycosylation consensus site at N38, with other ␣-subunits
containing similar sites at the homologous positions. The
GlyR ␣2-subunit contains additional consensus sites at
N45 and N76 (199). On the other hand, the ␤-subunit
contains consensus sites at N33 and N220 (137). The first
suggestion that the ␣1-subunit may be glycosylated was
the finding that mutations to N38 prevented surface expression of functional ␣1-GlyRs (10, 200). Recently, it was
found that glycosylation of ␣1-subunits is a necessary
prerequisite for homomeric receptor assembly and that
receptor assembly is required for transit from the endoplasmic reticulum to the Golgi apparatus and subsequently to the cell membrane (140). The question of
whether ␤-subunits are glycosylated remains to be addressed.
D. Large Intracellular Domain
As the large TM3-TM4 domain is poorly conserved
among LGIC members, both in terms of its length and
amino acid sequence, it is likely to exhibit considerable
structural variation as well. The only structural information to date suggests that the Torpedo nAChR intracellular
domains form a hanging gondola-type structure with
transverse holes (or “portals”) connecting the pore with
the cytoplasm (266). Because these portals are approximately the same size as a permeating ion plus its first
hydration shell (266), they are ideally suited to influence
ion permeation. Indeed, it has recently been shown that
the deletion of three positively charged residues in the
5-HT3AR TM3-TM4 domain dramatically increases the
pore unitary cation conductance (183), implying that
these residues may frame the portals. The homologous
region of the GlyR ␣1-subunit is denoted by gray shading
in Figure 2. The GlyR ␤-subunit has an unusually large
internal domain, comprising 130 residues, whereas the
␣1-subunit contains 86 residues (Fig. 2). The intracellular
domains of both ␣- and ␤-subunits contain a variety of
sites that mediate interactions between the GlyR and
cytoplasmic factors. These putative interaction sites are
now considered.
1. Ubiquitination domain
Under appropriate conditions, intracellular ubiquitin
molecules can covalently attach themselves to specific
lysine side chains on the cytoplasmic protein surface. In
fact, multiple ubiquitin molecules can attach themselves
Physiol Rev • VOL
end to end in a piggyback manner, resulting in a condition
termed “polyubiquitination.” Ubiquitination or polyubiquitination precipitates the internalization and degradation
of many protein types, including surface-expressed ␣1GlyRs (55). Following internalization, the ubiquitin molecules induce the 49-kDa GlyR ␣1-subunit to be proteolytically nicked into a glycosylated (i.e., NH2-terminal) 35kDa fragment and a 17-kDa COOH-terminal fragment.
These fragment sizes are consistent with the ubiquitination domain lying in the large intracellular domain. The
TM3-TM4 domain contains a total of 10 lysine residues
(Fig. 2), several of which probably need to be individually
ubiquitinated before GlyRs can be endocytosed (55). This
mechanism is likely to be important in regulating the
number of surface-expressed GlyRs per postsynaptic density.
2. SH3-binding motif
Because prolines induce kinks into peptide chains,
regular spacing of these residues can form helical structures known as polyproline (PII) helices. Circular dichroism studies reveal the GlyR ␣1-subunit to contain a significant fraction (9%) of this structure (58). A certain class
of protein-protein interaction sites, termed SH3 domains,
are formed from PII helices (290). As recently noted (58),
GlyR ␣1- and ␤-subunits both contain SH3 consensus
sequences in their large intracellular domains (Fig. 2).
Although the role of these domains has yet to be investigated, they may be involved in GlyR trafficking or cytoskeletal attachment.
3. Phosphorylation sites
The locations of phosphorylation consensus sites in
the ␣1- and ␤-subunits are shown in Figure 2. The evidence that phosphorylation of these sites is able to modulate GlyR function is considered in section VIIA.
4. Gephyrin binding domain
This important molecule has long been known to
copurify with the native GlyR as a 93-kDa protein (300).
Kirsch and Betz (188) showed that it mediates the clustering of GlyRs at postsynaptic sites. Gephyrin interacts
with a large and growing number of binding partners,
suggestive of a high degree of complexity in the regulation
of GlyR clustering. A description of the interactions of
gephyrin with molecules other than the GlyR is beyond
the scope of this review. Developments in this area are
moving rapidly, and recent progress has been covered in
several excellent reviews (189, 190, 219). The GlyR gephyrin contact site was isolated to an 18-amino acid domain
in the central region of the ␤-subunit TM3-TM4 loop (259)
(Fig. 2). Insertion of the gephyrin binding domain into the
␣1-subunit promotes the clustering of ␣1-homomeric
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
GlyRs (220, 259). A site-directed mutagenesis study isolated gephyrin binding activity to multiple hydrophobic
residues in this domain (191). A hydropathy plot suggests
that this region forms an irregular amphipathic helix, with
the putative gephyrin-binding residues located along the
hydrophobic side.
5. Basic cluster required for TM3 integration
A positively charged cluster, RFRRKRR, located
close to the intracellular boundary of TM3 in the ␣1subunit (Fig. 2), appears to be important for the correct
membrane insertion of TM3. It was found that neutralization of positive charges in this cluster prevented the correct translocation of TM3-TM4 into the lumen of the endoplasmic reticulum (328). This effect was rectified by
deleting positive charges from TM2-TM3. From these results, it was concluded that the basic cluster is necessary
to compensate for the positively charged residues in TM2TM3 which would otherwise preclude the correct membrane insertion of TM3 (328).
E. Receptor Assembly
1. Subunit stoichiometry and arrangement
The subunit composition of the GlyR was determined
by cross-linking its polypeptides with cross-linking reagents of various specificities and lengths (209). Because
the size of the largest cross-linked product totaled approximately five times the mean individual subunit size,
functional membrane GlyRs were concluded to comprise
pentamers. Of course, since a pentameric subunit arrangement was well-established in other LGIC members
(and now in AChBP), it is scarcely conceivable that the
GlyR quaternary structure would have been different.
Oddly, however, evidence for a trimeric ␣1-homomeric
subunit composition has recently been deduced on the
basis of both laser scattering and single particle electron
microscopic analyses (413). A substantial measure of
credibility must be granted to this study as the same
group also proposed a pentameric GABAAR structure using the same techniques. These are the only GlyR images
published to date, and further investigation into the basis
of these findings appears warranted. One possibility is
that the structure is distorted by a closely associated
protein.
Although GlyRs almost certainly comprise pentameric subunit complexes, the stoichiometry and subunit
arrangement of heteromeric receptors are less certain. An
invariant 3␣:2␤ stoichiometry was proposed based on the
observation that ␣-subunits predominated over ␤-subunits in all tissues examined (209). Although no more
direct evidence in favor of any particular stoichiometry
has ever been presented, the 3␣:2␤ ratio is a long-held
Physiol Rev • VOL
1063
dogma in the field. Even if this stoichiometry is correct,
there is no compelling rationale for distinguishing a sideby-side ␤-subunit arrangement from one whereby the
␤-subunits are separated by an ␣-subunit. Knowledge of
this is particularly important for understanding GlyR molecular pharmacology because binding sites of all types
are located at subunit interfaces (74), and the number of
␣-␣, ␣-␤, ␤-␣, and ␤-␤ interfaces per receptor cannot
currently be determined. A careful reanalysis of subunit
stoichiometry and arrangement in ␣␤-heteromeric GlyRs
is overdue.
2. Intersubunit contact points and assembly domains
GlyR ␣1- and ␣2-subunits appear to be able to coassemble in a random binomial manner that is dependent
only on the relative abundance of each subunit (200).
However, when ␤-subunits are present, the ␣:␤ subunit
stoichiometry appears to be invariant, as inferred from
the monotonic nature of the glycine dose-response (200).
The regions of the GlyR ␤-subunit responsible for this
behavior have been investigated in detail (140, 200). The
initial characterization involved coexpressing ␣1-subunits
with chimeric subunits made from ␣1- and ␤-subunits.
Fixed assembly was found to require the extracellular,
but not the TM or intracellular regions, of the ␤-subunit
(200). To further delineate the regions responsible for
subunit assembly, certain divergent domains (termed “assembly boxes”) in the ␤-subunit NH2-terminal domain
were mutated back towards the ␣1-subunit sequences to
see whether they permitted the individual chimeric subunits to express as homomers. Indeed, the introduction of
various combinations of boxes permitted homomeric assembly (200). The important boxes involved in this process are shown in Figure 2. As a minimum requirement,
box 1 has to combine with either box 3 or box 2 plus box
8 to result in ␣1-homomer formation (140, 200). The critical ␣1-subunit residues that must be set towards the
␣1-subunit sequence are as follows: box 1 (P35, N38, S40)
and box 3 (L90, S92), or box 2 (P79) and box 8 (N125,
Y128) (140). Note that N38 is a glycosylation site. The
intersubunit contact points between AChBP subunits and
their predicted counterparts in the GlyR ␣1-subunit are
shown in Figure 2. It is apparent that the boxes do not
directly form the interfaces, although boxes 3 and 8 lie
directly adjacent to interface sites. This suggests that the
box mutations act allosterically to modulate the interface
conformation. The roles of the interface residues themselves in controlling subunit assembly have yet to be
investigated.
3. Effects of high receptor density
The injection of increasing amounts of ␣1-subunit
cDNA into Xenopus oocytes results in a progressive increase in both maximum current (Imax) and glycine sen-
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JOSEPH W. LYNCH
sitivity (90, 362). A recent study has provided a possible
explanation for this. When ␣1-GlyRs containing introduced gephyrin binding domains were clustered using
gephyrin, they exhibited an additional extremely fast desensitizing current component (220). Although the glycine
EC50 of the peak current was similar to that of unclustered receptors, the EC50 of the plateau current was significantly reduced. Fast (submillisecond) solution application to small (HEK293) cells was required to see the fast
desensitizing component. Since such rapid solution exchange is difficult to achieve with Xenopus oocytes, it
seems possible that the fast desensitizing component was
missed in the earlier study. These effects could be the
result of direct interactions between adjacent receptors,
allosteric actions of gephyrin on the ␣-subunit, or depletion of a cytoplasmic cofactor necessary for normal receptor function.
4. Coassembly with other LGIC subunits
Functional evidence suggests that the GABAC ␳1subunit can coassemble with glycine ␣1- and ␣2-subunits
in vitro (287). This study showed that the recombinant
expression of a mutant ␳1-subunit with the ␣1- or ␣2subunits caused a change in the gating of GABA-induced
currents. Because homomeric GlyRs were not activated
by GABA, it was proposed that the change in pharmacology must have been due to coassembly of ␳1- with ␣-GlyR
subunits. This finding raises the intriguing possibility that
such heteromeric receptors might also exist in vivo.
IV. STRUCTURE AND FUNCTION OF THE PORE
A. Functional Properties of the Pore
1. Ionic selectivity
Although GlyRs are strongly selective for anions over
cations, they have a small but measurable permeability to
K⫹ and Na⫹ (45, 186). Native GlyRs expressed in cultured
neurons have a permeability sequence of SCN⫺ ⬎ NO⫺
3 ⬎
I⫺ ⬎ Br⫺ ⬎ Cl⫺ ⬎ F⫺ (45, 107). This sequence is in
proportion to the ionic hydration energies, implying that
the removal of waters of hydration is the major barrier to
ion channel entry. Because electrostatic interactions with
pore sites are, by inference, less important, this sequence
corresponds to a “weak field strength” binding site. The
GlyR pore also exhibits anomalous mole-fraction behavior, suggesting the presence of at least two interacting
binding sites (45, 107). The permeability has also been
probed with large organic anions. From space-filling models of the molecules tested, the narrowest part of the pore
is estimated to have a diameter of at least 5.2 Å in spinal
neuron GlyRs (45), 5.5– 6.0 Å in hippocampal neuron
GlyRs (107) and 5.22–5.45 Å in recombinant GlyRs (324).
Physiol Rev • VOL
By comparison, cationic LGICs also possess a weak field
strength binding site, but they have not been shown to
display anomalous mole fraction effects and have a significantly larger minimum pore diameter of at least 7.5 Å
(225).
2. Single-channel conductance
GlyRs display multiple unitary conductance states
(45, 46). A useful comparison of GlyR conductance states
in native neuronal GlyRs, as well as in various recombinant subunit configurations, is presented in Table 1 of
Reference 307. Recombinant ␣1-GlyRs exhibit five conductance states ranging between 20 and 90 pS, with the
90-pS state occurring with the greatest frequency. The ␣2and ␣3-GlyRs share the same conductance states but also
exhibit a frequently visited 110-pS conductance level. This
characteristic is conferred to the ␣1-GlyR by the G2⬘A
(␣1 3 ␣2) mutation (46). Coexpression of ␣1-subunits
with the ␤-subunit eliminates the highest conducting levels, leaving a 45-pS state as the most frequently occurring
state (46). Incorporation of the E20⬘S (␤ 3 ␣1) mutation
into the ␤-subunit confers ␣1-GlyR-like conductance levels to heteromeric GlyRs. Because neither mutation abolishes the preexisting conductance states, it is difficult to
determine whether their actions are mediated allosterically or via direct interactions with permeating ions. The
relative probabilities of entering the predominant conductance states do not vary with agonist concentration
(25, 374).
As mentioned above, elimination of a series of three
positively charged residues in the 5-HT3AR TM3-TM4 domain resulted in a dramatic increase in the single-channel
conductance (183). It was proposed these residues might
line a narrow portal that links the pore with the cytosol. It
is yet to be established whether the homologous residues
in the GlyR ␣- or ␤-subunits influence the unitary chloride
conductance. However, since both positively and negatively charged residues line the corresponding domain in
the GlyR ␣1-subunit (Fig. 2), and the precise alignment is
unknown, it is difficult to predict what this influence
might be.
B. Molecular Determinants of Ion Selectivity
and Conductance
Much of the groundwork for our current understanding of GlyR permeation mechanisms has come from studies on the nAChR. One classic experiment showed that
the open-channel blocker QX-222 reached as far as the 6⬘
position when applied externally in the open state (61,
224), implying that the most constricted section of the
pore was located more internally than this point. Because
residues in the narrowest part of the pore are more likely
to influence both unitary conductance and ion selectivity,
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
this in turn implied that the residues near the intracellular
boundary of TM2 controlled both processes. A recent
SCAM study has provided the most definitive functional
evidence to date for a drastic pore constriction between
the ⫺2⬘ to the ⫹2⬘ positions (404). It should be noted,
however, that the Miyazawa TM domain structure shows
the pore widening considerably from the ⫹2⬘ to ⫺2⬘
positions (267). The reason for this apparent mismatch is
not yet understood.
