Physiol Rev
82: 429 – 472, 2002; 10.1152/physrev.00001.2002.
TRP Channel Proteins and Signal Transduction
BARUCH MINKE AND BOAZ COOK
Department of Physiology and the Kühne Minerva Center for Studies of Visual Transduction,
The Hebrew University-Hadassah Medical School, Jerusalem, Israel
I. Introduction
A. Drosophila phototransduction is a model system for studying TRP
B. Inositol lipid signaling mediates signal transduction cascades
II. Historical Background
A. The trp phenotype
B. Mammalian TRP homologs
III. Molecular Analysis of the TRP Family
A. Molecular structure and localization of Drosophila TRP and TRPL
B. The trp gene is conserved throughout evolution
C. “TRP-homolog” group
D. “TRP-related” group
IV. Functional Analysis in the Native Tissue
A. Drosophila light-sensitive channels TRP and TRPL
B. Activation of TRP and TRPL channels
C. Role of TRPC3 in pontine neurons
D. Control of Ca2⫹ influx in vascular smooth muscle cells
E. TRPC2 regulates Ca2⫹ entry into mouse sperm triggered by egg ZP3
F. The TRP-related group
G. NOMPC is a channel that mediates mechanosensory transduction
H. Summary
V. Functional Analysis in Heterologous Systems
A. Heterologous expression of trpl, but not trp, leads to channels with similar properties
of the native channel
B. Mechanism of activation of TRPL
C. Heterologous expression of “TRP-homolog” channels
D. Heterologous expression of “TRP-related” channels
VI. TRP Localizes and Anchors the Transduction Signaling Complex of Drosophila
VII. Concluding Remarks
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Minke, Baruch, and Boaz Cook. TRP Channel Proteins and Signal Transduction. Physiol Rev 82: 429 – 472, 2002;
10.1152/physrev.00001.2002.—TRP channel proteins constitute a large and diverse family of proteins that are
expressed in many tissues and cell types. This family was designated TRP because of a spontaneously occurring
Drosophila mutant lacking TRP that responded to a continuous light with a transient receptor potential (hence TRP).
In addition to responses to light, TRPs mediate responses to nerve growth factor, pheromones, olfaction, mechanical, chemical, temperature, pH, osmolarity, vasorelaxation of blood vessels, and metabolic stress. Furthermore,
mutations in several members of TRP-related channel proteins are responsible for several diseases, such as several
tumors and neurodegenerative disorders. TRP-related channel proteins are found in a variety of organisms, tissues,
and cell types, including nonexcitable, smooth muscle, and neuronal cells. The large functional diversity of TRPs is
also reflected in their diverse permeability to ions, although, in general, they are classified as nonselective cationic
channels. The molecular domains that are conserved in all members of the TRP family constitute parts of the
transmembrane domains and in most members also the ankyrin-like repeats at the NH2 terminal of the protein and
a “TRP domain” at the COOH terminal, which is a highly conserved 25-amino acid stretch with still unknown
function. All of the above features suggest that members of the TRP family are “special assignment” channels, which
are recruited to diverse signaling pathways. The channels’ roles and characteristics such as gating mechanism,
regulation, and permeability are determined by evolution according to the specific functional requirements.
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0031-9333/02 $15.00 Copyright © 2002 the American Physiological Society
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BARUCH MINKE AND BOAZ COOK
I. INTRODUCTION
The role of plasma membrane channel proteins is to
regulate the flow of different ions between the cell and its
environment. Each channel has a typical permeability to
different ions, conductance, mechanism of opening and
closing (gating), typical agonists and antagonists, voltage
dependence, and additional regulatory characteristics.
The gating of channels may be direct by voltage or ligand
binding, or indirect via cascade of molecular events and
production of second messengers that lead to channel
opening. A large volume of data and detailed understanding has been accumulated on voltage- and ligand-gated
channels. However, channel proteins of the TRP family do
not strictly fit to any of the above categories. The activation and regulation mechanisms of TRP channels are
largely unknown and highly diverse. Channel activity is
affected by several physical parameters such as osmolarity, pH, mechanical force, as well as biochemical interactions with external ligands or cellular proteins.
The structure of TRP channels relates them to the
superfamily of voltage-gated channel proteins characterized by six transmembrane segments (S1-S6), a pore region (between S5 and S6), and a voltage sensor, although
the voltage sensor in S4 is missing in TRPs. Functionally,
they may be categorized as nonselective cation channels,
although they demonstrate large diversity in the characteristics of their permeability to ions and some of them
are highly selective for Ca2⫹. TRP channels have been
found in many organs, mainly in the brain but also in
heart, kidney, testis, lung, liver, spleen, ovaries, intestine,
prostate, placenta, uterus, and vascular tissues. They have
been found in many cell types, including both neuronal
cells, such as sensory, primary afferent neurons and nonneuronal tissues such as vascular endothelial cells, epithelial cells, and smooth muscle cells. Functionally, the
most prominent cellular signaling pathways in which
TRPs play a role are also mediated via PLC. (for reviews,
see Refs. 70, 107, 111, 118, 119, 152, 165).
A. Drosophila Phototransduction Is a Model
System for Studying TRP
The archetypal TRP protein was found and extensively studied with relation to Drosophila phototransduction (107, 111, 118, 165). The phototransduction cascade
provides a unique and powerful model system to study
functions of specific signaling proteins with relevance to
many transduction cascades. Studying phototransduction
in Drosophila offers many advantages. The small size of
the genome, ease of growth, and short generation make
Drosophila ideally suited for screening large numbers of
mutagenized individuals. Furthermore, the creation of
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powerful genetic tools such as balancer chromosomes
and P-element-mediated germline transformation combined with tissue-specific promoters allow the introduction of cloned genes back into the organism, providing a
way to study in vitro mutagenized genes in the living fly
(107, 175). In Drosophila it is thus easy to screen for
mutants that have physiological or morphological defects
(133). Studies of single cells using electrophysiology and
microfluorimetry allow characterizing different functional
aspects of responses in great detail. The high sensitivity to
light allows detection and characterization of responses
to activation of single receptor molecules. Moreover, responses to higher light intensities enable the study of fast
response kinetics and adaptation. Several important and
novel signaling proteins have been identified in Drosophila, and their role was established using the genetic dissection approach (107, 111, 118, 165). TRP is an illuminating example for a novel signaling protein of prime
importance whose function has been elucidated due to
the powerful genetic tools and functional tests that have
been developed in Drosophila. These studies have led to
identification and characterization of TRP as a light-sensitive and Ca2⫹-permeable channel (60, 126, 140), although the mechanism that leads to channel opening is
not yet clear. The studies of Drosophila phototransduction have thus indicated that TRP is permeable to Ca2⫹,
and it is the target of a phosphoinositide cascade, leading
to the suggestion that phototransduction in Drosophila
might be analogous to the general and widespread process of phosphoinositide-mediated Ca2⫹ influx (61).
B. Inositol Lipid Signaling Mediates Signal
Transduction Cascades
Inositol lipid signaling is one of the most widespread
signal transduction cascades, which exists in virtually
every eukaryotic cell. This transduction cascade that is
mediated via G protein-activated phospholipase C (PLC)
produces two second messengers: diacylglycerol (DAG)
and inositol 1,4,5-trisphosphate (InsP3) (14). This cascade
begins by activation of a surface membrane receptor,
followed by activation of InsP3 receptors in the endoplasmic reticulum (ER), leading to Ca2⫹ release, and ends by
the opening of surface membrane channels and Ca2⫹
influx, which activates a large variety of specific and
critical cellular functions (Fig. 1). A great deal is known
about the initial stages of this ubiquitous cascade; however, the molecular mechanism underlying activation and
gating of the channels is elusive (152).
Currently, the surface membrane nonexcitable Ca2⫹
influx channels are classified into two categories: storeoperated channels (SOC) (100, 152) and store-independent channels (70, 152). Both channels require PLC for
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FIG. 1. The phosphoinositide cascade of vision. Cloned genes (for all of which mutants are available) are shown in
italics, alongside their corresponding proteins. Upon absorption of light, rhodopsin (ninaE gene) is converted to the
active metarhodopsin state, which activates a heterotrimeric G protein (dGq). This leads to activation of phospholipase
C (PLC␤, norpA gene) and subsequent opening of two classes of light-sensitive channels encoded at least in part by trp
and trpl genes, by an as yet unknown mechanism. TRP and TRPL opening leads to the light-induced current (LIC).