Charged residues are obvious candidates as ionic
selectivity sites. As shown in Figure 1A, the nAChR ␣1subunit contains four charged residues in the vicinity of
TM2: a negatively charged aspartic acid at ⫺4⬘ (the “cytoplasmic ring”), negatively charged glutamic acids at ⫺1⬘
(the “intermediate ring”), and 20⬘ (the “outer ring”) and a
positively charged lysine at 0⬘. Three lines of evidence
indicate that K0⬘ faces away from the pore: 1) chargereversal mutations have no effect on single-channel conductance (168), 2) the pore has a strong negative electrostatic potential in this region (289), and 3) the Miyazawa
TM domain structure unequivocally shows K⬘0 facing
away from the pore (267). Although charge-reversal mutations to the cytoplasmic and outer rings significantly
affect single-channel conductance (168, 169), neither site
appears to significantly impede the access of negatively
charged molecules to deeper regions of the pore (404).
However, mutations to the intermediate ring strongly influence both single-channel conductance (168) and pore
effective diameter (387). In addition, mutations to 2⬘ residues also affect cation selectivity and effective diameter
(reviewed in Ref. 225). Thus residues at the ⫺1⬘ and ⫹2⬘
positions, which are separated by one ␣-helical turn in the
narrowest part of the pore, form the main selectivity filter.
Structural predictions indicate that both side chains have
extensive exposure to the pore (267).
By comparing TM2 sequences between the ␣7nAChR and the ␣1-GlyR, Galzi et al. (126) sought to
determine the minimum number of GlyR residues required to switch the nAChR pore from cation selective to
anion selective. The minimum requirement was found to
be the E-1⬘A and V13⬘T mutations as well as the insertion
of a proline between ⫺2⬘ and ⫺1⬘ (i.e., ⫺2⬘P). The same
mutations achieved a similar effect in the 5-HT3R (149).
Because the E-1⬘A and V13⬘T mutations alone do not
convert selectivity, they are considered to be “permissive”
rather than “essential” for anion permeability (73). Interestingly, the proline insertion site was not critical: selectivity reversal was also effected by inserting it into the
⫺4⬘ position (73). Together these results imply that selectivity reversal required a change in either the lumen geometry or in the exposure profile of side chains lining the
selectivity filter.
The reverse triple mutation (A-1⬘E, T13⬘V and deletion of ⫺2⬘P) was subsequently found to convert ␣1-GlyR
selectivity from anionic to cationic (185). However, the
Physiol Rev • VOL
1065
resultant channels had a low conductance (3 pS at ⫺60
mV) and converted the ratio of permeabilities (PCl/PNa)
from 24 to 0.3, implying that Cl⫺ permeation may have
been reduced without a concomitant increase in Na⫹
permeation. A stronger case for an increase in cation
permeability was made by the same group when they
showed that the reverse double mutation (A-1⬘E and deletion of ⫺2⬘P) decreased the PCl/PNa further to 0.13,
while increasing the unitary cation conductance to 7 pS at
⫺60 mV (186, 272). Furthermore, these mutations increased the effective pore diameter of this GlyR to approximately the value seen in the nAChR. Curiously, however, selectivity reversal was also achieved by incorporating only the A-1⬘E mutation into both the ␣1-GlyR and the
␳1-GABAAR (186, 406). From this result, it is tempting to
speculate that anion selectivity is associated with a net
positive electrostatic charge caused by both the elimination of the negative charge at E-1⬘ and an enhanced pore
exposure of R0⬘.
However, one problem with the “net positive charge”
model of anion permeation is that the anion selectivity of
the triple mutant ␣7-nAChR is not affected by the chargeeliminating K0⬘Q mutation (73). In addition, the E-1⬘A
mutation alone did not increase anion permeability (73).
Indeed, these two observations prompted Corringer et al.
(73) to suggest that the anion-selective nAChR pore may
be lined by polar groups from the peptide backbone.
Further insight into this issue was recently provided by
the finding that the ␳1-GABAAR anion-to-cation selectivity
switch was conferred by the charge-reversing mutation
R0⬘E, but not by the charge-eliminating mutations R0⬘C or
R0⬘M (406). It appears feasible to reconcile all these
findings by proposing that a net negative charge in the ⫺1⬘
region is associated with cation selectivity, whereas a net
neutral or positive charge is associated with anion selectivity. In the event of a neutral potential in this region,
positive dipoles of local polar groups may confer anion
permeability. A critical test of this proposal would be to
quantitate the GlyR pore electrostatic potential profile
using SCAM (289, 405). It would also be informative to
investigate the effects of a variety of substitutions at the
⫺1⬘ and 0⬘ positions to differentiate the effects of side
chain charge, size, hydrophilicity, and hydrophobicity on
ion selectivity and pore effective diameter. Unfortunately,
however, mutations to these residues have a habit of
precluding functional receptor expression.
The influence of ␤-subunits on ion permeation has
yet to be investigated in detail. As shown in Figure 1A, ␣and ␤-subunits have a low sequence homology throughout TM2. Of particular note, the ␤-subunit includes an
alanine-for-proline substitution at ⫺2⬘ and a proline-forglycine substitution at position 2⬘. These substitutions
suggest the ␤-subunit secondary structure may be different in the pore selectivity filter. However, as summarized
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JOSEPH W. LYNCH
above, experiments to date suggest the ␤-subunit does not
induce drastic changes in permeation characteristics.
The effect on permeation of the outer ring of charge,
formed by R19⬘, has also been investigated in the ␣1-GlyR.
Elimination of this charge by the human startle disease
mutations R19⬘L and R19⬘Q caused a decrease in the
unitary Cl⫺ conductance (208, 305). More recently, it was
shown that the R19⬘A and R19⬘E mutations increased the
unitary cation conductance in cation-selective GlyRs and
changed the rectification properties of the pore (272).
Together, these results are consistent with an electrostatic contribution of R19⬘ to ion permeation. However,
R19⬘ does not preclude the entry of positively charged
molecules into the GlyR pore (341, 343) and is more likely
to affect conductance by concentrating anions in the
outer vestibule (186). The ␤-subunit E20⬘S mutation,
which neutralizes a negative charge at the adjacent position, may also increase the single-channel conductance
via an electrostatic mechanism (46).
An impressive array of ␣1-GlyR pore functional properties was reconciled by a three-dimensional Brownian
dynamics simulation study (284). This study used a model
of the pore based on the TM2 primary structure and the
best available estimates of pore diameter prior to publication of the Miyazawa study (267). The selectivity filter
diameter was permitted to vary between 6 and 8.3 Å, but
the charge at R0⬘ was held constant at ⫹0.375e. Under
these conditions, the first Cl⫺ experiences a deep energy
minimum near R0⬘. A second Cl⫺ entering the pore experiences a weaker energy minimum near the pore midpoint. These two ions exist in equilibrium. A third entering
Cl⫺ abolishes these energy wells, allowing the innermost
ion to escape into the intracellular solution. In light of the
preceding discussion, it would be interesting to determine
the effects on permeation of variations to the charge at R0⬘.
C. The Channel Activation Gate
This gate refers to the physical barrier that stops ions
from traversing the pore in the unliganded state. There is
no information available to date as to its location in the
GlyR. There are, however, two schools of thought as to its
location in other LGIC members. One proposal, supported
by the structural analyses of Unwin, Miyazawa, and colleagues (266, 267, 376), is that the TM2 domains are
kinked inwards to form a centrally located gate near the
highly conserved L9⬘ residue. However, the results of
SCAM experiments on the GABAAR and nAChR are inconsistent with the gate lying more externally than the 2⬘
position (7, 289, 409). A particularly detailed study on the
nAChR, which probed the accessibility of cysteines introduced into TM2 to both sides of the membrane in the
closed and open states, delimited the gate to the same
narrow pore region (⫺2⬘ to ⫹2⬘) that houses the selectivity filter (404).
Physiol Rev • VOL
D. Molecular Mechanisms of Desensitization
Desensitization is a property whereby an agonistgated channel closes in the continued presence of agonist.
In general, the rates of onset and recovery from desensitization are important parameters governing the size, decay rate, and frequency of fast synaptic currents (175).
Until recently, it was considered that GlyRs desensitized
too slowly to influence these parameters (219). However,
a recent study using ultra-fast solution exchange identified a very rapid desensitization component (decay time
constant ⬃5 ms) that is induced by either the clustering of
␣1-GlyRs (220) or by coexpression of the ␣1- with the
␤-subunit (268). This rapid component could conceivably
influence the properties of repetitive glycinergic synaptic
currents. However, further research is required to clarify
the role, if any, of GlyR desensitization at glycinergic
synapses.
The location of the desensitization gate in the GlyR is
not yet known. However, SCAM evidence from the
nAChR suggests that the desensitized state is associated
with a crimping of the channel pore between the ⫺2⬘ and
9⬘ residues (403). In contrast, a similar approach showed
that the activation gate crimps the pore between only the
⫺2⬘ and ⫹2⬘ positions.
A growing body of evidence reveals that intracellular
domains can profoundly influence GlyR desensitization. A
functional comparison of human ␣3K- and ␣3L-GlyRs
showed that the removal of a 15-amino acid segment from
the large intracellular domain dramatically increased the
desensitization rate (280). Desensitization rate is also dramatically enhanced by the human startle disease mutations, I244N and P250T (also known as P-2⬘T), in the
␣1-GlyR TM1-TM2 loop (237, 334). Other mutations in this
region, including W243A, I244A, and A251E (also known
as A-1⬘E), have similar effects (186, 237). The relationship
between desensitization rate and the properties of side
chains introduced into the P250 position showed that
bulky hydrophobic residues yielded the fastest desensitization rates (51). Thus desensitization rate is exquisitely
sensitive to structural perturbations in the TM1-TM2 loop.
However, in the GlyR at least, such observations are of
phenomenological interest only until it can be demonstrated that events that alter desensitization in vivo do so
by varying the conformation of this domain.
V. AGONIST BINDING AND
RECEPTOR ACTIVATION
A. Introduction
This section considers the molecular basis by which
agonists bind to and activate (or gate) the GlyR. In particular, it will consider the following important questions.
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
What is the structure of the binding site and where is it
located? What is the molecular basis for binding site
selectivity? What structural changes underlie the activation of the receptor? One problem with addressing these
questions is that it is difficult to experimentally dissect
binding from gating mechanisms as the two processes are
tightly coupled (72). To understand how this separation
may be achieved, it is necessary to briefly consider the
theory of receptor activation.
1. Review of basic receptor theory
In its simplest form, the receptor activation process
can be represented as:
where A is the agonist, R is the vacant receptor, AR is
occupied but shut, and AR* is occupied and activated. The
equilibrium constant for binding (or binding affinity) is
given by:
(1)
where koff is the dissociation rate constant (units of s⫺1)
and kon is the association rate constant (units of M⫺1䡠s⫺1).
The equilibrium constant for gating (or efficacy) is given
by:
E ⫽ 关AR*兴/关AR兴 ⫽ ␤/␣
(2)
where ␤ is the opening rate constant and ␣ is the closing
rate constant (both with units of s⫺1). With the use of
classical receptor theory (along with some simplifying
assumptions), it can be shown (72) that the agonist concentration required for a half-maximal response:
EC 50 ⫽ K A/共1 ⫹ E兲
assumed to affect binding, either directly or allosterically. If a mutation affects mainly E, then the mutated
residue is assumed to affect the conformational change
leading to the open state, either directly or allosterically. Single-channel kinetic analysis can provide reasonably direct estimates of both KA and E. A more
convenient, but less precise, method involves measuring
relative changes in the peak whole cell current (Imax) activated by partial agonists. Since Imax ⫽ inPomax, where i is
single-channel conductance and n is number of activated
receptors, Imax provides a qualitative measure of changes in
E, assuming that i, n, and desensitization rate remain constant.
2. Concerted versus sequential models
of receptor activation
KA
E
A⫹RO
¢¡ AR O
¢
¡ AR*
K A ⫽ 关A兴关R兴/关AR兴 ⫽ koff/kon
1067
(3)
and the maximum fraction of receptors in the activated
state (or maximum open probability)
The above model of receptor activation is an oversimplification because ␣-GlyRs, as pentameric multimers, contain five agonist binding sites. Two starkly contrasting models have been proposed to describe multisubunit protein allosteric mechanisms. These are the
coupled Monod-Wyman-Changeux (MWC) model (60)
and the uncoupled or sequential Koshland-NemethyFilmer (KNF) model (193). In the simplest version of
the MWC model, all the subunits change conformation
simultaneously, and in consequence, the receptor can
exist in only the closed or entirely activated states. In
contrast, in the KNF model, each subunit can independently adopt a specific conformation change depending
on the number of bound agonist molecules, leading to a
series of intermediate protein conformational states.
Extended MWC models that allow for multiple functional states and subunit asymmetry can explain many
characteristics of nAChR behavior (60). In particular,
two simple observations that support an MWC model
over a KNF model are that 1) nAChR single-channel
conductance is independent of agonist concentration
and 2) spontaneous channel openings are occasionally
seen in the absence of agonist. As discussed in the
ensuing paragraphs, the information available to date
also favors an MWC model for the GlyR.
B. Kinetic Models of Glycine Receptor Gating
Pomax ⫽ E/共1 ⫹ E兲
(4)
Equation 3 tells us that the EC50 is a function of both
the binding and gating properties of the receptor. Equation 4 tells us that variations in E have no measurable
effect on maximum open probability unless they occur
within a limited range of ⬃0.1–10.
The structural basis of GlyR binding and gating has
mainly been investigated using site-directed mutagenesis coupled with functional (i.e., electrophysiological)
analysis. If a mutation affects mainly KA, then it is
Physiol Rev • VOL
The single-channel kinetic properties of GlyRs were
first characterized in native receptors from embryonic
mouse spinal neurons (374). This study revealed several
important features of GlyR kinetic behavior. First, unlike
nAChRs, GlyR channel openings did not occur in the
absence of agonist. Second, although the single-channel
conductance was not constant, it displayed no variation
with agonist concentration. The third and perhaps most
revealing feature was that there were at least three exponential components in the open period distributions.
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JOSEPH W. LYNCH
Short-lived channel bursts (corresponding to the faster
exponential components) predominated at low concentrations, whereas the relative frequency of longer lived
bursts (slower exponential components) increased at
higher concentrations. It was therefore concluded that
progressively longer-lived states were associated with increasing numbers of bound glycine molecules and that
maximal channel activation therefore required a minimum of three bound glycines (374). Because the molecular identity of the mouse GlyRs is not known, the number of glycine binding sites per receptor is uncertain.
Irrespective of this, the three-open-state model is in
broad agreement with several more recent studies on
recombinant ␣1-GlyRs (25, 123, 139, 212, 227). However,
one study that examined the equilibrium gating of recombinant ␣1-GlyRs identified two further burst-length components, namely, a particularly short-lived state that was
prominent at low glycine concentrations and a particularly long-lived state that was seen only at the highest
tested concentrations (25). The authors interpreted these
results by proposing that the shortest to longest lived
states correspond to receptors occupied by one to five
glycine molecules, respectively. These findings were reconciled into an MWC-like model where any one of five
possible liganded states can lead to channel opening. An
attractive feature of this model is that ␣1-GlyRs do indeed
possess five glycine-binding sites.
However, this model is controversial because it predicts that mono-liganded openings should add an instantaneous linear component to the onset of the glycinestimulated response, and experiments involving the rapid
application of glycine to both native and recombinant
GlyRs have revealed the distinct absence of such a component (128, 139, 218, 227). The shape of the glycine
activation response was consistent with a minimum of
two bound glycines being required to activate the native
GlyR (218) or a minimum of two or three bound glycines
for activation of the recombinant ␣1-GlyRs (128, 139,
227). Thus the GlyR kinetic models generated by analysis
of stationary and nonstationary receptor kinetics cannot
be resolved at present. Because such models are crucial
for understanding and predicting receptor behavior, it is
hoped that both stationary and nonstationary kinetic analyses will be included in the development and functional
testing of future models.
In contrast to the ␣1-GlyR, the ␣2-GlyR can be modeled as possessing only a single open state (246). It also
has an extraordinarily slow rate of receptor activation
that requires at least two simultaneously bound glycine
molecules (246). Mangin et al. (246) successfully modeled
these properties by proposing that the single open state
was linked to the fully liganded closed state.
Transitions to and from desensitized states have also
been modeled in ␣1- (128, 218, 246) and ␣2-GlyRs (246).
These models depict these states as being accessed from
Physiol Rev • VOL
the singly or doubly liganded closed states. Some kinetic
studies have ignored the effects of desensitization because it generally occurs at a relatively slow rate.
C. Agonist Binding
1. Agonist affinity and efficacy
Native and recombinant GlyRs are activated by
amino acid agonists with the following rank order of
potency: glycine ⬎ ␤-alanine ⬎ taurine ⬎ GABA. In ␣1GlyRs expressed in HEK293 cells, glycine, ␤-alanine, and
taurine all behave as full agonists, with glycine exhibiting
an EC50 value of 20 –50 ␮M (46, 237). However, when the
same ␣1-GlyRs are expressed in Xenopus oocytes, the
EC50 values of all agonists are increased by about an
order of magnitude, and the peak magnitudes of saturating ␤-alanine-, taurine- and GABA-gated currents are reduced relative to those activated by glycine (214). These
differences, shown in diagrammatic form in Figure 4,
imply variations in GlyR conformation between the two
expression systems. A recent detailed study of Xenopus
oocyte-expressed ␣1- and ␣2-GlyRs showed that the glycine, taurine, and GABA sensitivity varied in parallel from
cell to cell over a surprisingly large (10-fold) range (90). In
addition, the relative peak magnitudes of taurine- and
GABA-gated currents varied according to their EC50 values. The origin of this variability is not known, but such a
degree of variability has not been reported in GlyRs expressed in HEK293 cells.
The information available to date suggests that ␣2-,
␣3-, and ␣4-GlyRs exhibit similar agonist sensitivities to
the ␣1-GlyR (90, 130, 157, 201, 202, 246). The incorpora-
FIG. 4. Typical agonist sensitivity profiles of human ␣1 GlyRs
recombinantly expressed in HEK293 cells (top) and Xenopus oocytes
(bottom).
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
tion of ␤-subunits has little effect on receptor sensitivity
to glycine (154, 298, 268), although its effect on the EC50
values for other agonists has not been determined to date.
A combination of rapid agonist application techniques and equilibrium single-channel kinetic analysis
was used by Lewis et al. (227) to estimate the agonist
affinities and efficacies of glycine, ␤-alanine, and taurine
on ␣1-GlyRs expressed in HEK293 cells. The respective
KA values were estimated as follows: glycine, ⬃160 ␮M;
␤-alanine, ⬃360 ␮M; and taurine, ⬃460 ␮M. These differences were found to be entirely due to variations in the
agonist dissociation rates. The E values were predicted to
be 16, 8.4, and 3.4 for glycine, ␤-alanine, and taurine,
respectively (227). The differences among these values
were caused by variations in the channel opening rate,
with the channel closing rate remaining constant for all
three agonists. Earlier studies on homomeric ␣1-GlyRs
(128) and heteromeric ␣1␤-GlyRs (218) estimated comparable E and KA values for glycine. In contrast, the homomeric ␣2-GlyR was estimated to have a glycine E value of
at least 250 and a KA value near 40,000 ␮M (246).
2. Agonist binding domains
As discussed in detail in section IIIC, the ligand-binding pocket is formed as a cleft between adjacent subunits.
Three loops (domains A, B, and C) form the “principal”
ligand-binding surface on the ⫹ side of the interface (Fig.
3A) and three ␤-strands (domains D, E, and F) from the
adjacent subunit comprise the “complementary” (or ⫺)
face. The residues of AChBP that contribute to these
domains are indicated in Figure 2. The human GlyR ␣1and ␤-subunit residues that align with the AChBP ligandbinding site residues are also shown.
Kuhse et al. (202) observed that the GlyR ␣2*-splice
variant subunit had a glycine EC50 that was ⬃40 times
higher than those of the ␣2- or ␣1-subunits. Glycine sensitivity was restored by incorporating the E167G mutation
into the ␣2*-subunit, thus reversing the only nonconserved amino acid between the ␣2*- and ␣2-subunits. This
residue aligns with G160 in agonist binding domain B of
the GlyR ␣1-subunit (Fig. 2). It was subsequently shown
that the double mutant F159Y, Y161F ␣1-GlyR caused a
modest (12-fold) increase in glycine sensitivity, but surprisingly large (121- and 45-fold) increases in the sensitivities to ␤-alanine and taurine (338). Although EC50
changes alone do not provide evidence for binding interactions, domain B was considered likely to bind glycine
since 1) the mutation also dramatically affected GlyR
sensitivity to the competitive antagonist strychnine (see
below), and 2) this domain had already been identified as
an nAChR binding site (reviewed in Ref. 19). The domain
has since been implicated in agonist binding in the GABAA
and 5-HT3 receptors (14, 354).
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The agonist-binding role of the GlyR ␣1-subunit C
domain has been investigated by the Schofield group (309,
381, 382). The GlyR is unusual among LGICs in that its C
domain is contained within a second extracellular disulfide loop. It was shown that mutations to the even-numbered residues, L200, Y202, and T204, profoundly affect
receptor sensitivity to glycine or strychnine, or both (309,
381, 382). Of particular note, the conservative Y202F mutation caused a drastic increase (480-fold) in the glycine
EC50, while having little effect on strychnine sensitivity
(309). Due to the differential sensitivity of specific residues to glycine and strychnine and the fact that this
domain had been implicated in acetylcholine binding to
the nAChR (reviewed in Ref. 19), binding domain C is
likely to be involved in glycine binding. However, most
mutations to this domain also affected receptor gating, as
evidenced by their conversion of taurine from a full into a
partial agonist (309).
Residues I93 and N102 in domain A were identified as
agonist-binding elements on the grounds that conservative mutations affected agonist EC50 values only, without
affecting strychnine sensitivity or the agonist efficacies of
␤-alanine or taurine (151, 379). The R97G mutation, which
induced spontaneous gating in ␣1-GlyRs (32), may also
have exerted a direct or allosteric effect on this site.
The possible agonist-binding roles of the GlyR ␣-subunit complementary (D, E, and F) domains have not yet
been investigated. This constitutes a significant gap in our
understanding of glycine binding mechanisms, as it is
currently unclear whether bound glycine molecules are
coordinated by adjacent subunits.
The possible agonist-binding role of the ␤-subunit
has also received scant attention. A study on Xenopus
oocyte-expressed GlyRs found that the incorporation of
␤-subunits reduced the Hill coefficient for glycine activation from 4.2 in ␣1-homomers to 2.4 in ␣1␤-heteromers
(46). This implied that ␤-subunits did not contain glycine
binding sites. However, not all studies have observed a
change in Hill coefficient upon ␤-subunit incorporation
(e.g., Ref. 154). Interestingly, a single-channel study investigating the effect of the ␤-subunit startle disease mutation G229D found that it disrupted the agonist binding
affinity (314). Because G229 lies in loop C (Fig. 2), this
effect was most likely mediated by either a direct or
allosteric disruption of a ␤-subunit glycine binding site.
Clearly, further experiments are required to clarify the
agonist-binding role of the ␤-subunit.
3. Physical basis of agonist binding
There is abundant evidence that the ACh-nAChR
binding reaction is mediated mainly by noncovalent “cation-␲” electrostatic interactions (430). In this system, the
aromatic side chains of phenylalanine, tyrosine, or tryptophan contribute a negatively charged ␲ surface while
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JOSEPH W. LYNCH
the cation is provided by the agonist. In the ␣1-nAChR
subunit, the ACh quaternary nitrogen interacts directly
with Y149 by this mechanism (434). The corresponding
GlyR ␣1-subunit residue Y161 is conserved in all GlyR
subunits. By analogy with the nAChR, Y161 could conceivably form a cation ␲ interaction with the glycine
amine nitrogen.
Glycinergic agonists have been subjected to a number of structure-activity investigations, with the most notable being that of Schmieden and Betz (336). This study
tested the agonist and antagonist potencies of a range of
␣- and ␤-amino acids. Agonist activity was found exclusively to be a property of those molecules where the
amino and acidic moieties existed in a cis-conformation
(Fig. 5). One molecule (nipecotic acid) that was locked
into a trans-conformation exhibited only antagonist activity. However, ␤-amino acids (e.g., taurine) that randomly flicker between both conformations displayed both
agonist and antagonist activity (Fig. 5). This model predicts a specific antagonist recognition site in a common
ligand binding pocket that is accessible only by transisomers (336). By binding in the trans-conformation,
␤-amino acids may either stabilize the closed state or
sterically prevent glycine from binding in the pocket, or
both. This model also predicts that the partial agonist
activity of taurine results from a fraction of molecules
binding as antagonists and another fraction binding as
agonists at the same receptor. Although attempts to identify a putative antagonist contact site have proven inconclusive to date (151, 339), this interesting model is certainly worthy of further consideration.
D. Structural Changes Accompanying Activation
1. The ligand-binding domain
Functional data suggest that nAChR activation is mediated by global intersubunit movements (74). A recent
structural analysis of the nAChR by Unwin et al. (377) has
provided more direct support for such a model. This
group interpreted low-resolution electron diffraction images of Torpedo nAChRs obtained in both the closed and
open states using the AChBP crystal structure as a template. In modeling the structural changes, they divided the
␣-subunit into inner and outer parts and considered each
separately. The inner part (which includes all residues up
to the conserved cysteine loop) is so-called because it
faces the channel vestibule and contains most of the
intersubunit contact points plus agonist-binding domain
A. The outer part (which comprises ␤-sheets 7–10) includes the agonist-binding domains B and C. Upon agonist
binding, the outer part was found to undergo an upwards
tilt around an axis parallel with the membrane plane.
Simultaneously, the inner part rotated ⬃15° in a clockwise direction (when viewed from the synapse) around an
axis perpendicular to the membrane plane. Being essentially a combination of rigid body movements, the largest
residue displacements (up to 2 Å) occurred at the subunit
interfaces. The rotation of the inner sheets is viewed as
the crucial event in transmitting agonist-binding information to the channel gate (377). The inner sheets contain
two loops, the conserved cysteine loop and loop linking
the first and second ␤-sheets (referred to hereafter as loop
2), that are ideally located to interact directly with the
TM2-TM3 domain (52, 267). Interactions between these
loops and the TM2-TM3 domain appear crucial to the
GlyR activation process, and the extensive body of evidence implicating these regions in receptor gating will
now be considered.
2. The TM2-TM3 domain
FIG. 5. Structural comparison of GlyR agonists and antagonists.
Ligands (glycine and L-alanine) that display only agonist activity are
shown on the left. Nipecotic acid, which displays only antagonist activity, is shown on the right. Taurine, which displays both agonist and
antagonist activity, flickers randomly between the two indicated conformations. In the cis-conformation, the distance between amino and
acidic groups is similar to that found in glycine. In the trans-conformation, the distance is similar to that seen for nipecotic acid.
Physiol Rev • VOL
The structural changes initiated in the ligand-binding
domain are transmitted to the membrane-spanning domains, where they culminate in a change in conformation
of TM2. As stated above, one such contact point is the
“TM2-TM3 domain” which extends from R271 to D284 in
the GlyR ␣1-subunit. Although originally thought to comprise an extended extramembranous loop, the Miyazawa
TM structure now reveals this domain to comprise the
extramembranous portions of the TM2 and TM3 ␣-helices
plus a short connecting loop (267).
A signal transduction role for single residues in this
domain was first suggested by studies on the GABACR
(204), the GlyR (305), and the nAChR (56). A systematic
study subsequently proposed a structural role for the
entire ␣1-GlyR TM2-TM3 domain in the gating process
(237). This conclusion was based on the observation that
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
several naturally occurring human disease mutations,
plus several more alanine-substitution mutations, scattered throughout this domain acted similarly in reducing
the agonist efficacies of taurine and ␤-alanine relative to
glycine. Because ␤-alanine and taurine retained high potency as antagonists, their agonist binding affinities were
considered to be little affected. Thus, because a number
of mutations throughout this domain predominantly impaired E, the whole domain was hypothesized to comprise a structural element of the receptor activation pathway (237). A subsequent single-channel study on one of
the tested mutants (K276E) supported this interpretation
(228). A more direct test of this theory required an investigation into whether the region moves during activation.