Deactivation of channel activity is regulated by protein kinas C (PKC, inaC gene). PLC, PKC, and the TRP ion channel
form a supramolecular complex with the scaffolding protein INAD (inaD gene, not shown). PLC catalyzes the hydrolysis
of phosphatidylinositol 4,5-bisphosphate (PIP2) into the soluble second messenger inositol 1,4,5-trisphosphate (IP3) and
the membrane-bound diacylglycerol (DAG). DAG is recycled to PIP2 by the phosphatidylinositol (PI) cycle shown in an
extension of the smooth endoplasmic reticulum called submicrovillar cisternae (SMC, shown on the bottom). DAG is
converted to phosphatidic acid (PA) via DAG kinase (DGK, rdgA gene) and to CDP-DAG via CD synthetase (not shown).
After conversion to PI, PI is presumed to be transported back to the microvillar membrane by the PI transfer protein
(PITP encoded by the rdgB gene). Both RDGA and RDGB have been immunolocalized to the SMC. At the base of the
rhabdomere, a system of submicrovillar cisternae has been proposed by analogy to other insects, to represent Ca2⫹
stores endowed with IP3 receptors (IP3R), which is an internal Ca2⫹ channel that opens and releases Ca2⫹ upon binding
of IP3.
their activation. SOC channels are activated by the reduction in stored Ca2⫹. Store-independent channels include
channels that are activated by Ca2⫹ elevation in the cytosol due to Ca2⫹ release and channels that are activated
by the DAG branch of the inositol-lipid signaling pathway.
In both cases the mechanism of channel activation is
unknown (107). The Ca2⫹ release activated current
(ICRAC) represents activation of the classical SOC channels (134), and recently a TRP-related channel was suggested as a molecular candidate for ICRAC channels (215).
The paradigm that has been used to demonstrate SOC
channel activation rather than store-independent channels is based on a bypass of InsP3 production. Thus
depletion of the internal stores by a specific Ca2⫹-ATPase
inhibitor, thapsigargin, which is known to deplete InsP3sensitive stores (83) or by the Ca2⫹ ionophore ionomycin
in Ca2⫹-free medium in the presence of strong Ca2⫹ buff-
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ers in the cell, activates SOC channels. Accordingly, after
store depletion, application of Ca2⫹ to the external medium results in Ca2⫹ influx. The store-independent channels are activated by signaling mechanisms not directly
related to the Ca2⫹ stores and, therefore, are not sensitive
to thapsigargin or other Ca2⫹-mobilizing agents.
At present, the only candidates for the inositidemediated Ca2⫹ influx channels, which include both SOC
channels and store-independent channels, are proteins of
the TRP family. In this review we emphasize the importance of studying TRP functions and properties in native
cells and, therefore, first outline the research on TRP and
TRP homologs that have been performed on Drosophila
photoreceptors. Then we describe the fast-growing literature on a wide variety of TRP homologs that have
been recently found in other species. The in vivo properties and possible functions of TRP channels and their
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BARUCH MINKE AND BOAZ COOK
relevance to signal transduction processes are the focus
of this review.
II. HISTORICAL BACKGROUND
A. The trp Phenotype
A spontaneously occurring Drosophila mutant was
designated transient receptor potential (trp) by Minke et
al. (113) because of its unique phenotype. In this mutant
the response to prolonged illumination declines to baseline during light (38, 106, 113) (Fig. 2). Drosophila photoreceptors of wild-type and trp mutants have become a
preparation of extensive research because of the ability to
study TRP within the well-characterized phototransduction cascade in the native tissue (63, 107, 116, 157).
The trp phenotype was originally explained by the
hypothesis that some critical factor, which is required for
excitation and needs to be constantly replenished during
illumination, is in short supply in the mutant and cannot
be replenished fast enough due to a mutation in the trp
gene. Indeed, several lines of evidence indicate that the
decline of the light response in trp mutants is due to
exhaustion of excitation. Accordingly, the response to
continuous intense light becomes equivalent to a response to dim light and then to darkness during illumination (106, 113).
A correlation between the trp phenotype and exhaustion of cellular Ca2⫹ was provided by exposure of isolated
Drosophila ommatidia to Ca2⫹-free medium for a long
(1 h) period of time in cells buffered with EGTA to ⬃50
nM intracellular Ca2⫹. The typical trp phenotype was
obtained under such conditions (60). Similar apparent
cellular Ca2⫹ deprivation could be obtained in the isolated ommatidia of wild-type (WT) Drosophila during a
critical period of development. At this developmental
stage (P14), no response to light can be observed unless
Ca2⫹ (␮M) is applied by the whole cell recording pipette.
Interestingly, the light response under such conditions
has the typical characteristics of the trp phenotype (65).
Presumably under such conditions cellular Ca2⫹ is the
limiting factor of excitation as in La3⫹-treated or Ca2⫹deprived WT cells.
A recent study has proposed that the transient photoresponse of the trp mutants is due to Ca2⫹-dependent
inactivation of the TRP-like (TRPL) channels rather than
exhaustion of Ca2⫹-dependent excitation (164). According to this model, light-induced influx of Ca2⫹ through the
TRPL channels (which remain operative in the trp mutant
and have a considerable permeability to Ca2⫹) activates
calmodulin (CaM), which in turn binds to CaM binding
sites of TRPL (147) and deactivates the channels. In contrast, other experiments revealed that the trp phenotype
is exacerbated in Ca2⫹-free medium, thus indicating that
the negative feedback hypothesis probably does not underlie the trp phenotype (37). This conclusion was
strongly supported by Hardie et al. (67), who found that
trp decay and the associated inactivation are correlated
with a drastic Ca2⫹-dependent reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels in the microvilli,
while recovery from inactivation is abolished in mutants
of the PIP2 recycling pathway (rdgB and cds).
A turning point in understanding the trp phenotype
and the possible role of TRP in phototransduction was
brought about by the discovery that lanthanum (La3⫹)
mimics the trp phenotype in WT flies (72, 181).
FIG. 2. The trp phenotype. Light-induced currents in response to prolonged intense orange lights were recorded in
voltage-clamped photoreceptors of wild type (WT), the trp mutant, and WT treated with La3⫹. A peak response and a
plateau characterize the light response of WT. The rapid peak-plateau transition is a manifestation of Ca2⫹-dependent
light adaptation. The response of the trp photoreceptor decays close to baseline during light due to exhaustion of
excitation. A similar decay of the light response close to baseline is obtained by application of 10 ␮M La3⫹ to the
extracellular medium of WT photoreceptors. The horizontal bar indicates the light monitor. The intensity of the orange
light stimulus is 10-fold dimmer in WT than in the trp mutant or WT treated with La3⫹. [From Minke and Selinger (111).]
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1. La3⫹ mimics the trp phenotype in WT
3⫹
Application of La in micromolar concentrations to
the extracellular space of several species of WT flies
accurately mimics the trp phenotype (Fig. 2) (60, 72, 181).
The rate of occurrence of the single photon responses
decreases in La3⫹-treated WT flies with incrementing intensity of long light stimulation in a similar manner to the
effect of light found in the mutant. Furthermore, application of La3⫹ to the mutant has virtually no effect (181).
The effect of La3⫹ is reversible upon removal of La3⫹ from
the external solution. Because La3⫹ is a known nonspecific Ca2⫹ channel blocker, it was suggested that exhaustion of excitation in La3⫹-treated WT flies or in the trp
mutant, which underlies the trp phenotype, is Ca2⫹ dependent. Thus, with the assumption that cellular Ca2⫹ is
required for sustained excitation, it has been suggested
that a TRP-mediated mechanism is responsible for replenishing cellular Ca2⫹ fast enough during strong illumination and, therefore, TRP is a Ca2⫹ channel/transporter
(109, 110).
2. TRP and TRPL are light-activated channels
Whole cell voltage-clamp recordings applied to isolated Drosophila ommatidia (55, 156) revolutionized the
field of Drosophila phototransduction and made it possible to examine the hypothesis that TRP is a Ca2⫹ channel/
transporter (109). The experiments of Hardie and Minke
(60) revealed that the fundamental defect in the trp mutant is a change in the ionic selectivity of the light-sensitive conductance. The relative Ca2⫹ permeability of the
light-sensitive conductance in trp mutant was reduced by
a factor of ⬃10 (60, 151, 158), along with a significant
change in the relative permeability to different monovalent ions. The reduced Ca2⫹ permeability in the trp mutant has also been corroborated by the demonstration of
a reduced light-induced Ca2⫹ influx using Ca2⫹ indicator
dyes (59, 140) or Ca2⫹-selective microelectrodes (139).
The effect of the trp mutation on ionic selectivity could be
mimicked in WT photoreceptors by bath application of
La3⫹ in micromolar quantities (60, 181).
The effects of the trp mutation on the permeability
properties of the light-sensitive channels and the fact that
in null trp mutants a significant response to light is preserved led Hardie and Minke (60, 61) to propose that there
are at least two light-sensitive channels. Accordingly, one
channel has the permeation properties of the channels
remaining in the trp mutant, and a second class has high
Ca2⫹ permeability, which is absent in trp mutants and
blocked by La3⫹.