Accordingly, SCAM was employed to probe for changes in
its surface accessibility between the closed and open
states. The results did reveal an increased surface accessibility of the NH2-terminal (␣-helical) half of the domain
in the open state (234), consistent with a conformational
change during gating. Several approaches have since been
used to implicate various TM2-TM3 residues in the gating
of other LGICs including the nAChR (75, 141, 142, 320,
321) and GABAAR (37, 44, 113, 181, 285).
Recent studies have begun to shed light on the specific structural role of this domain. The Auerbach group
examined the linear free-energy relationships (LFERs) of
nAChRs incorporating mutations in various positions
(142). They showed that the energy transition experienced by a TM2-TM3 residue was intermediate between
those experienced by residues at the binding site and the
activation gate. This was interpreted to mean that the
TM2-TM3 domain is positioned midway along the agonistinduced “conformational wave” that proceeds from the
binding site to the activation gate. Another study, undertaken on the GABAAR, employed a mutant cycle analysis
approach to identify an electrostatic attraction between
the negatively charged residue D149 and the positively
charged residue K279 (181). D149 lies in the conserved
cysteine loop, whereas K279 lies in the TM2-TM3 domain.
A cysteine cross-linking approach further supported the
idea of a closer association between these residues in the
open state (181). Together, the results suggested that
channel activation is accompanied by an increased electrostatic attraction between these two residues. However,
the electrostatic attraction between the corresponding
charged residues (D148 and K276) in the ␣1-GlyR is
weaker, suggesting other interactions may also contribution to channel activation (1, 340).
Structural and functional analyses have also revealed
a close interaction between the TM2-TM3 domain and
loop 2 of the ligand-binding domain (181, 267). Indeed, the
Miyazawa TM structure suggests that one loop two hydrophobic side chain fits into the end of the nAChR TM2
helix like “a pin in a socket.” Charged loop 2 residues have
also been implicated in gating the ␣1-GlyR (1, 340) and the
Physiol Rev • VOL
1071
GABAAR (181). Clearly, substantial gaps remain in our
understanding of how agonist signals are transduced from
the binding site to the activation gate of the GlyR. Although it is possible that different LGIC members employ
different coupling mechanisms, a common coupling
mechanism for ␳1-GABACRs and ␣1-GlyRs is suggested
by the chimeric studies of Mihic and colleagues (262).
3. The TM1-TM2 domain
Systematic mutagenesis of residues in the intracellular TM1-TM2 domain suggested this domain may also be
involved in ␣1-GlyR gating (237). As discussed in section
IVD, several mutations in this region also affect desensitization. These observations imply that GlyR gating is very
sensitive to structural perturbations in this domain. It
seems likely, therefore, that this region might also move
during GlyR activation. Further experiments are required
to test this hypothesis.
4. The membrane-spanning domains
Investigations into the structural rearrangements of
TM2 have revealed two major features that will be considered in turn. The first feature originally stemmed from
the observation that the TM2 domain of the Torpedo
nAChR incorporated a centrally located kink that appeared to form the channel gate (376). Concurrent SCAM
studies (7) also provided evidence for a discontinuity in
the ␣-helix around the central (L9⬘) position. Such a discontinuity is likely to introduce a degree of conformational flexibility because some of the H-bonds responsible
for maintaining the ␣-helical structure may be broken.
This discontinuity may therefore serve as a swivel joint to
permit the outer half of TM2 to move asynchronously with
the inner half. In other words, gating may involve a backbone rearrangement in the vicinity of L9⬘. This has indeed
been suggested by SCAM studies (7), LFER analysis (83),
molecular dynamics simulations (216), and a study that
introduced unnatural, backbone-altering mutations into
TM2 (104).
The second major feature of TM2 gating also concerns the 9⬘ position. The muscle nAChR L9⬘T mutation
dramatically affects the desensitization rate, the size of
spontaneous leakage currents, and the effects of allosteric modulators (reviewed in Ref. 74), implying complex
(possibly global) effects on channel gating mechanisms.
Furthermore, mutating the 9⬘ leucines to small polar residues (serines or threonines) had an equal effect on the
ACh dose-response regardless of which subunit was mutated (110, 206). As binding sites exist at only two of the
five subunit interfaces, these results implied that neighboring nAChR subunits interact allosterically via their
respective L9⬘ residues. Recent evidence suggests that
GlyR ␣- and ␤-subunits interact in a similar manner (344).
The Miyazawa TM domain structure (267) provides some
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JOSEPH W. LYNCH
insight into how these interactions might occur. The
structure suggests the existence of hydrophobic bonds
between the 9⬘ and 10⬘ residues of adjacent subunits.
These bonds appear to “balance” the L9⬘ residues into a
fivefold radially symmetrical arrangement that holds the
channel closed. It is likely that agonist-induced conformational changes asymmetrically disrupt some of these
bonds, leading to a collapse of symmetry and a simultaneous conversion of all TM2 domains to the activated
state. Mutations to one or more L9⬘ residues may achieve
a similar effect.
In summary, the main features of agonist-induced
TM2 movements are that 1) its midpoint acts as a swivel,
and 2) this swivel point mediates interactions between
adjacent subunits. Agonist-induced backbone rearrangements at this position may thereby lead to a concerted
conformational change at either a centrally located or an
intracellularly located gate (376, 404).
Functional evidence suggests that TMs 1, 3, and 4
may also be involved in LGIC gating. Single-channel kinetic analysis of mutations incorporated into each of
these domains suggests that some residues may have
specific roles in gating the nAChR (47, 89, 104, 388). SCAM
analysis has also provided evidence for state-dependent
structural rearrangements of TM1 and TM3 in the nAChR
and GABAAR, respectively (6, 400 – 402, 432). However,
insufficient information is available to date on any LGIC
member to form a coherent picture of how these domains
contribute to activation. The recently elucidated nAChR
TM domain structure (267) now permits the design of
more specific experiments to investigate these mechanisms.
5. The ␤-subunit
The role of the ␤-subunit in ␣1␤-GlyR gating was
recently investigated by incorporating mutations into corresponding positions in ␣1- and ␤-subunits and comparing
their effects on receptor function (344). Although cysteine-substitution mutations to residues in the NH2-terminal half of the ␣1-subunit TM2-TM3 loop dramatically
impaired gating efficacy (234), the same mutations exerted little effect when incorporated into corresponding
positions of the ␤-subunit (344). Furthermore, although
␣1-subunit TM2-TM3 loop cysteines were modified by
cysteine-specific reagents (234), the corresponding ␤-subunit cysteines showed no evidence of reactivity (344).
These observations suggest structural or functional differences between ␣1- and ␤-subunits. However, the incorporation of the L9⬘T mutation into the ␤-subunit dramatically increased the glycine sensitivity (344), suggesting
an allosteric modulatory effect on the ␣1-subunit. Thus
␤-subunit conformational changes do contribute to the
activation of the GlyR, although their involvement in this
process is significantly different to that of the ␣1-subunit.
Physiol Rev • VOL
VI. GLYCINE RECEPTOR MODULATION
A. Phosphorylation
Phosphorylation can cause long-term changes in the
functional properties of ion channels, and an abundance
of evidence implicates such mechanisms in various forms
of synaptic plasticity (177). Intracellular signaling pathways determine the phosphorylation state of proteins by
coordinating the activities of protein kinases, which induce phosphorylation, and phosphatases, which reverse
it. All of the functional phosphorylation sites on LGICs
have been mapped to the major intracellular loop (360).
As discussed above, LGIC subunits exhibit a low degree
of sequence homology in this region, and this underlies
the subunit-specific distribution of phosphorylation consensus sites (Fig. 2). It has recently become apparent that
clustering and cytoskeletal anchoring proteins can influence the proximity of kinases and phosphatases to the
LGIC intracellular domains (226, 360). Thus the ability of
an LGIC to be phosphorylated depends not only on its
subunit composition but also its proximity to the appropriate enzymes. In addition, kinases and phosphatases
may have indirect effects on the GlyR by controlling the
phosphorylation state of modulatory proteins. This diversity could lead to tissue-specific differences in the propensity of a given GlyR isoform to be phosphorylated.
1. Protein kinase A
Contrasting effects of cAMP-dependent phosphorylation on GlyR current magnitudes have been reported in
neurons from various parts of the brain (219). Although
the differences may be related to the phosphorylation of
GlyR modulatory proteins (or possibly to tissue-specific
or nonspecific effects of pharmacological probes), biochemical experiments strongly suggest that spinal cord
GlyR ␣-subunits themselves are directly phosphorylated
in vitro (378). However, most GlyR ␣-subunit isoforms
do not contain protein kinase A (PKA) phosphorylation
consensus sequences. The exception to this is the ␣1ins
splice variant which contains an eight-amino acid insert
(. . . SPMLNLFQ. . . ) in the large intracellular domain, and
the first residue of this insert may serve as a phosphorylation site (244). As shown in Figure 2, the ␤-subunit also
contains a PKA consensus site at a different position
(T363) in the TM3-TM4 domain (137). However, it is yet to
be determined whether the ␣1ins or the ␤-subunit sites are
directly phosphorylated and, if so, whether phosphorylation induces changes in receptor function. As noted by
Legendre (219), it is possible that the contradictory effects of cAMP-dependent phosphorylation may be explained by the differential expression of ␣1ins- and ␤-subunits throughout the central nervous system (245).
Whether this is true, and whether cAMP-dependent phos-
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1073
phorylation has a physiological role at glycinergic synapses, are important questions for future research.
tween the phosphorylation site and the alcohol binding
site.
2. Protein kinase C
3. Protein tyrosine kinase
Physiologically, protein kinase C (PKC) is activated
by increases in intracellular calcium or diacylglycerol.
The GlyR ␣1-subunit contains a PKC phosphorylation
consensus sequence at S391 in the TM4 domain (323).
Consistent with this, the spinal cord GlyR ␣-subunit was
shown to be phosphorylated by PKC in an in vitro assay
(378). Support for a functional role for S391 is provided by
the observation that the homologous residue in the
GABAAR ␤-subunit is phosphorylated by PKA and PKC
(274). Surprisingly, however, the corresponding residue in
the Torpedo nAChR appears inaccessible to the protein
surface (267). The GlyR ␤-subunit also contains a putative
PKC phosphorylation consensus site at position 389 in the
TM3-TM4 domain (137), although its functional status is
yet to be confirmed. Pharmacological manipulations
aimed at stimulating or inhibiting PKC have revealed contrasting effects on glycine-activated currents in a variety
of neuron types (219). As discussed above, these differential effects may be due to a multitude of causes, including, in some cases, nonspecific effects of pharmacological
agents (e.g., Ref. 281). Our current understanding of PKCdependent phosphorylation is further complicated by the
fact that differences have also been observed in supposedly identical recombinant GlyRs. For example, PKC was
found to potentiate glycine currents in Xenopus oocyteexpressed ␣1- and ␣2-homomeric GlyRs (281). In apparent contrast, PKC activators did not affect current magnitude in ␣1-GlyRs expressed in either Xenopus oocytes
(253) or HEK293 cells (128). However, the later study
reported that PKC activation accelerated the onset of and
slowed the recovery from desensitization (128). A definitive understanding of PKC-dependent phosphorylation
processes in the GlyR will require functional assays involving site-directed mutagenesis of putative phosphorylation sites and biochemical assays to directly determine
the receptor phosphorylation state.
Interestingly, PKC activation has been shown to increase the potentiating effects of ethanol in recombinant
␣1-GlyRs (253). Ethanol was shown to potentiate, inhibit,
or have no effect on glycine-activated responses in 35, 45,
and 20%, respectively, of a large sample of ventral tegmental area (VTA) neurons (422, 423). In the population
of VTA neurons where ethanol induced inhibition, PKC
activators seem to compete with ethanol for a common
inhibitory site (364). A pharmacological study on those
VTA neurons where ethanol induced potentiation, activation of the PKC epsilon isoform was found to increase
potentiation magnitude (173). Together, these findings
raise the possibility of a specific allosteric linkage be-
Lavendustin A, a protein tyrosine kinase (PTK) inhibitor, reduced glycine currents in hippocampal and spinal
neurons, whereas intracellular application of c-Src, an
endogenous tyrosine kinase, increased glycine current
magnitudes (57). The current increases, which were mediated by a leftward shift in the glycine EC50, were accompanied by an enhanced desensitization rate (57). Similar effects of c-Src were observed on recombinant ␣1␤GlyRs expressed in HEK293 cells, but not in recombinant
␣1-GlyRs expressed in the same cells. Mutation of a putative tyrosine phosphorylation site (Y413F) in the large
intracellular loop of the ␤-subunit (137) abolished the
effects of several tyrosine phosphorylation modulators,
suggesting this site is functionally phosphorylated (57).
Although these experiments provide strong circumstantial evidence for ␤-subunit phosphorylation, biochemical
confirmation is required to eliminate the possibility of
allosteric effects induced by the Y413F mutation.
Physiol Rev • VOL
B. Modulators of Possible Physiological Relevance
1. Zinc
A) A PUTATIVE PHYSIOLOGICAL ROLE. Zinc is concentrated
into round clear presynaptic vesicles in the central nervous system and is released into the synaptic cleft by
nerve terminal stimulation (20, 120, 164). During synaptic
stimulation, zinc is thought to reach a peak external concentration of ⬎100 ␮M (20, 120, 385). At such concentrations, zinc is able to modulate a wide variety of pre- and
postsynaptic ion channels (349). Low (0.01–10 ␮M) concentrations of zinc potentiate glycinergic currents by increasing the apparent glycine affinity without changing
the saturating current magnitude, whereas higher concentrations (⬎10 ␮M) inhibit the current by reducing the
apparent glycine affinity (213). This pattern of zinc action
is seen in native receptors (42, 63, 152, 359, 365) as well as
in recombinant ␣1-, ␣2-, and ␣1␤-GlyRs (213). In addition
to increasing the magnitude of glycinergic IPSCs, low
concentrations of zinc have also been shown to prolong
their duration and frequency (211, 359), with both effects
presumably being due to the increased glycine sensitivity.
The role of zinc has been most thoroughly investigated at the mossy fiber glutaminergic synapse in the
hippocampus (120). However, some suggestions have recently emerged that zinc may also have a physiological
role at glycinergic synapses. First, an ultrastructural study
has found evidence for zinc and glycine colocalization in
individual presynaptic terminals in the spinal cord (39).