A significant step forward in the study of TRP became possible after the trp gene was cloned and sequenced by Montell and Rubin (121) and described as
encoding a novel membrane protein. Subsequent reanalysis of the sequence by Kelly and colleagues (147) rePhysiol Rev • VOL
vealed significant homologies of the transmembrane domain to voltage-gated Ca2⫹ channels, the dihydropyridine
receptors, and thus raised the hypothesis that TRP and its
homolog, TRPL, may be channel proteins (147). A second
putative channel gene, trp-like (trpl), was isolated using a
screen for calmodulin binding proteins and was found to
show ⬃40% overall identity with trp (147; for review, see
Refs. 111, 116). This homology suggested that trpl might
encode a second class of light-sensitive channel, responsible for the light-induced current recorded in the trp
mutant (60, 147). Subsequent isolation of a null mutant of
the trpl gene strongly supported the hypothesis that TRPL
functions as a second light-sensitive channel (126). Under
physiological conditions, the trpl mutation has a relatively
small but distinct influence on the light response (92).
Importantly, eliminating TRP-dependent current in trpl,
either by La3⫹ or by examining the double mutant trpl;trp,
results in complete abolishment of the response to light.
Therefore, TRP and TRPL channels make up all the lightactivated channels or are required for their activation (for
details, see Refs. 158, 164; for review, see Refs. 107, 111,
116). The isolation of the null trpl mutant allows investigating whether the trp phenotype is a property inherent in
the TRPL channels (164). An answer to this question was
provided by the creation of the double mutants in which
only a small amount of functional TRP channels remain
(126, 158). Interestingly, these mutant alleles show the
typical trp phenotype (92), indicating that the trp phenotype does not arise only from a specific property unique to
the TRPL channel, as previously suggested (164), but also
from low level of TRP in the absence of TRPL.
B. Mammalian TRP Homologs
The first report that TRP-related proteins might also
be found outside invertebrate photoreceptor came from
Petersen et al. (143), who identified partial sequences of
TRP homologs from Xenopus oocyte and murine brain
cDNA libraries. Shortly thereafter, the full sequence of a
human homolog (TRPC1) was reported following homology
searches of EST databases (199, 216). Meanwhile, a large
number of vertebrate TRP homologs have been cloned
and sequenced (see Table 1). The functional role in the
native tissues of vertebrate TRP homologs that have high
similarity to Drosophila TRP is largely unknown. They
show very widespread tissue distribution, but their properties have been mainly inferred from heterologous expression studies. The function of other members of the TRP
family with low identity to Drosophila TRP can be hypothesized from their specific tissue distribution and production of knockout mice, which suggests important functions in both neuronal and nonneuronal cells (Table 1).
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BARUCH MINKE AND BOAZ COOK
TABLE
1. Molecular properties of the “TRP-homolog” and “TRP-related” channels
Channel
Number of
Amino Acids
Identity
to TRP, %
Expression Pattern
Cloned From the
Following Species
Reference Nos.
TRP-homolog channels
TRP
1,275
TRPL
1,124
39
TRP␥
1,128
54
TRPC1
759 (␤-Isoform)
793 (␣-Isoform)
778 (Xenopus)
1,172 (Mouse)
40 (Human)
42 (Rat)
TRPC2
Photoreceptors of
Drosophila; antenna of
Drosophila during
development
Photoreceptors of
Drosophila
Photoreceptors of
Drosophila
Brain, heart, ovary, testes
(change during
development)
Bovine: testes, spleen,
liver; mouse: testes;
rat: vomeronasal organ
Brain mainly, also in low
amount in intestine,
lung, prostate,
placenta, testis
Brain, but also in lung,
ovary
Heart, lung, eye, but also
in lower amount in
brain, spleen, testes
Brain, but also in adrenal,
heart, lung, liver,
spleen, kidney, testis,
uterus, aorta
Brain, olfactory bulb
25–30 (Mouse)
TRPC3
848 (Human)
836 (Mouse)
34
TRPC6
37 (Mouse)
TRPC7
929 (Rat)
930 (Mouse)
862 (Mouse)
33 (Mouse)
TRPC4
974 (Mouse)
41 (Rabbit)
TRPC5
975 (Mouse)
974 (Rabbit)
36 (Mouse)
OSM-9
937
VR1 (TRPV1)
828 (Rat)
23 to OSM-9
VRL-1
761 (Rat)
78 to VR1
24 to OSM-9
GRC (TRPV2)
OTRPC4 (TRPV4)
756
871 (Mouse)
ECaC (ECaC1)
730 (Rabbit)
80
41
39
30
30
to
to
to
to
to
VRL-1
VR1
VRL-1
ECaC
VR1
CaT2 (TRPV5)
723
CaT1 (ECaC2)
727 (Rat)
CaT-L (TRPV5)
725
84
73
75
34
26
90
77
to
to
to
to
to
to
to
ECaC
CaT1
ECaC
VR1
OSM-9
CaT1
ECaC
Polycystin-2 (TRPP)
968 (Human)
PCL (TRPP)
Mucolipin-1 (TRPML)
805
580 (Human)
NOMPC (TRPN)
1,619
LTRPC7 (TRP.PLIK)
(TRPM7)
1,862
LTRPC2 (TRPM2)
1,503
Drosophila, Calliphora
32, 79, 80, 94, 121, 126,
148, 164, 176, 187, 188,
214
Drosophila
Drosophila
126, 147, 164, 187, 198,
208
207
Xenopus, mouse, rat,
bovine, human
30, 50, 98, 99, 173, 185,
200, 205, 206, 218, 221
Bovine, rat, mouse
84, 96, 114, 192, 218
Human, rat, mouse
19, 51, 95, 114, 193, 217,
218
Rat, mouse, rabbit
16, 19, 81, 114, 218
Mouse
131
Bovine, rat, mouse
122, 143, 144
Rabbit, mouse
132, 145
C. elegans
36
Human, mouse, rat
25, 27
Rat, human, mouse
26
Mouse
Mouse
86
177
Intestine, kidney,
placenta
Kidney
Rabbit
73
Rat
137
Intestine, brain, thymus,
adrenal gland
Rat
138
Placenta, exocrine
pancreas, salivary
gland, prostate cancer
Kidney
Human
202
Human
54
Kidney
Ubiquitous
Mouse
Human
11, 31
10, 12, 179
Drosophila bristles
Drosophila
196
Brain, hematopoietic
cells
Mouse
123, 159
Brain, bone marrow,
spleen, heart,
leukocytes, liver, lung,
kidney, prostate, testis,
skeletal muscle,
ubiquitous
Human
141, 160
TRP-related channels
23 to CaT1 within amino
acids 596–877
50 to polycystin-2
38 to TRP within amino
acids 331–521
⬃20% to TRPC channels
over ⬃400 amino acid
residues of TMD
⬃20 to TRP over ⬃325
amino acid residue
region including, TMD,
TRP domain
⬃20 to TRP over ⬃325
amino acid residues
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Olfactory,
mechanosensory and
osmosensory neurons
Dorsal root ganglia,
trigeminal nerve
Spinal cord, spleen, lung,
Brain, intestine, vas
deferens
Spleen, lung, brain
Kidney, heart, liver
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rescued the mutant phenotype (120), indicating that the
gene is responsible for the trp mutant phenotype. The
molecular structure of the TRP proteins as depicted in
Figure 3 shows multiple domains that are likely to play
important roles in cellular functions.
III. MOLECULAR ANALYSIS
OF THE TRP FAMILY
A. Molecular Structure and Localization
of Drosophila TRP and TRPL
The trp gene encodes a 1,275-amino acid membrane
protein (121). Expression of trp genomic DNA in a trp
mutant (trpCM) background by germ-line transformation
1. Ankyrin repeats may regulate TRP activity
At the NH2 terminal of TRP there are three or four
ankyrin repeats (147). Ankyrin repeats are 33-amino acid
FIG. 3. Putative domain structure and topology of Drosophila TRP, TRPL, and human
TRPC1. The protein sequences contain six putative transmembrane helices S1-S6 (TMD;
A) and a putative pore region between S5 and
S6 (shaded sphere between S5 and S6 in B and
C); S3-S6 shows sequence fragments identical
to the equivalent regions of the dihydropyridine receptor (a vertebrate voltage-gated Ca2⫹
channel). Both TRP and TRPC1 have four consensus ankyrin motifs (ankyrin) toward the
NH2 terminus. In TRP there is one putative
calmodulin-binding site (CaM) in the sequence. This site is missing in TRPC1. The
TRP protein has a curious and unique hydrophilic sequence near the COOH terminus consisting of nine repeats of the sequence DKDKKPG/AD (8 ⫻ 9). The other features of TRP
are PEST sequence, a proline-rich region with
the dipeptide KP repeating 27 time (KP), and
an INAD binding domain at the end of the
COOH terminus. The special structural features of TRPC1 are the coiled-coil (cc) domain
near the NH2 terminus and a dystrophin domain (dystrophin). [Modified from Minke and
Selinger (111).]
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BARUCH MINKE AND BOAZ COOK
residue motifs that mediate specific protein-protein interactions with a diverse repertoire of macromolecular targets (166). Ankyrin has been shown to inhibit InsP3 and
ryanodine receptor-mediated Ca2⫹ release from the internal stores (21, 22). Ankyrin also provides a mechanism for
linking membrane proteins to the cytoskeleton and plays
a role in subunit interactions of proteins with two or more
subunits (105). If TRP is a subunit of an oligomeric channel, the ankyrin repeat may play a role in subunit interactions or in TRP localization. Importantly, the ankyrin
repeat is highly conserved throughout evolution in most
members of the TRP family, strongly suggesting that it
plays an essential, although still unknown, role in TRP
function.
2. TRP displays transmembrane region typical
for voltage-gated channels
The most prominent structure of TRP is the transmembrane segments S1-S6 and the pore region loop between transmembrane segments S5 and S6, which is typical of voltage-gated channels. The identification of this
region was the first molecular confirmation of the hypothesis that TRP may function as a channel (147). Furthermore, parts of this sequence are the most conserved
region of TRP in all members of this family (see below).
3. The TRP domain
A highly conserved region of TRP is found COOH
terminal to S6 and includes 25 amino acids, six of which
(Glu-Trp-Lys-Phe-Ala Arg) are referred to as the TRP box
because of their invariant sequence. The function of this
unique region is still unknown (119).
4. TRP contains a CaM binding domain
TRP contains a putative CaM binding domain somewhere in the region of the COOH terminal (residues 628 –
977) (147). A more defined fragment of the TRP sequence
has been reported to bind CaM in a Ca2⫹-dependent manner (32). Sequence analysis suggests that the most likely
CaM binding site in this region would be residues 705–
725, which constitute the equivalent location to the CaM
binding site in TRPL, even though there is no amino acid
identity in this region between TRP and TRPL (see below). Drosophila photoreceptors contain an abundant
amount of CaM and have specific proteins (NINAC) that
stock-pile large amounts of CaM in the cell (149), yet the
function of CaM in Drosophila phototransduction seems
to be diverse and not entirely clear (6, 164).
5. TRP contains a PEST region but has a slow
turnover rate
The COOH-terminal region near the putative CaM
binding domain contains a PEST sequence, which is a
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signal for protein degradation by the calcium-dependent
protease calpain, typical of CaM binding proteins. Thus it
has been expected that TRP will show a fast turnover
rate. In contrast to this expectation, recent studies that
measured the turnover of TRP in flies between 1.5 and 9.5
days old revealed only 25% turnover of TRP during that
time, indicating a slow turnover of TRP (94).
6. TRP has special structural features
at the COOH terminus
At the COOH-terminal region there is also a prolinerich sequence in which the dipeptide KP is repeated 27
times (KP, in Fig. 3). Near the end of the COOH terminal,
TRP contains the following amino acid sequence repeated
in tandem nine times: D K D K K P G/A D. This 8 ⫻ 9
repeats region is likely to be important for protein-protein
interactions. This region is unique to TRP and has not
been found in any other member of the TRP family.
At the end of the COOH terminal there is a binding
domain for the PDZ scaffold protein INAD (169, 187, 208)
(see sect. VI and Fig. 3).
7. TRPL is a second light-activated channel with CaM
binding domains
The TRP homolog protein TRP-like (TRPL) was isolated using a screen to detect CaM binding proteins and
shared an overall 40% identity with TRP with much
greater similarity (70%) in the putative transmembrane
regions. Two regions that contain a typical CaM binding
site were found in the trpl-encoded protein (Fig. 3A). A
region homologous to the first putative CaM binding site
in TRPL is not present in TRP. One putative CaM binding
regions is capable of forming amphiphilic helices, which
is typical for CaM bindings sites (147, 169, 187). Localization of the two CaM binding sites (designated CBS1 and
CBS2) was determined by CaM overlays on fusion protein
fragments (198). CBS1 is localized within residues 710 –
728, while CBS2 lies within residues 865– 895, where there
is no predicted amphiphilic helix. CBS2 is also unconventional in binding CaM in the absence of Ca2⫹ (198). The
COOH termini of TRP and TRPL show very little homology (17%); nevertheless, both proteins show a large number of proline residues and thus contain a proline-rich
domain at the COOH terminus part.
8. Are the light-activated channels homomers
or heteromultimers?
Both TRP and TRPL have weak but significant homologies to a known channel subunit of the vertebrate
voltage-gated Ca2⫹ channel, the dihydropyridine receptor.
However, TRP and TRPL contain only one of the four
channel motifs with the six putative transmembrane domains S1-S6. By analogy with voltage-gated K⫹ channels
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and the cyclic nucleotide-gated channels, both TRP and
TRPL represent subunits of putative tetrameric channels.
Because null trp and trpl mutants both respond to light,
each can function without the other. However, heterologous coexpression studies and coimmunoprecipitation
experiments led to the suggestion that in WT flies the
light-dependent conductance was mediated by at least
three types of channels, two of which were TRP homomultimers and TRP-TRPL heteromultimer (209). Biophysical measurements of WT, trp, and trpl mutants questioned this conclusion, but these experiments did not
exclude the possibility that heteromultimeric TRP-TRPL
channels are important for specific, still uninvestigated
features of the response to light (158).
A recent study has revealed an additional channel
subunit called TRP␥ (207), which is highly enriched in the
photoreceptor cells. The NH2-terminal domain of TRP␥
dominantly suppressed the TRPL-dependent transient receptor potential on a trp mutant background, suggesting
that TRPL-TRP␥ heteromultimers contribute to the photoresponse. Furthermore, TRPL and TRP␥ coimmunoprecipitate, suggesting that a physical interaction between
these proteins forms a heteromeric channel (207). Further
studies will be required to unequivocally determine the
subunit composition of the light-sensitive channels.
9. TRP homologs of the blowfly Calliphora and squid
The trp gene of the closely related blowfly Calliphora (79) and a more distantly related squid, Loligo
(115), have been cloned and sequenced. The Calliphora
sequence shows 77% sequence identity with Drosophila
TRP, indicating that it is the ortholog of TRP rather than
TRPL. The greatest difference is in the COOH terminal in
which the KP proline-rich sequence in Calliphora is
somewhat shorter (79). The squid sequence is more distantly related, showing only 46% identity to TRP and
TRPL, and thus is equally similar to both proteins. As in
TRP and TRPL, squid TRP and Calliphora TRP have CaM
binding domains on the COOH terminus, multiple ankyrin
repeats are found in the NH2 termini, which are common
feature of all TRP homologs, including those in vertebrates (see below).
10. TRP and TRPL are localized to the rhabdomere
Immunogold labeling indicates that the putative lightsensitive channels TRP and TRPL are localized to the
microvillar membrane (187). One immunofluorescence
study (148) indicates that TRP is particularly localized to
the base of the microvilli in contrast to other studies (32,
187, 214). The reason for these differences in immunofluorescence localization of TRP is not clear. In addition,
TRP is found in photoreceptor axons up to the lamina and
medulla regions in the brain (148). Functional TRP is
found in the antenna of the developing but not in the
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mature olfactory system of Drosophila (176). The possible localization of TRP to the base of the microvilli in
juxtaposition to the putative InsP3-sensitive Ca2⫹ stores
bears important implications on the possible gating mechanism of TRP (see below). It should be noted that TRP is
an abundant protein with amounts equal to that of PLC
(i.e., ⬃107 copies per cell, Ref. 79). This is unusual for
channel proteins that are usually expressed in low
amounts. Because the maximal light-induced current
(LIC) in response to very intense light is ⬃20 nA (at ⫺60
mV holding potential) and single-channel conductance of
TRP is ⬃4 pS, it follows that ⬃7 ⫻ 104 channels are
sufficient to account for the maximal LIC. Accordingly, a
small fraction (i.e., ⬍1%) of TRP channels is sufficient to
mediate the maximal LIC measured during very intense
light. A possible reason for the unusually high levels of
TRP is the second function of this protein as an anchor
that localizes signaling complexes to the microvillar membrane (see sect. VI). A support for the notion that the large
amount of TRP is not required for its function as a channel stems from experiments in which TRP was dissociated genetically from the INAD scaffold protein in the
inaDP215 mutant. In inaDP215 mutants there is a large
reduction of TRP in the microvilli, with minor effects on
the response to light that manifests in somewhat slow
response termination (32). Similarly, transgenic Drosophila trp⌬1272, in which the binding site of TRP with INAD
was deleted, shows a gross mislocalization of TRP in
young flies. However, there was no effect on the response
to light (94, 169). These data implicate that a large portion
of TRP proteins are not necessary for production of the
response to light and thus may have additional long-term
roles (see below).