Second, at glycinergic synapses of the intact zebrafish
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JOSEPH W. LYNCH
hindbrain, zinc chelators decreased the amplitude, duration, and frequency of glycinergic IPSCs, whereas zinc
application had the opposite effect (359). However, it
remains to be established whether zinc is coreleased with
glycine at concentrations high enough to modulate GlyR
function.
It is important to note that even strongly inhibiting
zinc concentrations (ⱖ30 ␮M) causes an initial transient
potentiation followed by the slowly developing inhibition
(235). The duration of this transient potentiation, which is
of the order of 1 s (203, 235), easily exceeds that of a
typical glycinergic IPSC. However, zinc inhibition stabilizes much more rapidly in the absence of agonist (235) so
that if an inhibiting concentration of zinc reaches the
GlyR before glycine does, then only inhibition is seen
(211, 235). Together, these observations mean that high
zinc concentrations may have opposite effects on glycinergic IPSC magnitude depending on whether the zinc
reaches the receptor before or after glycine. This should
be considered in models of zinc action on glycinergic
synapses.
No other metal ion has convincingly been shown to
mimic the biphasic action of zinc. However, the potentiating site is recognized by several metal ions with the
potency sequence: zinc ⬎ lanthanide ⬎ lead ⬎ cobalt (96,
203), whereas the inhibitory site exhibits the potency
sequence: zinc ⬎ copper ⬎ nickel (96, 203).
B) MOLECULAR MECHANISM OF ACTION. In most proteins,
zinc ions are coordinated by nitrogen, sulfur, or oxygen
atoms found in the side chains of histidine, cysteine,
aspartic acid, and glutamatic acid residues (131). Zinc
binding sites are usually comprised of two to four residues, and the affinity of a site depends on the number of
residues, their relative positions, and the local electrostatic environment (21, 317). Several lines of evidence
suggest that the GlyR zinc potentiating and inhibitory
binding sites are physically discrete.
C) INHIBITION. In contrast to the rapid onset of potentiation, zinc inhibition is slow to develop (235). However,
as noted above, this inhibition develops much more rapidly in the absence of agonist (235). This result suggests
that glycine-induced activation is accompanied by a structural change in this location and that zinc acts by stabilizing the closed conformation. A single-channel study
found high (50 ␮M) zinc concentrations reduced ␣1-GlyR
open probability by reducing mean channel open time and
the relative abundance of long channel bursts (212). It
concluded that zinc increases the rate at which the channel exits from the open state, supporting the view that
zinc stabilizes the closed state.
Zinc inhibition of the recombinant ␣1-GlyR was
found to be selectively abolished by either reducing the
pH or by pretreatment with diethylpyrocarbonate, a histidine-specific modifying agent (158). Because both treatments effectively reduce the ability of zinc to bind with
Physiol Rev • VOL
histidine imidazole rings, histidines were implicated in the
complexation of zinc at its inhibitory site. Mutations to
either H107 or H109 were subsequently shown to abolish
zinc inhibition, strengthening the case that these residues
formed an inhibitory binding site (158). Histidine ␣-carbonyl oxygen atoms need to be within 13 Å of each other
to permit their imidazole rings to coordinate a zinc ion
(21). Because the histidines are separated by only one
residue, it is certainly feasible that the zinc ions could be
coordinated within individual ␣-subunits. However, the
␣-carbonyl oxygens of the homologous residues in adjacent AChBP subunits are separated by 7.7 Å (52). With the
assumption that this structure is reasonably well conserved in the GlyR, it is plausible that zinc ions could be
coordinated by adjacent ␣-subunits. This possibility was
tested by coexpressing ␣1-subunits containing the H107A
mutation with ␣1-subunits containing the H109A mutation
(276). Although sensitivity to zinc inhibition is markedly
reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the
coexpression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild-type ␣1-homomeric GlyR. This
constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent ␣1-subunits, but
does not rule out the possibility that zinc may also be
coordinated within ␣1-subunits. No evidence was found
for ␤-subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding
sites per heteromeric receptor is sufficient for maximal
zinc inhibition (276). This region of the ␣1-subunit aligns
poorly with AChBP due to the existence of three additional ␣1-subunit residues. Homology models were constructed using several alignments, and only one of these
produced a plausible zinc binding site (276). The successful alignment is depicted in Figure 2, and the modeled
structure of this site is shown in Figure 3B.
D) POTENTIATION. Low (5 ␮M) zinc concentrations increase the open probability of the ␣1-GlyR by increasing
both the opening frequency and the mean burst duration
(212). The authors concluded that the channel opening
and closing rates were not significantly affected and that
zinc acted primarily by slowing the rate of glycine dissociation from the binding site. This suggests an allosteric
effect of zinc that improves the fit of glycine to its site. In
contrast, zinc had a dual effect on taurine-gated currents.
It not only slowed the rate of taurine dissociation from its
binding site, but increased the rate at which bound taurine could activate the channel (212). Another group
showed that mutations to various residues in the TM1TM2 and TM2-TM3 domains abolished zinc potentiation
of glycine currents, while leaving zinc potentiation of
taurine currents intact (235). These results suggest that
the allosteric linkage between the zinc potentiating site
and the glycine binding site or transduction pathway was
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
selectively disrupted. The results can be reconciled with
those of Laube et al. (212) by proposing that the mutations selectively disrupted the ability of zinc to enhance
the glycine affinity.
Analysis of chimeric constructs of ␣1- and ␤-subunits
implicated D80 as a specific determinant of zinc potentiation (213). Indeed, mutations (D80A, D80G) to this residue disrupted zinc potentiation of glycine currents (212,
235), whereas mutations to neighboring aspartic acid residues (D81A, D84A) had no effect (212). However, because D80A did not abolish zinc potentiation of taurinegated currents (235), its putative role as a zinc binding site
must be queried. The potentiating effect of zinc was also
abolished by the H109A mutation (158) as well as several
mutations in the TM1-TM2 and TM2-TM3 linker domains
(235). Unfortunately, this widespread distribution of sitedirected mutations that abolish zinc potentiation does not
bode well for the use of this approach in identifying the
GlyR zinc potentiating site. The lack of zinc voltage dependence and rapid reversibility of the potentiating effect
(212, 235) indicate that the potentiating binding site resides in an extracellular domain.
2. Calcium
Calcium current influx through glutamate-activated
channels causes a rapid (⬍100 ms) and transient elevation of glycine current magnitude (124, 411, 412) that may
have an important physiological role in modulating the
gain of glycinergic transmission. Based on a pharmacological analysis on GlyRs expressed on rat spinal sensory
neurons, Xu and colleagues (411, 412) proposed that the
effect was mediated by calcium activation of calmodulindependent protein kinase II and calcineurin. However,
Fucile et al. (124), who examined a similar effect in both
cultured spinal neurons and recombinant ␣1-GlyRs, found
it was resistant to a variety of manipulations designed to
disrupt phosphorylation, dephosphorylation, and G protein-dependent processes. These differences may relate to
the different origins of the neurons studied and could be
indicative of multiple mechanisms contributing to this
effect. Another recent study, conducted on VTA neurons,
found the calcium-dependent potentiation to be antagonized by ethanol (438).
In the effect seen by Fucile et al. (124), single-channel
analysis suggested the calcium-dependent factor exerted
complex effects on both the glycine binding affinity and
the gating rate (124). Because the calcium-induced increase did not reverse in inside-out patches, it was considered likely to be mediated by the calcium-induced
removal of a soluble cytoplasmic intermediate from the
receptor internal surface. Although the identity of this
intermediate remains elusive, it appears to be endogenously expressed in HEK293 cells as well as in spinal
neurons (124).
Physiol Rev • VOL
1075
3. pH
Transient increases in extracellular pH occur in response to the activation of anionic LGIC receptors (65).
The mechanism is most likely related to the high HCO⫺
3
permeability of anionic LGICs. When the pore opens it is
likely that HCO⫺
3 exit the cell, causing intracellular acidification and extracellular alkalization (65). In recombinant ␣1- and ␣1␤-GlyRs, the glycine EC50 is significantly
increased as the pH is lowered from 7.5 to 6.0 (64). This
effect appears to be mediated by a specific interaction
with the GlyR extracellular domain as it is abolished by
the ␣1-subunit mutations H109A, T112A, and T112F, but is
not affected by other mutations to T112 or by mutations
to neighboring negatively charged residues (64). Mutation
to the ␤-subunit residue that corresponds to T112 (i.e.,
T135A) was also found to reduce proton sensitivity (64).
Thus pH sensitivity appears to be specific to the receptor
region that houses the zinc inhibitory binding site. However, as threonines are not ionizable, it is unlikely that
they directly form the proton acceptor site.
4. Neurosteroids
Neurosteroids are hormones that are synthesized in
central nervous system glia and neurons from cholesterol
or blood-borne steroidal precursors (24, 357, 372). Although neuroactive steroids produced in the peripheral
steroidogenic glands can easily access the brain, it is
thought that steroids produced in the central nervous
system have important paracrine roles (207). Although
the complex behavioral effects of neurosteroids have
been attributed primarily to GABAA and NMDA receptors
(24, 103, 207, 357), potent neurosteroid actions have been
observed on native and recombinant GlyRs. Antagonistic
effects of progesterone, its precursor pregnenalone
(PREG), and pregnenolone sulfate (PREGS) were first
shown on glycine currents in cultured spinal neurons
(407, 408). Another early report showed that glycine sensitivity in the rat optic nerve was increased by several
corticosteroids at concentrations of 1–10 ␮M (299). On
the other hand, both ␣1- and ␣1␤-GlyRs were found to be
insensitive to the action of 5␣-pregnen-3␣-ol-20-one (295).
Using a more systematic approach, Maksay et al. (243)
examined a battery of neurosteroids on recombinant ␣1-,
␣2-, ␣4-, ␣1␤-, and ␣2␤-GlyRs. Both PREGS and dehydroepiandrosterone sulfate (DHEAS) inhibited all of these
receptors with inhibitory constant (Ki) values of 2–20 ␮M,
with the compounds showing only a modest degree of
subunit specificity. Of particular note, DHEAS inhibited
the ␣2-GlyR with an IC50 of 4.2 ␮M, whereas its IC50 at the
␣1␤-GlyR was 21.2 ␮M. PREG caused an ⬃25% increase in
the ␣1-GlyR current with an EC50 of 1.4 ␮M, but had no
effect on ␣1␤- or ␣2-GlyRs (243). On the other hand,
progesterone inhibited the ␣2-GlyR current by 23% with
an IC50 of 20 ␮M, while exerting no effect on the ␣1- and
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JOSEPH W. LYNCH
␣1␤-GlyRs (243). Given the ␣2 3 ␣1␤ subunit switch that
occurs after birth in the rat, the differential effects of
DHEAS and progesterone on the ␣2- and ␣1␤-GlyRs may
have physiological relevance for neuronal development.
Although the molecular determinants of neurosteroid
action at the GlyR have not yet been determined, some
progress has been made on the GABAAR. On the basis of
a chimeric study on alphalaxone-sensitive and -insensitive
GABAAR subunits, a necessary determinant of neurosteroid action was isolated to the NH2-terminal half of TM2
(318). Consistent with this, the V2’S mutation in the
GABAAR ␣1-subunit reduced the potency of PREGS block
by 30-fold (11). Particularly since the GlyR 2⬘ residue is
known to affect the potencies of other GlyR modulators
(see below), it is a promising candidate as a specific
determinant of neurosteroid action in the GlyR.
Finally, it has long been known that that the synthetic
steroid RU 5135 displaces [3H]strychnine binding with a
remarkably high (5 nM) affinity (48). Because the mechanism of action of this ligand has never been investigated,
it is not known whether it shares a similar mode of action
to the endogenous neurosteroids.
5. G protein ␤␥-subunits
The irreversible activation of G proteins by nonhydrolyzable GTP analogs has recently been shown to exert
potent effects on the GlyR. These reagents cause both a
leftward shift in the glycine EC50 of recombinant ␣1GlyRs and a prolongation of glycinergic synaptic currents
in cultured spinal neurons (425). These effects are most
likely mediated by a direct interaction of G protein ␤␥subunits with the GlyR ␣1-subunit because 1) ␤␥-subunits
coimmunoprecipitate with the GlyR ␣1-subunit, and 2)
addition of ␤␥-subunits to the intracellular membrane
surface reversibly increases GlyR channel activity (425).
These results suggest that G protein-coupled receptor
activation may be important in vivo for regulating the gain
of glycinergic synaptic transmission.
C. Molecular Pharmacology
1. Strychnine and analogs
The plant alkaloid strychnine (Fig. 6) is a highly
selective and extremely potent competitive antagonist of
glycine, ␤-alanine, and taurine with a dissociation constant of 5–10 nM (81, 427, 428). Strychnine sensitivity is
currently the most definitive means of discriminating glycinergic from GABAergic synaptic currents. Because of
its high affinity and specificity, [3H]strychnine has been
widely used in radioligand displacement studies to investigate the potencies and allosteric actions of other GlyR
ligands (241). Strychnine has been subjected to several
structure-activity investigations (162, 239, 249). However,
Physiol Rev • VOL
no analog has ever been shown to possess a higher apparent affinity than strychnine. Similarly, no strychninelike ligands have yet been shown to exert agonist or
allosterically enhancing properties. Surprisingly, however, strychnine has been shown to behave as an agonist
in modified ␣7-homomeric nAChRs (286).
Early evidence from GlyR protein modification experiments indicated that the strychnine and glycine binding sites were mutually interactive but not identical (249).
This interpretation has also been reached as a result of
site-directed mutagenesis experiments. The first of these
experiments stemmed from the observation that the ␣2*splice variant subunit had a strychnine IC50 that was ⬃560
times higher than those of the ␣2- or ␣1-subunits (202).
Strychnine sensitivity was restored by incorporating the
E167G mutation into the ␣2*-subunit, thus reversing the
only nonconserved amino acid between the ␣2*- and ␣2subunits (202). This residue aligns with G160 in the GlyR
␣1-subunit. As expected, strychnine insensitivity was conferred to the ␣1-subunit by the reverse G160E mutation
(381). Mutation to the adjacent ␣1-subunit residue Y161A
also abolished strychnine sensitivity (381), although the
more conservative mutation Y161F did not (338). Photoaffinity labeling experiments localized another crucial
strychnine binding element to either Y197 or Y202 of the
␣-subunit (322). Subsequently, Vandenberg and colleagues (309, 381, 382) identified K200 and Y202 as strychnine binding sites on the basis of functional analysis of
␣1-GlyRs incorporating site-directed mutations at these
positions. Because glycine also binds to common or adjacent residues in both of these strychnine-binding domains (see above), it is reasonable to conclude that
strychnine and glycine share overlapping but nonidentical
binding sites in principal ligand binding domains B and C.
This, of course, provides a structural basis for the competitive antagonist behavior of strychnine (382).
Substances that act purely as competitive antagonists are comparatively rare. Generally, it would be expected that any substance that binds to a receptor would
also bias the conformational equilibrium towards a particular state. However, the possible allosteric effects of
strychnine have yet to be considered. One way of doing so
would be to determine the effects of low strychnine concentrations on modified GlyRs that spontaneously gate in
the absence of agonist.
2. Picrotoxin and analogs
Derived from plants of the moonseed family, the
convulsant alkaloid picrotoxin is also widely used to discriminate GABAergic from glycinergic currents (348). Picrotoxin (PTX) strongly inhibits GABAARs at 1–10 ␮M
concentrations, whereas GlyRs in vivo are considerably
less sensitive. PTX comprises an equimolar mixture of
picrotoxinin and picrotin. As shown in Figure 6, these
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1077
FIG. 6. Structures of representative
compounds that exhibit bioactivity at the
GlyR.
compounds differ only in the structure of the terminal
isoprenyl group, which in the case of picrotin is hydrated
to remove the double bond. Picrotoxinin potently inhibits
the GABAAR, whereas picrotin is generally inactive at this
receptor. This behavior correlates well with the relative
systemic toxicities of the two substances. PTX inhibition
of the GABAAR is use dependent (i.e., inhibition reaches
steady-state at a faster rate in the open state) and noncompetitive, and its inhibitory potency is highly sensitive
to TM2 mutations (see below). This combination of properties has frequently led to PTX being classified as a
channel blocker. However, several lines of evidence (e.g.,
Refs. 277, 296) have firmly established PTX as an allosteric inhibitor of the GABAAR.
In 1992, Pribilla et al. (298) reported that ␣␤-heteromeric GlyRs were much less sensitive to PTX inhibition
than were ␣-homomeric GlyRs. This result was independent of which ␣-subunit (␣1, ␣2, or ␣3) was investigated.
They also showed that inserting the ␣1-subunit TM2 into
the ␤-subunit bestowed high PTX sensitivity to ␣1␤Physiol Rev • VOL
GlyRs. These findings were important for two reasons.
First, by establishing TM2 as a crucial determinant of PTX
sensitivity, they prompted an intensive investigation into
the molecular basis of PTX action in various LGIC members (106, 109, 150, 389, 410, 433). Second, they established PTX sensitivity as a standard pharmacological tool
for identifying the presence of ␤-subunits in recombinant
and native GlyRs. Although several other compounds also
display subunit sensitivity (see below), there is as yet no
better tool than PTX for this purpose.
It was subsequently shown that picrotin and picrotoxinin were equally efficacious in inhibiting ␣1-GlyRs
(236). The same study demonstrated that PTX inhibition
was not use dependent and that its inhibition was “competitive,” meaning that its potency decreased as agonist
concentration increased. Both behaviors are uncharacteristic of channel blockers. An allosteric mode of action
was confirmed by the findings that the R19⬘L and R19⬘Q
startle disease mutations transformed PTX into an allosteric potentiator at low concentrations (⬍3 ␮M) and a
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JOSEPH W. LYNCH
noncompetitive, slow-onset inhibitor at higher concentrations (236).
A series of studies on the GABAAR, GABACR, and
GluClR established the 2⬘ and 6⬘ residues as crucial determinants of PTX sensitivity (106, 109, 150, 389, 410, 433).
A common feature in all of these studies was that a ring of
6⬘ threonines was invariably required for high PTX sensitivity. Although all GlyR ␣-subunits contain a threonine at
this position, the ␤-subunit contains a phenylalanine. As
anticipated, a range of mutations to T6⬘ in the GlyR ␣1subunit, including the ␣3␤ substitution T6⬘F, greatly diminished the inhibitory potency of PTX and related compounds (341, 343, 356). In addition, incorporating a threonine into the ␤-subunit 6⬘ position restored high PTX
sensitivity to the ␣1␤-GlyR (341), although a range of
other ␤-subunit 6⬘ mutations had no such effect (343).
Does this highly specific requirement mean that T6⬘ is the
PTX binding site? Despite a molecular modeling study
finding that PTX can fit into this part of the pore and that
T6⬘ hydrogen bonds could plausibly coordinate a PTX
molecule (435), it is premature to draw this conclusion.
The PTX-competitive compound ␣-ethyl-␣-methyl-␥thiobutyrolactone (␣EMTBL; Fig. 6) was found to potentiate glycine responses in ␣1-GlyRs but inhibit them in
␣3-GlyRs (356). The TM2 domains of these subunits are
conserved with the exception of the 2⬘ residue: in the
␣1-subunit it is a glycine, but in the ␣3-subunit it is an
alanine. The inhibition seen in the ␣3-GlyR is abolished by
the T6⬘F mutation, although the potentiation seen in the
␣1-GlyR is not affected by this mutation (356). Moreover,
incorporating the ␣1 2⬘ residue into the ␣3-subunit (via
the A2⬘G mutation) converts ␣EMTBL inhibition into potentiation. To interpret these results in terms of binding
sites, one would have to postulate a site that toggles
between inhibitory and potentiating depending on the
identity of residues at the 2⬘ and 6⬘ positions. The existence of two discrete sites (an inhibitory site in the pore
and a potentiating site elsewhere) seems equally implausible. This would require that a mutation that abolishes
the PTX or ␣EMTBL inhibitory sites should simultaneously render functional a distant potentiating site.
Thus, although they provide little support for a PTX or
␣EMTBL binding site at T6⬘, the above results argue
strongly for a close allosteric coupling between the 2⬘ and
6⬘ residues.
The 15⬘ residue has also been implicated into this
scheme. When the GlyR ␣1-subunit S15⬘ was mutated to a
glutamine or asparagine, inhibition became use dependent and noncompetitive (92). This is similar to the effect
previously seen with R19⬘ mutations (236). Because S15⬘
and R19⬘ mutations abolished the rapid-onset PTX effect
that is also abolished by T6⬘ mutations, it suggests an
allosteric interaction between R19⬘, S15⬘, and T6⬘. Does
this imply that S15⬘ is the PTX-binding site? After all,
Physiol Rev • VOL
evidence summarized below strongly implicates S15⬘ as
an alcohol and volatile anesthetic binding site.
The current picture regarding the effects of PTX is
complex because mutations to residues at the 2⬘, 6⬘, 15⬘,
and 19⬘ positions can each affect the mode or potency of
PTX action. Since all of these mutations have been shown
to exert allosteric effects on PTX binding or effector sites,
it is uncertain which, if any, is a PTX contact site. It
appears that an imaginative approach will be required to
convincingly resolve this issue.
Single-channel analysis also reveals complexities in
the actions of PTX. In homomeric ␣-GlyRs, it reduced the
predominant conductance state from ⬃80 to 40 pS, with
the probability of entering the lower conductance state
progressively increasing as the PTX concentration was
raised from 1 to 30 ␮M (217). In contrast, 30 ␮M PTX had
no effect on ␣␤-heteromeric GlyRs, which (perhaps not
coincidentally) also exhibited a 40-pS predominant conductance level (217). Higher (100 ␮M) PTX concentrations induced flickery kinetics in both ␣- and ␣␤-GlyRs
(217, 426). These observations support the conclusion
that PTX acts in an allosteric manner.
In summary, experiments to date have revealed an
unusually complex mode of action for PTX. It seems they
brought us little closer to formulating testable hypotheses
concerning the binding site or mechanism of action of this
enigmatic compound.
3. Cyanotriphenylborate
Negatively charged cyanotriphenylborate (CTB; Fig.
6) was chosen as a potential GlyR pore blocker due to its
structural similarity with triphenylmethylphosphonium
bromide, a classical nAChR pore blocker. CTB was duly
shown to act in the predicted manner: its inhibition of the
␣1-GlyR was potent (IC50 ⬃1.3 ␮M), use dependent, voltage dependent, and noncompetitive (324). Furthermore, it
was not a potent inhibitor of the ␣2-GlyR or ␣3-GlyRs, and
replacing the ␣1-subunit 2⬘ residue with the ␣2-subunit
residue (via the G2⬘A mutation) abolished block. Together, these observations provide a strong case for CTB
binding in the pore. However, it is not straightforward to
conclude that CTB binds to the 2⬘ glycine, as it also
potently blocked an ␣2␤-GlyR in which the ␤-subunit TM2
domain had been entirely replaced by that of the ␣2subunit. This paradoxical result demonstrates that CTB
sensitivity can reside in a receptor containing only alanines at the 2⬘ position, thereby directly refuting the idea
that the CTB binding site requires 2⬘ glycines. One possibility is that regions apart from the TM2 domain may also
contribute to CTB sensitivity (324). Alternatively, ␤-subunits may serve the same role as ␣-subunit 2⬘ glycines in
creating a favorable geometry for the binding of CTB at
some level in the pore. Progress in characterizing the CTB
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
mechanism of action is currently limited due to its lack of
commercial availability.
4. Ginkgolides
Isolated from the leaves, roots, and bark of the
Gingko tree, these macrocyclic terpeine compounds are
widely used in herbal medicine. They also share common
structural features with picrotoxinin (170). Ginkgolide B,
which is well known as a platelet activating factor antagonist (Fig. 6), is also a specific and potent blocker (IC50
⬃0.27 ␮M) of glycine-gated currents in dissociated rat
hippocampal pyramidal neurons (192). It is noncompetitive and use dependent, and its inhibitory potency is not
affected by the competitive antagonist strychnine. Importantly, when applied externally, its blocking ability increased with cell depolarization, as expected for a negatively charged compound binding in the pore (192). These
characteristics establish ginkgolide B as a classical pore
blocker. Its molecular determinants of action and subunit
specificity have yet to be investigated.
More recently, the effects of several ginkgolides were
compared on GlyRs expressed in rat embryonic cortical
neuron slices (170). As these GlyRs were insensitive to
PTX, they may have comprised predominantly ␣2␤-heteromers. Ginkgolides B, C, and M were found to be more
potent than ginkgolide A, ginkgolide J, or bilolabide (a
related compound from the same tree). In agreement with
the earlier study (192), ginkgolide B inhibited the GlyR in
a use-dependent, noncompetitive manner and showed
specificity for the GlyR over a GABAAR expressed in the
same tissue (170).
5. Tropisetron and other 5-HT3R antagonists
Several 5-HT3R antagonists have potent effects on
the GlyR. The most potent of these compounds are those
which contain tropeine groups (i.e., esters and amides of
3␣-hydroxytropane) and include tropisetron, LY-278,584,
zatosetron, and bemesetron. The muscarinic acetylcholine receptor antagonist atropine also belongs in this
structural group. Some representative structures are
given in Figure 6. This review focuses on tropisetron, the
most widely characterized of these compounds.
In 1996, Chesnoy-Marchais found that tropisetron potentiated glycine currents in cultured spinal neurons at
low concentrations (0.01–1 ␮M) but caused inhibition at
higher concentrations (66). Its potentiation was mediated
by a leftward shift in the glycine dose-response curve.
Because the potentiation was additive with the potentiating effects of zinc, ethanol, and propofol (67), tropisetron
appeared to act via a novel mechanism. In radioligand
binding studies on membrane extracts from the spinal
cord and brain stem, tropisetron displaced [3H]strychnine
with a Ki of 2 ␮M (240). In addition, 0.1 ␮M tropisetron
increased the ability of glycine to displace [3H]strychnine
Physiol Rev • VOL
1079
(240), suggesting an allosteric enhancing effect on glycine
binding. Turning then to an electrophysiological analysis,
Maksay et al. (242) subsequently failed to detect tropisetron potentiation in ␣1- or ␣2-GlyR homomers or in ␣1␤or ␣2␤-GlyR heteromers, although the lower apparent
affinity inhibitory effect was observed. In apparent contradiction, Supplisson and Chesnoy-Marchais (358) reported tropisetron potentiation in the ␣1-homomeric and
the ␣1␤- and ␣2␤-heteromeric GlyRs, but not in the ␣2homomer where only inhibition was seen (358). The discrepancy was convincingly resolved by the demonstration
that potentiation required a low (EC10) glycine concentration, whereas higher (EC50) glycine concentrations, as
used by Maksay et al. (242), uncovered only inhibition
(358). The molecular determinants of tropisetron action
have not been elucidated. Because ␤-subunits are needed
to confer tropisetron potentiation to ␣2-subunits (358),
the potentiating binding site may be located at the interface of these subunits.
Tropisetron suppresses glycine-gated currents when
applied at high (⬎10 ␮M) concentrations to both native
neuronal and recombinant GlyRs. At least in the ␣2-GlyR,
it seems to behave in a noncompetitive manner. The
tropisetron inhibitory potency is modestly affected (IC50
increased by a factor of 2) by the T112A mutation (242), a
mutation that completely abolishes the inhibitory potency
of zinc. The increased tropisetron inhibitory potency of
the ␣2-GlyR is not transferred to the ␣1-GlyR via the A2⬘G
mutation (358), the only TM2 domain residue which is not
conserved between these subunits. However, as previously seen for CTB and PTX, it may be misleading to infer
locations of binding sites on the basis of site-directed
mutations at this position in the pore.
A structure-activity study concluded that the tropeine structure itself was required for potentiation (240).
However, atropine, which shares the tropeine moiety,
does not increase glycine displacement of strychnine
binding, although its inhibitory potency is only marginally
weaker than that of tropisetron (240, 242). Thus the tropeine moiety appears to be no guarantee of a potentiating
effect. These results are broadly consistent with another
structure-activity analysis, which concluded that an aromatic ring, a carbonyl group, and a tropane nitrogen are
required for glycinergic potentiation (70).