The localization of TRPL has not been extensively
studied. Nevertheless, TRPL was reported to localize to
the rhabdomere of Drosophila (126). Interestingly, TRP is
10-fold more abundant than TRPL (52, 209), and according to Hardie and colleagues (158), TRP and TRPL contribute about equally to the LIC under certain conditions.
B. The trp Gene Is Conserved
Throughout Evolution
By searching Caenorhabditis elegans genome database using the sequence that encodes the sixth putative
transmembrane segment (S6) and part of the adjacent
COOH-terminal cytoplasmic domain of Drosophila TRP,
Harteneck et al. (70) identified 13 C. elegans TRP members. These were divided into three groups: short, long,
and osm. The “short” group has a considerable similarity
to Drosophila TRP and TRPL and therefore will be referred to in this review as the “TRP-homolog” group or
TRPC. This group was recently designated classical TRPs
by Montell to indicate the very significant similarity of the
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mammalian members to the original Drosophila member
of this group (119). Full-length murine homologs, which
constitute most of the known mammalian members of the
TRP-homolog group have been cloned and characterized
by Birnbaumer and colleagues (218) and Schultz and colleagues (70). Recently, Montell (119) has proposed to
classify the nonclassical TRP channels into five subfamilies, which include the members of the “long” and “osm”
groups. These five subfamilies are distantly related to the
Drosophila TRP and TRPL and are referred to as the
“TRP-related” group in this review.
Based on the released Drosophila genomic sequence,
which accounts for ⬃95% of the euchromatic sequences,
a total of 14 members of the TRP family have been reported (2). However, it appears that only TRP, TRPL, and
TRP␥ belong to the TRP-homolog group while the rest are
TRP-related genes (207).
C. “TRP-Homolog” Group
On the basis of similarity to Drosophila TRP and
TRPL sequences, new TRP isoforms have been cloned
using database searches of expressed sequence tags
(EST), RT-PCR, or expression-cloning strategies. Seven
major groups termed TRPC1–7 have been cloned and
sequenced (Table 1; for review, see Ref. 152). The TRPC
homologs have been cloned from human, mouse, rat,
rabbit, bovine, and Xenopus. Five characteristic features
of the Drosophila TRP and TRPL proteins have been
found to be common to most members of the TRP-homolog group. 1) The predicted topology of six (but see
Ref. 203) transmembrane segments (S1-S6) includes the
typical pore region loop between transmembrane regions
S5 and S6. 2) The charged residues in the putative S4
helix, which usually underlies voltage gating, are replaced
by noncharged residues. 3) Three or four ankyrin repeats
are found at the NH2 terminal. 4) A proline-rich sequence
is found in the COOH-terminal domain (70). 5) The TRP
domain exists in all members of this group (119).
Birnbaumer and colleagues and Schultz and colleagues (16, 70, 218) have classified the vertebrate members of the TRP-homologs group into four subgroups
according to their primary amino acid sequence (see Fig.
4). Type 1 includes all isoforms of TRPC1. Type 2 includes
the TRPC2 homologs, which have the lowest similarity to
the other groups. Type 3 includes TRPC3, TRPC6, and
TRPC7 channel proteins. Type 4 includes TRPC4 and
TRPC5 channels and has a higher similarity to the TRPC1
group relative to the other groups.
1. TRPC1
TRPC1 was the first mammalian homolog of TRP that
was cloned independently by two laboratories using EST
database of human fetal brain cDNA library. It was initially called TRPC1 (199) or hTRP-1 (216). This TRP isoform is the shortest of all members of the TRP-homolog
group (but see TRPC2 pseudogene, below). It has 38 – 40%
identity and 58 – 62% sequence similarity to Drosophila
TRP, TRPL, and the C. elegans isoform of TRP, which
was identified during sequencing of chromosome III of
C. elegans. This homology does not allow determination if
TRPC1 is more similar to Drosophila TRP or to TRPL. In
addition to three putative ankyrin repeats, the NH2 terminal was also predicted to have a coiled coil structure at
amino acids 256 –300. Ninety amino acids at the COOH
terminal of the human TRPC1 showed significant homology to dystrophin (199) (Fig. 3).
A spliced variant of TRPC1 (called TRPC1A) was
cloned independently (221). This variant and TRPC1 were
expressed in Chinese hamster ovary (CHO; Ref. 221) cells,
in Sf9 (173, 221) cells, where they were studied functionally (see below), and in Xenopus oocytes (185). TRPC1
FIG. 4. Pairwise similarity phylogenetic tree of the
TRP family. Two distinct subgroups can be identified by
their phylogenetic relationship. The top group (except for
Drosophila TRP and TRPL) shows murine members of the
“TRP homolog” (TRPC) group. The bottom group includes
several members of the “TRP-related” group. The scale
represents evolutionary distance calculated by Clustal
analysis. [Modified from Strotmann et al. (177).]
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channels have also been found in other mammalian species: mouse, bovine, and rat (for review, see Ref. 152).
Recently, a Xenopus isoform of TRPC1 was cloned (called
Xtrp). The amino acids sequence of Xtrp was 82% identical to the mouse TRPC1, 81% identical to bovine TRPC1,
and 84% identical to rat TRPC1 (18). The cloning of a
Xenopus isoform of TRPC1 and its localization to the
oocyte surface membrane is important, since the native
inositol lipid signaling of Xenopus oocyte has been an
important model system to investigate SOCs (142).
The expression pattern of TRPC1 made by several
groups (30, 50, 173, 199, 216) shows that TRPC1 is most
abundantly expressed in the brain, heart, testes, ovary,
bovine aortic endothelial cells, and salivary glands and is
barely detectable in the liver or adrenal gland. Interestingly, the rat ortholog (941 amino acid long with 52%
identity to TRP) changes its expression pattern during
brain development (50), suggesting that it has a role in
developmental signaling. Injection of antisense oligodeoxyribonucleotide for mammalian TRPC1 inhibited the
native SOC response of the Xenopus oocytes (185); however, the expression pattern of TRPC1 and its physiological properties suggest that it is not ICRAC that was described in blood cells. Since a variety of SOC channels
have been described and some of them are TRPs (46, 142),
it is possible that specific TRP homologs mediate ICRAC or
other SOCs with still unidentified molecular identity. Support for this possibility was found in the report of Birnbaumer and colleagues (218) on carbachol-stimulated
store-operated Ca2⫹ influx seen in murine Ltk⫺ cells expressing the muscarinic M5 receptor. These cells show
loss of Ca2⫹ influx upon coexpression of a mixture of
murine TRPC1– 6 fragments in the antisense orientation.
Several additional studies have reported that a reduction
of TRPC1 expression using antisense approach led to
inhibition of endogenous SOCs (98). Similar results were
obtained in studies of TRPC3 (205) and TRPC4 (146) (for
review, see Ref. 46).
2. TRPC2
The human TRPC2 (also designated Trp2), which
was the first cloned TRPC2 gene (199), is probably a
pseudogene since several independent ESTs show mutations introducing early stop codons. A bovine homolog of
this pseudogene, which is mainly expressed in the testes,
spleen, and liver, was cloned and sequenced (203). Later,
a full-length 1,172-amino acid TRPC2 isoform from mouse
(192) and a spliced TRPC2 isoform from bovine (GenBank) were cloned. Thus TRPC2 is the longest of all
vertebrate TRP-homolog group, having a longer cytoplasmic NH2-terminal domain. In contrast to the TRPC1,
TRPC2 is tissue specific and in the mouse it was found
only in the testes and vomeronasal organ (VNO). In contrast to other TRPC proteins of this group, each having
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several consensus glycosylation sites within the putative
transmembrane domain of the protein, TRPC2 has only
one glycosylation site located in the putative pore region.
All other sites are localized to the cytosolic NH2 terminal.
TRPC2 shows only 25–30% identity to Drosophila TRP,
and it is closer to the TRP-related group than any other
TRP member of the TRP-homolog group (see Table 1 and
Fig. 4). This is also reflected in the existence of a segment
in the NH2-terminal domain (amino acid 155 to amino acid
238) of the type 2 polycystic kidney disease gene (PKD2).
PKD2 gene product is categorized as TRP-related channel
(see below). This sequence is unique to TRPC2 and is
absent in the other members of the TRP-homolog group.
The function of the PKD sequence of TRPC2 is unknown
(192).