6. Ivermectin
Ivermectin (22,23-dihydroavermectin B1a) is a naturally occurring macrocyclic lactone (Fig. 6) that is widely
used as an antiparasitic agent in agriculture, veterinary
practice, and human medicine (98, 319). Although the
target of its antiparasitic action is believed to be a GluClR
that exists in a number of invertebrate phyla (78, 178), it
also has direct activating or potentiating effects on
GABAARs and nAChRs (2, 87, 197, 198). At low (0.03 ␮M)
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JOSEPH W. LYNCH
concentrations, ivermectin potentiates subsaturating glycine responses, but at higher (ⱖ 0.03 ␮M) concentrations
it irreversibly activates recombinant ␣1- and ␣1␤-GlyRs
(342). Because ivermectin-gated currents have a different
pharmacology to glycine-gated currents, and glycine binding site mutations do not drastically affect its sensitivity
(342), ivermectin appears to activate the GlyR via a novel
mechanism. Apart from cesium (352), ivermectin is the
only non-amino acid agonist of the GlyR to be identified to
date.
7. Alcohols, anesthetics, and inhaled drugs of abuse
Traditionally, anesthetics have been depicted as nonselective agents that act by partitioning into and disordering lipid bilayers. However, in recent years it has become
increasingly apparent that specific binding sites on ligandgated ion channels are among their major molecular targets (36, 117, 196, 260, 418). Because alcohol and some
anesthetic effects on the GlyR are observed at pharmacologically relevant concentrations, it is possible that at
least part of their acute effects are mediated by this
receptor.
Exposure to pharmacologically relevant (50 –100
mM) concentrations of ethanol was first shown by Celentano et al. (59) to produce a persistent increase in the
glycine sensitivity of spinal neurons. Most subsequent
studies on GlyRs natively expressed in neurons have observed similar potentiating effects. Neonatal ventral tegmental area neurons appear to constitute an exception to
this rule: ethanol (0.1–10 mM) potentiated, inhibited, or
had no effect on glycine-activated responses in 35, 45, and
20%, respectively, of an impressively large sample of these
neurons (422, 423).
Because ethanol potentiation is achieved without disordering the membrane lipid bilayer (366), it is likely to be
acting via a specific site at the GlyR. When expressed in
Xenopus oocytes, ␣1-GlyRs were more sensitive to ethanol than were ␣2-GlyRs (251). This increased sensitivity
was abolished by incorporating the naturally occurring
spasmodic mouse mutation A52S into the ␣1-subunit
(251). However, the ␣1-subunit ethanol sensitivity was
largely lost upon recombinant expression in mammalian
Ltk⫺ or HEK293 cells (380). Because the GlyR ␣1-subunit
contains phosphorylation consensus sites, the phosphorylation status of the receptor was considered a possible
cause of this anomaly. Indeed, PKC-mediated phosphorylation selectively increases ethanol potentiation of Xenopus oocyte-expressed ␣1-GlyRs while having no effect on
the enhancement induced by the inhalation anesthetic
halothane or the intravenous anesthetic propofol (253).
On the other hand, PKA-mediated phosphorylation was
without effect (3).
The inhalation (or volatile) anesthetic isoflurane was
first shown by Harrison et al. (155) to potentiate recomPhysiol Rev • VOL
binant ␣2-GlyRs. For both recombinant ␣1-GlyRs and the
native GlyR in medullary neurons, the degree of potentiation increased in the rank order methoxyflurane, sevoflurane ⬍ halothane, isoflurane, enflurane, F3 (97, 250). The
potentiation was induced primarily by a leftward shift in
the glycine EC50 (97). Because the leftward shift is more
pronounced at low glycine EC values (97), the effects of
alcohols and volatile anesthetics have generally been investigated using glycine concentrations that activate 2–5%
of the saturating current magnitude.
In contrast to the GlyR, the GABACR is inhibited by
both classes of agents (261). By constructing a series of
chimeras between these two receptors, a region of 45
amino acid residues was identified as necessary and sufficient for mediating potentiation by both alcohol and
volatile anesthetics (262). Site-directed mutagenesis of
the nonconserved residues in this region identified S15⬘ in
the TM2 domain and A288 in the TM3 domain as crucial
determinants of alcohol and volatile anesthetic sensitivity
(262). Because both residues are located towards the
extracellular end of their respective TM segments, it was
hypothesized that these two residues faced each other to
form a pocket to accommodate an alcohol molecule. Lying within the membrane, this putative water-filled pocket
is thought to be inaccessible from the ion channel pore.
The existence of such a water-filled pocket lined by TM2
and TM3 domains is supported by SCAM data in the
GABAAR and structural evidence from the nAChR (see
sect. IIIB).
It was already known that alcohol potentiation of the
␣1-GlyR increases with the length of the side chain of a
series of n-alcohols until a cut-off is reached, after which
further increases in molecular size decrease alcohol potency (250). Increasing the size of the amino acid side
chain by the S15⬘Q mutation decreased the cutoff from
n ⫽ 10 –12 to 7, consistent with the expected reduction in
the volume of the binding pocket (399, 424). The converse
experiment on the GABACR, in which a smaller side chain
was introduced at the corresponding position, increased
the alcohol size cutoff (399). Using a similar idea, the
molecular volume and hydropathy of the side chain at the
288 position were revealed as crucial determinants of
volatile anesthetic sensitivity (420). Together, these experiments supported the view that S15⬘ and A288 might
form the binding pocket for both alcohols and volatile
anesthetics. Alternatively, the mutations might impose
structural changes that perturb the allosteric potentiation
mechanisms of these compounds. Indeed, because these
mutations affect glycine EC50 values (262, 420, 424), this
alternative is a distinct possibility. More direct evidence
for these residues forming a binding pocket came from a
SCAM-type approach (252). Current flux through the ␣1GlyR incorporating the S15⬘C mutation was irreversibly
enhanced by the sulfhydryl-containing anesthetic propanethiol under conditions of oxidation by iodine, or
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directly by propyl methanethiosulfonate. After modification by either compound, the GlyR could no longer be
potentiated by alcohols or anesthetics (252). This constitutes strong evidence that these compounds bind specifically in a pocket lined by S15⬘.
A recent investigation has found that ethanol potentiation of recombinant ␣1-GlyRs was antagonized by increased atmospheric pressure (85). Because the increased pressure did not affect the actions of glycine,
strychnine, or zinc, it is likely to exert a selective effect at
the alcohol site. It will be of interest to understand the
molecular basis of this phenomenon.
Recently, several commonly abused inhalants were
investigated in terms of their action at recombinant ␣1GlyRs. Toluene, 1,1,1-trichloroethane, trichloroethylene,
and chloroform potentiated ␣1-GlyRs by reducing the
glycine EC50 values (31, 33). Because these compounds
exhibited different patterns of sensitivity to S15⬘ mutations than did ethanol and enflurane, and also showed
competition with these compounds, it was proposed that
their binding sites shared some overlap with the alcohol/
anesthetic binding site (31, 33). However, the possibility
that the respective binding sites may interact allosterically cannot yet be ruled out.
The gaseous anesthetics nitrous oxide and xenon
weakly potentiate submaximal glycine currents in the
␣1-GlyR at clinically relevant doses (84, 419). Other anesthetics including propofol, thiopentone, pentobarbitone,
alphaxalone (a steroid), etomidate, and ketamine also
exert potentiating effects on some GlyR isoforms (35, 84,
250, 295). Because more dramatic effects of all these
compounds are seen at other receptors (36, 116, 118, 196,
418), their effects on GlyRs are unlikely to be clinically
relevant.
8. Miscellaneous bioactive compounds
Dihydropyridine antagonists of L-type calcium channels also inhibit GlyR currents in spinal cord neurons at
micromolar concentrations (69). Their effects are stronger at higher glycine concentrations and increase with
time during glycine application, reminiscent of an open
channel block mechanism. In addition, low (1–5 ␮M)
concentrations of nitrendipine and nicardipine exert potentiating effects that were additive with those of zinc,
implying discrete mechanisms of action (69).
The estrogen receptor modulator tamoxifen has recently been shown to have a particularly dramatic potentiating effect on submaximal glycine responses in cultured spinal neurons (68). A 5 ␮M concentration caused a
6.6-fold reduction in the glycine EC50. Several controls
were used to rule out possible effects of tamoxifen on cell
signaling cascades. The magnitude of the potentiation
was increased when tamoxifen was applied to the cells
before glycine application, although maintenance of the
Physiol Rev • VOL
1081
potentiation seen upon glycine application required continued tamoxifen application (68). The potentiation persisted in the presence of 10 ␮M zinc, implying a discrete
site of action. The same study also noted an apparently
direct potentiating effect of 4 ␮M dideoxyforskolin on
GlyRs, a finding that may be relevant to studies designed
to investigate phosphorylation mechanisms.
Clinical concentrations of the neuroprotective drug
riluzole were reported to accelerate the ␣1␤-GlyR desensitization rate while having no effect on the maximal
current magnitude (269). Paradoxically, however, the
time course of currents activated by brief (2 ms) applications of glycine were prolonged by riluzole (269). Similar
effects were also observed on the GABAAR. The prolongation of the simulated synaptic currents may help explain the sedative and anticonvulsant side effects of this
drug (95).
Colchicine, a microtubule-depolymerizing reagent,
competitively antagonizes glycine-induced currents with
IC50 values of 64 and 324 ␮M for the ␣1- and the ␣2-GlyRs,
respectively. Its effect is instantaneous and independent
of the microtubule depolymerization process (238). Interestingly, colchicine also prevents ethanol potentiation of
GABAAR currents (394, 397), which perhaps provides a
starting point for investigating its mechanism of action.
The tyrosine kinase inhibitor genistein has been
shown to have a direct inhibitory effect on native GlyRs in
neurons isolated from the hypothalamus and VTA (165,
437). This effect is not a pharmacological effect on PTK
activation as it was effective only from the external membrane surface (165). It inhibits in a noncompetitive and
use-dependent manner, suggesting that it binds in the
pore.
Reasoning that the pharmacology of the GlyR glycine
site may have structural similarities with the glutamate
(NMDA) receptor glycine site, Schmieden et al. (337) used
a potent NMDA receptor glycine antagonist, 2-carboxy-4hydroxyquinoline, as a lead compound for the development of novel GlyR antagonists. The most potent compound thus identified, 5,7-dichloro-4-hydroxyquinoline-3carboxylic acid, inhibited recombinant ␣1-GlyRs with an
IC50 of 20 ␮M. Although this compound was not active at
the NMDA receptor, the results imply some degree of
similarity in the glycine pharmacophores of the GlyR and
NMDA receptors. Another glutamate (KA/AMPA) receptor antagonist, NBQX, has recently been shown to inhibit
recombinant ␣1- and ␣2-GlyRs with an IC50 of 4 ␮M (256).
Effects of ginsenosides, the active ingredients of the
Panax ginseng plant, have been investigated on recombinant ␣1-GlyRs. The most active of these compounds, ginsenoside-Rf, potentiated submaximal glycine-gated currents with an EC50 of 50 ␮M (282).
Glycine-activated currents in recombinant ␣1- and
␣2-GlyRs were both inhibited by 3-[2-phosphonomethyl[1,1-biphenyl]-3-yl]alanine (PMBA) with an IC50 of
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JOSEPH W. LYNCH
⬃0.5 ␮M (163). When applied at higher (10 ␮M) concentrations, PMBA had differential effects on ␣2- and ␣1GlyRs. In the ␣1-GlyR, its main effect was to impose a
rightward shift on the glycine dose-response. However, in
the ␣2-GlyR, it also lowered the Hill coefficient for glycine
activation (163). Furthermore, preincubation with PMBA
delayed the rate of glycine-gated current activation in the
␣2-GlyR but not the ␣1-GlyR. Thus the mode of PMBA
binding or activation may differ between the ␣1- and
␣2-subunits.
Opioid alkaloids have long been known to exhibit
selectivity for the GlyR over the GABAAR (82). The relative efficacy of these compounds in antagonizing glycineinduced currents in intact spinal neurons is thebaine ⬎
morphine ⬎ codeine (79, 82), whereas thebaine was by far
the most potent of a series of opioid agonists in displacing
[3H]strychnine binding in spinal cord neurons, with an
IC50 of 1 ␮M (132).
Finally, as reviewed previously (307), a wide range of
compounds with structural similarities to various GABAAR
ligands have been investigated in terms of their [3H]strychnine displacement potencies. Benzodiazepines and their antagonists were among the most potent compounds thus
identified, with IC50 values of ⬃1–10 ␮M (48, 249, 429).
Interestingly, a recent report has identified a low-affinity
benzodiazepine inhibitory site on ␣2-containing GlyRs (367).
VII. GLYCINE RECEPTOR CHANNELOPATHIES
A. Human Startle Disease
Human hereditary hyperekplexia, or startle disease,
is a rare neurological disorder characterized by an exaggerated response to unexpected stimuli (15, 308). The
response is typically accompanied by a temporary but
complete muscular rigidity often resulting in an unprotected fall. This behavior is graphically illustrated in a
published videotape of the bovine form of the disorder
(161). Susceptibility to startle responses is increased by
emotional tension, nervousness, fatigue, and the expectation of being frightened. Unprotected falls in turn lead to
chronic injuries that are also characteristic of this disorder. Symptoms of this disease are present from birth, with
infants also displaying a severe muscular rigidity (or hypertonia) that gradually subsides throughout the first year
of life. Some affected infants die suddenly from lapses in
respiratory function (129). This disorder has a history of
being misdiagnosed as epilepsy, although hyperekplexia
is readily distinguished by an absence of fits and a retention of consciousness during startle episodes. The symptoms are successfully treated by benzodiazepines, with
clonazepam being the current drug of choice (436).
Hyperekplexia is caused by heritable mutations that
reduce the magnitude of glycine-gated chloride currents.
Physiol Rev • VOL
As summarized in Table 1, this is achieved by either
disrupting GlyR surface expression or by reducing the
ability of expressed GlyRs to conduct chloride ions. The
disease mutations thereby impair the efficiency of glycinergic neurotransmission in reflex circuits of the spinal
cord and brain stem, thus increasing the general level of
excitability of motor neurons. Patients are apparently
able to cope with this reduced inhibitory tone during
normal tasks (perhaps due to developmental compensations, see below), although they are unable to cope with
the increased demand required to dampen strong, unexpected excitatory commands.
Genetic linkage analysis of startle disease pedigrees
first localized this disorder to the distal portion of chromosome 5q (327), where the GlyR ␣1-subunit gene is found. It
was subsequently shown that an autosomally dominant
form of startle disease was due to either the R271L or R271Q
substitutions in the human ␣1-subunit (346). Although numerous other startle mutations have since been identified,
the R271 mutations seem to be the most common cause of
this disorder. When incorporated into recombinantly expressed GlyRs, these mutations caused a reduced glycine
sensitivity and single-channel conductance (208, 306). Other
autosomal dominant startle mutations, including Y279C,
K276E, Q266H, and P250T, have generally similar effects
(Table 1). In addition, the P250T mutation causes an enhanced desensitization rate (334), which may also contribute to the disease phenotype. As discussed previously in this
review, each of these mutations acts by impairing the receptor activation mechanism, and closer analysis of the mutation phenotypes has provided valuable insights into the
structural basis of GlyR function. The effect of the autosomally dominant V260M and S270T startle disease mutations
(88, 210) has yet to be characterized.
Autosomally recessive forms of startle disease have
been described in sporadic cases, generally in the offspring of consanguineous parents. One such patient exhibited homozygosity for a point mutation, I244N (312), in
the ␣1-subunit. When incorporated into recombinant ␣1homomeric GlyRs, this mutation resulted in a reduced
glycine current magnitude, a reduced glycine sensitivity,
and an enhanced desensitization rate (237). Two recessive forms of startle disease have also been described that
must have resulted in the complete nonexpression of the
GlyR ␣1-subunit. In one case, the mutation caused a homozygous deletion encompassing exons 1– 6 (53),
whereas the other involved a base pair deletion resulting
in a frameshift and a premature stop codon prior to the
TM1 domain (315). Despite the complete absence of ␣1subunit expression, the symptoms experienced by these
patients were no more severe than in those where the
␣1-subunit function was only mildly impaired. Another
recently identified ␣1-subunit recessive point mutation,
S231R, caused a reduced membrane insertion of functional receptors (166).
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
TABLE
1.
GlyR mutations underlying startle syndromes in various species
Mutation and Subunit
Inheritance Mode
Effect on GlyR Function
Reference Nos.
Human forms
␣1 P250T
Autosomal dominant
␣1
␣1
␣1
␣1
Autosomal
Autosomal
Autosomal
Autosomal
V260M
Q266H
S270T
R271L/Q
␣1 K276E
␣1 Y279C
dominant
dominant
dominant
dominant
␣1 I244N
Autosomal dominant
Autosomal dominant, variable
penetrance
Autosomal recessive
␣1 Deletion of exons 1-6
␣1 S231R
␣1 Stop codon at Y202
Autosomal recessive
Autosomal recessive
Autosomal recessive
␣1 G342S
Compound heterozygous?
␣1 R252H ⫹ ␣1 R392H
␤ G229D ⫹ ␤ exon 5 loss
Compound heterozygous
Compound heterozygous
␤ Line-1 intronic insertion
Autosomal recessive
(Spastic)
Autosomal recessive
(Spasmodic)
Autosomal recessive
(Oscillator)
Reduced single-channel conductance, reduced glycine
sensitivity, increased desensitization rate
As yet unknown
Reduced open probability, reduced glycine sensitivity
As yet unknown
Reduced glycine sensitivity, reduced single-channel
conductance
Reduced glycine sensitivity, reduced open probability
Reduced glycine sensitivity, reduced whole cell current
magnitude
Reduced glycine sensitivity, reduced whole cell current
magnitude, increased desensitization rate
Presumed nonfunctional
Reduced membrane insertion
Reduced surface expression, possible heterozygosity
with ␣1 V147M
No effect of individual mutation, possible
heterozygosity with other mutations
Reduced membrane insertion
Reduced glycine sensitivity, reduced surface expression
50, 334
88
263, 271
210
208, 306, 327, 346
101, 228, 237
205, 237, 345
237, 312
53
166
315
315
311, 384
314
Murine forms
␣1 A52S
␣1 Stop codon
Reduced surface expression
156, 187, 275
Reduced glycine sensitivity
335
Reduced surface expression
54
Bovine form
␣1 Stop codon
Autosomal recessive
(Myoclonus)
Reduced surface expression
Compound heterozygous forms of startle disease
have also been described. In one case, two distinct recessive ␣1-subunit mutations, R252H and R392H, caused
startle disease when present in different alleles (384).
However, patients possessing either single mutant allele
were healthy. Consistent with this observation, coexpression of the two mutant subunits resulted in a reduced
surface expression of functional GlyRs, whereas the individual mutations had no effect (311). The recessive
M147V and G342S ␣1-subunit mutations have also been
identified in patients exhibiting startle disease symptoms
(315). These mutations are presumed to result in compound heterozygous forms of startle disease, but if this is
the case their partner mutations remain to be identified.
Finally, compound heterozygous mutations have recently
been identified in the ␤-subunit. A ␤-subunit missense
mutation (G229D) and a splice site mutation (resulting in
the excision of exon 5) occurred simultaneously in a
compound heterozygote with a transient startle disease
phenotype (314). The G229D mutation alone induced a
modest (4-fold) decrease in the glycine sensitivity of recombinant ␣1␤-GlyRs (314), whereas the removal of exon
5 would presumably have precluded the expression of
functional ␤-subunits.
Physiol Rev • VOL
147, 294
To date, no startle mutations have been identified in
the human ␣2- or ␣3-subunits. It remains to be determined
for any species whether expression of these subunits is
upregulated to compensate for the loss of ␣1-subunit
expression or function. The effectiveness of clonazepam
in treating startle disease is presumably due to its potentiation of the GABAAR. Indeed, evidence from spastic
mice (26) (135) and myoclonic cattle (233) suggests that
spinal cord GABAergic neurotransmission may be upregulated during development in compensation for the
loss of glycinergic tone.
It would not be surprising if startle syndromes resulted from mutations that disrupt the function of other
proteins involved in the formation, maintenance, and
function of glycinergic synapses. Indeed, a hereditary mutation in the GlyR clustering protein gephyrin results in a
hyperekplexia phenotype in humans (313) and targeted
deletion of the glycine transporter subtype 2 gene produces a startle phenotype in mice (133).
B. Murine Startle Syndromes
Three naturally occurring murine startle syndromes
have been identified to date (Table 1). The symptoms of
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JOSEPH W. LYNCH
each are largely similar to those observed in human hyperekplexia (308). An exception is that mouse startle
symptoms commence at around the 20th postnatal day,
corresponding to the time when the replacement of ␣2homomers by ␣1␤-heteromers is complete. The most
thoroughly characterized murine syndrome is the spastic
mouse, which was first identified in the 1960s (255). This
autosomally recessive disorder is characterized by a reduction in the number of expressed GlyR ␣1-subunits,
although the function of the expressed receptors is normal (26, 28). The spastic phenotype was found to be
caused by the insertion of a 7.1 kb Line-1 repeating element into intron 5 of the ␤-subunit (187, 275). This element leads to aberrant splicing of the ␤-subunit transcripts resulting in an accumulation of prematurely terminated protein and a concomitant reduction in the
surface expression of ␤-subunits (187, 275). Because
␤-subunits are required to anchor ␣1-subunits to synaptic
scaffolding proteins, the disease phenotype is presumably
caused by a loss in the efficiency of GlyR recruitment to
postsynaptic densities. Introduction of low levels of a
wild-type ␤-subunit transgene effectively reverses this
disease phenotype (156).
As expected, the magnitudes of glycinergic IPSPs in
motor and sensory spinal neurons of spastic mice were
significantly reduced relative to wild-type controls (135,
386). However, the same two studies observed contradictory effects on the size of GABAergic IPSPs in the same
neurons. Graham et al. (135), who measured the magnitude of spontaneous mini-IPSPs in dorsal horn sensory
neurons, reported an increase in GABAergic IPSP magnitude. On the other hand, Von Wegerer et al. (386), who
recorded electrically stimulated IPSPs in ventral horn
motor neurons, reported a reduction in GABAergic IPSP
magnitude. The difference may relate to the identity of the
neurons studied or to the different IPSPs (evoked versus
spontaneous) measured.
The spasmodic mouse contains the homozygous
point mutation A52S in the ␣1-subunit. This mutation
results in a moderate (6-fold) reduction in glycine sensitivity (326, 335). Spinal inhibitory neurotransmission has
yet to be characterized in this mouse. An allelic variant of
spasmodic, termed oscillator, is the only lethal form of
startle disease identified to date. This syndrome is caused
by a frameshift mutation in the TM3-TM4 domain resulting in the translation of an incomplete form of the ␣1subunit protein (54). With the assumption that the majority of spinal glycinergic synapses comprise ␣1␤-heteromers by postnatal day 20, homozygous oscillator mice
should have virtually no glycinergic tone. Consistent with
this, membranes isolated from oscillator homozygote spinal cords display a 90% reduction in strychnine binding,
indicating a drastic loss in the functional expression of
the ␣1-subunit (54). Surprisingly, however, the magnitude
of strychnine-sensitive synaptic currents was found to be
Physiol Rev • VOL
reduced by only 50% in dorsal horn sensory neurons of
mice homozygous for this mutation (135). It would be of
interest to examine the pharmacology of the currents that
remain: is it possible that the strychnine-insensitive ␣2*subunit is upregulated to compensate for the loss of ␣1subunits?
It is noteworthy that lethality does not result from
human and bovine startle mutations that act similarly to
completely preclude the functional expression of ␣1-subunits (53, 294, 315). A possible reason for the difference is
that the ␣2 3 ␣1␤ subunit switch in the mouse occurs
after birth resulting in an inability to feed. In humans and
cattle, the switch is likely to occur before birth, thereby
not affecting the ability to feed, and affording time for the
development of synaptic adaptations before parturition.
Two transgenic mice strains have been generated
with a view to developing an animal model of human
hyperekplexia. The first approach mimicked the spastic
mouse mutation by engineering a transposon insertion
into the ␤-subunit (29). Homozygous mice displayed a
startle phenotype that resembled human hyperekplexia
(30). The second approach incorporated the dominant
human ␣1-subunit R271Q mutation into transgenic mice.
Transgenic mice that were heterozygous for the R271Q
human startle mutation displayed a pronounced startle
phenotype, and mice homozygous for the mutation were
not viable (30). Knock-in mice bearing the ␣1-subunit
S267Q mutation (which eliminates the alcohol binding
site and reduces peak current magnitude) displayed a
reduced sensitivity to alcohol and an oscillator-type phenotype (111, 112). Finally, as noted above, knock-out of
the glycine transporter subtype-2 gene produces a lethal
oscillator-type phenotype (133).
C. Bovine Myoclonus
A congential recessive startle syndrome, called myoclonus, has been identified in Poll Hereford cattle (160).
This disorder was recently shown to be due to a single
base pair deletion in the ␣1-subunit gene, leading to a
frameshift and a premature stop codon before TM1 (294).
As would be expected, this mutation induced a dramatic
reduction in the surface expression of functional GlyRs
(147). A similar syndrome may also exist in Peruvian Paso
horses (148).
VIII. OUTLOOK
GlyRs have important roles in a variety of physiological processes, especially in mediating inhibitory neurotransmission in the spinal cord and brain stem. Although
recent progress in understanding the molecular functional architecture of these receptors has been rapid,
many gaps remain in a number of critical areas. The
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THE GLYCINE RECEPTOR CHLORIDE CHANNEL
following are considered to be among the most pressing
research priorities at the present time.
1) A high-resolution structure of the entire GlyR complex will permit the design of much more precise experiments to understand receptor structure and function.
Unfortunately, however, crystal structures are notoriously difficult to obtain for membrane-spanning proteins,
and complete structures have yet to be resolved for any
LGIC member.
2) It is essential to establish the exposure pattern of
residues in TM2 and in the pore selectivity filter of the
GlyR ␣- and ␤-subunits. This is a prerequisite for understanding the ion-permeation and channel-opening mechanisms. It is also essential to probe the surface electrostatic potential in the pore selectivity filter to further our
understanding of the ionic charge discrimination mechanism.
3) Because binding sites are located at subunit interfaces, it is necessary to resolve the GlyR subunit stoichiometry and arrangement so that the number and type of
subunit interfaces can be defined. This is an important
first step toward resolving the structural and functional
basis of ligand binding.
4) There are large gaps in our knowledge of glycine
binding mechanisms. In particular, the role of the ␣-subunit complementary binding domains in coordinating glycine needs to be investigated. This is a prerequisite for the
structural modeling of the agonist binding pocket.
5) The role of ␤-subunit in channel in agonist binding
and channel activation has received scant attention.
Again, this information is necessary for the structural
modeling of ligand binding sites and the understanding of
activation mechanisms.
6) The modeling of GlyR kinetics remains controversial. Because kinetic models are crucial for understanding
and predicting receptor behavior, it is hoped that future
studies will carefully readdress this situation.
7) The diversity of GlyR subtypes at least partially
underlies the diversity in glycinergic neurotransmission
properties throughout the central nervous system (219).
Understanding the GlyR structural variations that underlie this synaptic functional diversity is an important question for future research.
8) The role of ␣2-homomeric GlyRs in embryonic
neurons remains to be clarified.
9) Extrasynaptic GlyRs are present on many central
nervous system neurons. In addition, GlyRs have been
found in a number of nonneuronal tissues. The physiological roles of these GlyRs need to be investigated in more
detail.
10) The therapeutic possibilities of GlyR modulatory
agents warrant further investigation. As noted in a recent
review (215), the fact that GlyRs are involved in motor
reflex circuits and nociceptive sensory pathways suggests
that GlyR modulators could have therapeutic potential as
Physiol Rev • VOL
1085
analgesics and muscle relaxants. This review describes
the effects of a number of molecules with potential to be
lead compounds for the development of such therapeutics. Further therapeutic possibilities may emerge as the
roles of extrasynaptic and nonneuronal GlyRs are characterized in more detail.
I gratefully acknowledge the patience of my laboratory
members during the writing of this review. Professor Peter
Barry and the anonymous reviewers are acknowledged for providing valuable insights and helpful suggestions. Dr. Brett Cromer and Prof. Michael Parker are thanked for kindly providing
the unpublished image in Figure 3B.
Research in the author’s laboratory is funded by the Australian Research Council and the National Health and Medical
Research Council of Australia.
Address for reprint requests and other correspondence: J.
W. Lynch, School of Biomedical Sciences, Univ. of Queensland,
Brisbane QLD 4072, Australia (E-mail: j.lynch@uq.edu.au).
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