A rat ortholog of the mouse and bovine TRPC2 genes
was cloned by Liman et al. (96). Importantly, the rat
ortholog is highly localized and heavily expressed in
the VNO. This organ plays a key role in the detection of
pheromones, which are chemicals released by other rats,
and elicits stereotyped sexual behavior (96). Turning
TRPC2 gene into a pseudogene in humans fits well with
the hypothesis that the VNO is no longer functional
in apes.
Recent studies revealed that InsP3 is a second messenger of VNO receptor neurons of snake (184). This fact
fits with TRPC2 as a channel, which is activated by the
inositol-lipid signaling cascade.
A TRPC2 isoform is highly enriched in the sperm and
seems to have an important function in the fertilization of
mice (see below and Ref. 84).
3. TRPC3, TRPC6, and TRPC7
This group, especially the TRPC3 homologs, has been
thoroughly studied by a variety of functional tests and,
therefore, significantly contributes to our understanding
of TRP function and gating mechanism (see below). The
molecular structure of TRPC3 shows the characteristic
five features of the TRP family listed above. Human,
mouse, and rat orthologs of TRPC3 and TRPC6 have been
cloned as well as a mouse ortholog of TRPC7.
TRPC3 homologs are predominantly expressed in the
brain and at much lower levels also in ovary, colon, small
intestine, lung, prostate, placenta, and testes (51, 114,
218). TRPC6 expression is highest in the brain, but it is
also expressed in the lungs and ovaries. Interestingly, the
development of tumors is associated with downregulation
of TRPC6 isoform in a mouse model for an autocrine
tumor (24).
TRPC7 from mouse turned out to be very similar
(81% identity and 89% similarity) to TRPC3 and also to
TRPC6 (75% identity and 84% similarity) while only 33%
identical (53% similar) to Drosophila TRP and TRPL.
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TRPC7 is mainly expressed in the heart, lung, and eye and
at a lower level in the brain, spleen, and testes (131).
4. TRPC4 and TRPC5
TRPC4 and TRPC5 channels are similar in structure
to TRPC1. Therefore, several investigators consider these
three channels types as one group (15, 152), which also
share similarity in function (see below). Petersen et al.
(143) first cloned a partial sequence of the mouse TRPC4
isoform. Then, full-length mouse bovine and rat orthologs
were described (122, 144). Another TRP homolog with
high similarity to TRPC4 was cloned and designated
TRPC5 (132, 145). The rabbit and mouse orthologs of
TRPC5 show 69% sequence identity to bovine TRPC4 and
41% identity to Drosophila TRP. The expression patterns
of TRPC4 and TRPC5 are very different. TRPC5 mRNA is
expressed predominantly in the brain (145), while TRPC4
is expressed in the brain but also in the adrenal gland and
at a much lower level in the heart, lung, liver, spleen,
kidney, testes, thymus, aorta, and uterus (132). Of special
interest are TRPC4 isoforms, which are expressed in vascular endothelial cells of various species (mouse, human,
and bovine) (49; see sect. IVD).
D. “TRP-Related” Group
In contrast to members of the TRP-homolog group
that were discovered due to sequence homology to Drosophila TRP, the various members of the “TRP-related”
group were found following studies aiming to explore
specific sensory transduction pathways or specific genetic diseases. These pathways include olfaction and osmolarity in C. elegans (36), defects in mechanosensory
transduction in C. elegans and Drosophila (196), defects
in pain mechanism in primary afferents of dorsal root
ganglia (27), Ca2⫹ transport in Ca2⫹ transporting epithelial cells (73), polycystic kidney disease in cells expressing the polycystin genes PKD (31), tumor suppression in
the skin (reviewed in Ref. 119), and mucolipidosis type IV
in cells expressing defective mucolipin (10, 179). The
different approaches resulted in a wealth of functional
data about TRP-related members, in contrast to most
mammalian members of the TRP-homolog group, whose
functions in most of the native tissues are largely unknown (but see below). In contrast to mammals, none of
the five C. elegans members of the TRP-related group or
other four C. elegans members of the TRP-homolog subfamily has been characterized at the cellular level, neither
in the native cells (but see CUP5, below) nor in heterologous systems.
The TRP-related group (also referred to as nonclassical TRP by Montell) has been subdivided into five subfamilies by Montell (119). All members of this group share
significant homology to Drosophila TRP in the transmemPhysiol Rev • VOL
brane segments. However, the degree of structural similarity to TRP varies considerably among the various subfamilies. The TRPV subfamily is the closest to Drosophila
TRP (see below). Some of these subfamilies lack the
ankyrin repeats or the TRP domain. Nevertheless, they
share some similar structural features and some of them
also share similar unusual functional features such as
permeability to Mg2⫹ and sensitivity to metabolic stress
(see below).
1. TRPV subfamily
This subfamily has all the structural features of the
TRP-homolog group and is more similar to this group than
any other subfamily of the TRP-related group (see Fig. 4).
The sequence identities to Drosophila TRP are primarily
limited to homology within ⬃50 amino acid residues at
the COOH-terminal ends of S1-S6, the adjacent COOHterminal region, and conservation of the TRP domain
(119). A gene that functions in a subtype of olfactory
neurons of C. elegans and designated osm-9 (36) was the
first member of this subfamily.
A) THE C. ELEGANS OSM-9. Six recessive alleles of osm-9
have been identified on the basis of defective response to
odorants, high osmotic strength, or light touch to the nose
(36). A fusion portion between osm-9 and green fluorescent protein (GFP) revealed that OSM-9 is expressed in a
subset of chemosensory, mechanosensory, and osmosensory neurons. There is no overlap in expression pattern
between osm-9 and the C. elegans TRP (Z C21.2), which
belongs to the TRP-homolog group (199) and is expressed
in motor neurons, interneurons, vulval muscles, and intestinal smooth muscles (36). Similarly, it appears that
there is no overlap in expression pattern between the C.
elegans NOMPC and the above TRPs of C. elegans (196).
Osm-9 encodes a predicted protein of 937 amino
acid. As in the other TRP-related members, the similarity
of OSM-9 and the TRP-homolog group (including Drosophila TRP and TRPL) is very significant in the vicinity
of S6 and the putative pore region. The osm-9 homolog
also contains ankyrin motifs in their NH2 terminus. These
ankyrin motifs do not show higher similarity to those of
TRP than to the ankyrin consensus motif. One of the
characteristic features of the TRP-homolog group, the
proline-rich motif at the COOH terminus, is missing in
osm-9 and other members of the TRP-related group.
B) THE MAMMALIAN VANILLOID RECEPTOR 1, VR1, AND ITS HOMOLOG
The VR1 member of the TRP-related group was
cloned following a search for pain (noxious)-initiated receptor in the peripheral terminals of subgroup of sensory
neurons of the dorsal root ganglia. Julius and colleagues
(27) used a functional screening strategy based on capsaicin-induced Ca2⫹ influx to transfected HEK 293 cells.
Capsaicin belongs to chemical group called vanilloids that
are known to induce noxious stimuli. The receptor was
VRL-1.
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discovered using the above screening library and was
therefore named vanilloid receptor 1 (VR1; also designated TRPV1). The cloned VR1 cDNA encodes a protein
of 828 amino acid with the six putative transmembrane
segments S1-S6. It also has a typical short hydrophobic
region between S5 and S6 assumed to form the pore
region. Three putative ankyrin repeats are found in the
NH2 terminus (27) and a TRP domain near the COOH
terminus (119). VR1 is expressed in a subset of sensory
ganglion cells of the dorsal root ganglia (DRG) and trigeminal ganglia. When tested in rat DRG, ⬃40% of all
neurons express VR1, predominantly in a population with
small cell bodies. VR1 is only 23% identical to C. elegans
osm-9 (Table 1).
A structurally related gene to VR1 designated VRL-1
(vanilloid receptor-like protein) was cloned after a search
of the GenBank database. Human and mouse orthologs of
VRL-1 have been found in EST databases with 78% identity and 86% similarity to one another and 49% identity and
66% similarity to rat VR1 (26). VRL1 (also designated
TRPV2) is only 24% identical to the C. elegans osm-9.
Interestingly, heterologously expressed VRL-1 is not activated by vanilloids or low pH (like VR1) but only by heat.
VRL-1 is expressed in only 16% of all DRG cells of rat, in
cells of medium or large diameter, while a similar pattern
of expression was found in trigeminal ganglia. VRL-1 is
also expressed in tissues other than DRG and spinal
cord including lung, spleen, intestine, and brain where it
is probably activated by stimuli other than noxious
heat (26).
C) VARIATIONS OF VR1 HAVE BEEN REPORTED THAT DIFFER DUE TO
Other members of the TRPV subfamily have been reported to have highly similar structure
but distinct mode of activation. An interesting mouse
member of this subfamily, which has ⬃80% sequence
identity to rat VRL-1, was designated “growth factor-regulated channel” (GRC). GRC translocates between the ER
and the plasma membrane upon stimulation of cells by
insulin-like growth factor I (IGF-I). The translocation of
endogenous GRC was demonstrated in MIN6 insulinoma
cells and in heterologous system in CHO cells upon coexpression with GFP (86). Electrophysiological and fluorimetrical measurements showed that IGF-I augmented
calcium entry through translocation of GRC to the plasma
membrane in CHO cells. However, since the CHO cells
were marked by GFP, which is toxic to cells, it was not
excluded that the observed currents and Ca2⫹ influx did
not arise from activation of endogenous channels, which
are sensitive to stress.
D) EPITHELIAL CALCIUM CHANNELS. Epithelial calcium channel (ECaC; also designated ECaC1, CaT2, or TRPV5) (73)
and calcium transporter 1 (CaT1, also ECaC2 or TRPV6)
(119, 138) were cloned from rabbit kidney and rat duodenum, respectively. These are members of the TRPV subfamily found in 1,25-dihydroxyvitamin D3-responsive epiALTERNATIVE MRNA SPLICING.
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thelial kidney cells (ECaC1) (73) and in Ca2⫹-transporting
cells of the small intestine (CaT1) (138). ECaC1 is a
putative apical Ca2⫹ channel in epithelia. In kidney it is
abundantly present in the apical membrane of Ca2⫹-transporting cells and it colocalized with 1,25-dihydroxyvitamin D3-dependent calbindin-D28K. The ECaC1 cDNA encodes a 730-amino acid protein and shows ⬃30%
homology to the VR1 channel. It has all the molecular
features of the TRP-related group including the S1-S6
transmembrane segments with the putative pore region
between S5 and S6, three ankyrin binding repeats, and
two N-linked glycosylation sites in S1-S6 region and a TRP
domain. The protein is predominantly expressed in the
kidney, small intestine, and placenta (Table 1).
The cDNA of the calcium transport protein, CaT1,
encodes a protein of 727 amino acid residues which has
all the structural features of ECaC1 except that four
ankyrin repeats have been identified in the NH2 terminus
and the N-glycosylation sites are missing. Nevertheless,
CaT1 shows 75% amino acid sequence identity to ECaC of
rabbit (138). Functional studies on CaT1 have suggested
that CaT1 is a promising candidate of the ICRAC channel
(see below and Ref. 215). CaT1 shows 34 and 27% identities to VR1 and osm-9, respectively. CaT1 is mainly distributed in the small intestine of rat, but it was also found
in small amounts in brain, thymus, and adrenal gland.
However, it was not found in the kidney, in striking
difference to ECaC1 (138). This difference in expression
pattern is surprising in light of the high homology between the two genes that normally indicates a common
functional role. By screening a rat kidney cortex library
with CaT1 probe, a cDNA encoding additional channel
designated CaT2 was isolated (137). CaT2 has 84 and 73%
amino acid identities to ECaC1 and CaT1, respectively
(137). Unlike ECaC1, CaT2 is kidney specific in rat. A new
member of this subfamily designated CaT-like (CaT-L)
has been recently isolated from human. CaT-L is abundantly expressed in the placenta, pancreatic acinar cells,
and salivary glands. Interestingly, the CaT-L transcripts
are undetectable in benign prostate tissue but are present
at high level in locally advanced prostate cancer, metastatic lesions, and recurrent androgen-insensitive prostate
adenocarcinoma, suggesting a possible link between Ca2⫹
signaling and prostate cancer progression (202).
E) THE PUTATIVE OSMORECEPTOR OTRPC4. OTRPC4 (now designated TRPV3) has structural similarity to VR1, osm-9,
and ECaC, and it is expressed in kidney, heart, and liver
(177). The protein is composed of 871 amino acid residues. The hydropathy profile predicts the typical six
transmembrane segments S1-S6, pore-forming loop between S5 and S6, and three ankyrin repeats at the NH2
terminal. Like other members of this group, OTRPC4
lacks the proline-rich motif at the COOH terminal. Sequence alignment with VR1, VRL-1, and ECaC channels
reveal 41, 39, and 23% overall similarity, respectively. In
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situ hybridization analysis revealed high levels of
OTRPC4 transcripts in the inner cortex of the kidney,
while higher resolution microscopy indicated a high density in the distal convoluted tubule. This specific localization and functional tests in heterologous system strongly
suggest that OTRPC4 is involved in cellular osmoreception (177).
NOMPC was identified following a screen for Drosophila
mutants with behavioral defects in coordinated movement. The mechanosensory organs of mutants showing
the behavioral defects were examined electrophysiologically, leading to the discovery of the nompC gene. This
mutant displays a large loss of mechanoreceptor current
relative to control flies. Cloning of the nompC gene was
carried out after mapping and rescue of the nompC mutant. NOMPC cDNA encodes a putative channel protein of
unusual structure composed of 1,619 amino acid residues.
The 1,150 NH2-terminal amino acid residues consist of 29
ankyrin repeats. The remaining 469 amino acid residues
share a significant similarity to the TRP family with S1-S6
transmembrane segments and putative pore region between S5 and S6. Pairwise comparison between NOMPC
and the various members of the TRPC family in the channel domain reveals ⬃20% identity and ⬃40% similarity.
Thus NOMPC is a distant member of the TRP-homolog
subfamily. NOMPC is expressed in the mechanosensory
organs of Drosophila in agreement with its function as a
mechanosensory sensitive channel (196). A C. elegans
homolog to NOMPC was identified and revealed high
expression in mechanosensitive neurons of the nematode
(196). Because no human ortholog of NOMPC has been
reported, it would be interesting to see in the future
whether the ability to respond to mechanical stimuli is a
general feature of TRPs or this activity is specific for
invertebrate mechanotransduction.
A) POLYCYSTIN. Polycystin-1 (encoded by PKD1) and
polycystin-2 (encoded by PKD2) are membrane proteins
that are defective in human autosomal dominant polycystic kidney disease (204). Members of this subfamily are
conserved through evolution from C. elegans to human.
Studies in C. elegans reveal that homologs of the polycystins act together in signal transduction pathway in
sensory neurons (11). Structural analysis of the amino
acid sequence predicts that the human polycystin-2 protein, which is composed of 968 amino acids, has putative
six transmembrane domains S1-S6 with putative poreforming loop between S5 and S6 segments, typical of the
TRP-related group. However, the ankyrin repeats typical
for the TRP family proteins are missing in the polycystin
proteins (31). Although PKD-1 and PKD-2 gene products
have structural similarities, only polycystin-2 seems to be
a cationic channel while polycystin-1 seems to be a larger
structural protein (of 4,572 amino acids in human) that
interacts directly with polycystin-2 (11; see also Ref. 204).
An interaction between polycystin-1 and polycistin-2 was
revealed by coexpression of the two channel proteins in
CHO cells. This interaction appears to be crucial for
polycystin-2 function as a channel because only in the
presence of polycystin-1, polycystin-2 translocates to the
plasma membrane and function as a channel (54). Thus
neither polycystin-1 nor polycystin-2 alone is capable of
producing currents. Moreover, disease-associated mutant
forms of either polycystin protein that are incapable of
heterodimerization do not result in new channel activity
(54). In C. elegans, there is a homolog of PKD-1 called
LOV-1 that is composed of 3,125 amino acids and a PKD-2
homolog with an overall identity of 27% to the human
PKD-2 (11). Vertebrate polycystins have the Ca2⫹ binding
motif, EF hand, and a coiled coil domain near the COOH
terminal. Heterologous expression of a human polycystinlike (PCL) protein, which shares 50% identity and 71%
homology to PKD-2 in Xenopus oocytes, confirms that
PCL forms a nonselective cation channel that can be
activated by Ca2⫹ (31) (see sect. VD2). PKD2 has modest
similarity to the Ca2⫹ transporting channel, CaT1, in the
region between amino acid residues 596 – 687 of CaT1,
where 23% identity between these proteins has been
found (138).
3. TRPP subfamily
4. TRPM subfamily
This subfamily is also distantly related to Drosophila
TRP, because it neither has the ankyrin repeats domain
nor the TRP domain (in the mammalian members) (119).
The founding member of this subfamily was discovered in
many cases of polycystic kidney disease (PKD, Ref. 205).
TRPP channels share ⬃25% amino acid identity with two
members of the TRP-homolog group TRPC3 and TRPC6
in the S5-S6 transmembrane segments and in the pore
region (119).
This subfamily is also distantly related to the TRPhomolog group. The TRPM subfamily is conserved
through evolution and exists in C. elegans (CED-11,
GON-2) and Drosophila. Members of this subfamily are
sharing ⬃20% amino acid identity to Drosophila TRP,
over ⬃325 residue region that includes S2-S6 transemembrane segments and the TRP domain. This subfamily also
lacks the ankyrin repeats (119). Originally this subfamily
was designated long TRP (LTRPC) (70) because of the
2. TRPN subfamily
This subfamily is distantly related to Drosophila
TRP, it does not contain the TRP domain, it is characterized by a large number of ankyrin repeats, and so far it has
been found only in invertebrate species.
A) THE NO MECHANORECEPTOR POTENTIAL C CHANNEL, NOMPC.
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unusually long NH2-terminal region of ⬃750 amino acid
residues relative to the TRP-homolog group. The total
length of this subfamily is 1,000 –2,000 amino acids, and it
varies considerably because of large diversity of the
COOH-terminal domain. Members of the TRPM subfamily
have a potential role in cell cycle regulation and in regulation of Ca2⫹ influx in immunocytes (160). Thus C. elegans members of TRPM function in programmed cell
death (CED-11) and mitotic cell division (GON-2; reviewed in Ref. 119). Importantly, the melastatin member
of this subfamily (TRPM1) has been related to skin cancer
as a putative tumor suppressor protein (for review, see
Ref. 119). An interesting mode of regulating TRPM gating
was recently described for another member of the TRPM
subfamily, MLSN (210). It was found that an alternatively
spliced short form of MLSN (MLSN-S) interacts directly
with and suppressed Ca2⫹ entry of full-length MLSN
(MLSN-L) in HEK293 cells. This suppression appears to
result from inhibition of translocation of MLSN-L to the
plasma membrane (210).
A very interesting member of this subfamily is the
bifunctional channel protein designated phospholipase C
(PLC)-interacting kinase (TRP-PLIK or TRPM7), which
constitutes the first example of a channel with putative
kinase activity. This protein has an NH2-terminal domain
⬎50% identical over 1,250 amino acids residues to that of
TRPM1 fused to COOH terminal with protein kinase domain (123, 159). Another member of this subfamily now
designated TRPM2 (or LTRPC2) also seems to be bifunctional, and in addition to the channel-forming domain,
which is similar to the channel domains of other members
of this subfamily, there is a NUDT9 homology domain
(NUDT9-H) at the COOH terminal. Sequence analysis of
NUDT9 revealed the presence of a signal peptide and
Nudix box sequence motif. Nudix boxes are found in a
family of diverse enzymes that catalyze the hydrolysis of
nucleoside diphosphates derivates (141). This motif is
also highly conserved in the C. elegans protein EEED8.8.
On the basis of these findings combined with heterologous expression of TRPM2 in HEK-293 cells, Scharenberg
and colleagues (141) concluded that TRPM2 functions
both as a cation channel activated by ADP ribose (ADPR)
and a specific ADP-ribose pyrophosphatase. TRPM2 is
mainly expressed in the brain but also in lower amounts
in bone marrow, spleen, heart, leukocytes, liver, and lungs
(141). A potentially important function of LTRPC2 in the
native cells has been recently elucidated when LTRPC2
was identified in immunocytes. Detailed experiments
have demonstrated that LTRPC2 mediates Ca2⫹ influx
into immunocytes where cellular ADPR and NAD directly
activate LTRPC2 and enable Ca2⫹ influx. Importantly,
NAD-induced activation was suppressed by ATP, suggesting that NAD activates TRPC2 when ATP is depleted,
while the activated LTRPC2 causes Ca2⫹ influx and apoPhysiol Rev • VOL
ptosis in these cells linking apoptosis with the metabolism
of ADPR and NAD (160).
5. TRPML subfamily
A) MUCOLIPIN-1. Mucolipin-1 (also called mucolipidin, encoded by the MCOLN1 gene) is a novel membrane protein
that is defective in mucolipidosis type IV disease, which is
a developmental neurodegenerative disorder characterized by lysosomal storage disorder, abnormal endocytosis
of lipids, and accumulation of large vesicles (10, 12, 179).
The predicted full-length mucolipin-1 is a 580-amino acid
protein that has the typical six transmembrane S1-S6
domains with the putative pore-forming loop between S5
and S6. Structure analysis revealed similarity to the TRP
family between amino acids 331 and 521, which corresponds to domains S3-S6, while the pore region is located
between amino acids 496 and 521. Unlike most members
of the TRP family, two proline-rich regions were identified
near the NH2 terminal. A leucine zipper motif is located at
S2, a nuclear localization motif at amino acids 43– 60, and
a serin lipase motive. In the region that shows similarity
to TRP there is 38% identity, whereas in the pore region
there is 58% identity (179). Mucolipin-1 has no ankyrin
repeats or TRP domain. Unlike other subfamilies, no functional analysis of mucolipin is available, and it is still not
clear if it functions as a channel protein. Members of the
TRPML subfamily are conserved in C. elegans (40% amino
acid identity) and Drosophila (44% identity, Ref. 48). A
recent study on a C. elegans homolog of mucolipin-1
designated CUP-5 showed that a loss-of-function mutation in CUP-5 resulted in an enhanced rate of fluid-phase
markers, decreased degradation of endocytosed protein,
and accumulation of large vacuoles (48). Overexpression
of the normal CUP-5 caused the opposite effects, thus
indicating that CUP-5 is a promising model system for
studying conserved aspects of structure and function of
mucolipin-1 and mucolipidosis type IV (48).
6. Summary
The TRP family of channel proteins reveals a high
structural diversity. This family can be divided on the
basis of primary amino acid sequence into six subfamilies.
The founding member of this family, the Drosophila TRP,
shares structure homology with all subfamilies mainly at
the region of the ion pore, strongly suggesting that the
fundamental function of all members of this family is to
constitute cation channels. Some members without clear
pore region may function as regulators of the channels or
they may be responsible for channel translocation into the
plasma membrane. Additional structural features that are
common to most, but not all, subfamilies are the ankyrin
repeats at the NH2 terminal and the TRP domain at the
COOH-terminal side of the transmembrane domain. Because ankyrin repeats are important regions for protein-
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protein interactions, such interactions seem to be fundamental for TRP function, although the actual function of
these regions in TRP is still unknown. More mysterious is
the function of the TRP domain, which characterizes most
members of the TRP family. In addition, one subfamily
(TRPM) includes proteins with dual function of channel
and enzyme, a property unique to the TRP family. In
general, the diverse structural features of members of the
TRP family strongly suggest that members of this family
are involved in a wide range of cellular functions.
IV. FUNCTIONAL ANALYSIS IN THE
NATIVE TISSUE
The discovery of vertebrate TRP channels in a large
variety of cells and tissues each having a putative specific
function suggests that the channels of the TRP family also
have diverse and elaborate gating mechanisms that
largely depend on their cellular environment. This fact
makes the few cases in which channel proteins have been
analyzed in the native cellular environment highly valuable. Genetic manipulations allow conducting studies of
structure-function relationship of channel proteins while
the other cellular components are intact. Such studies in
the native tissue in conjunction with studies in heterologous systems provide powerful tools for studying the
function of channel proteins. Nevertheless, due to technical difficulties, heterologous expression is still the main
method for studying the function of a large number of
TRP isoforms. In the TRP-homolog group, so far only
Drosophila TRP, TRPL, and mammals TRPC2, TRPC3,
and TRPC4 channels have been studied in detail in the
native cells. A few studies in which the effects of knockout or partial knockout using the antisense approach of
native TRPs were carried out suggested that some of the
TRP homologs contribute to SOC (98, 146, 160, 205, 218).
The native Xenopus TRPC1 isoform has been suggested to
contribute to SOC activity of the oocytes, since knockout
of TRPC1 isoform reduces SOC activity (18).
A. Drosophila Light-Sensitive Channels TRP
and TRPL
1. TRP has a small single-channel conductance
The subcellular localization of TRP and TRPL to the
microvilli imposes a considerable difficulty for access
with patch electrodes. Accordingly, only very recently it
has become possible to record single-channel activities in
patches of Drosophila microvilli (53), as previously reported for Limulus (8) and mollusk photoreceptors (125).
The light-sensitive conductance in Drosophila has been
studied mainly under whole cell voltage-clamp conditions
using the dissociated ommatidial preparation (55, 156).
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The newly developed preparation of rhabdomeral
membranes from Drosophila photoreceptors has been
used only under the restrictive conditions of excised
patches in the absence of Ca2⫹. Under these conditions
the channels are constitutively open with a wide range of
conductance amplitudes. Conductance events as large as
144 pS seem to arise from coordinated gating of many
small individual events of 4 pS. This large conductance
seems to arise from openings of TRP and not TRPL channels, since they were blocked by 10 ␮M La3⫹ and were
completely absent in a null trp mutant (53). It is not clear
if the coordinate openings represent a physiological phenomenon, since quantum bumps, which represent coordinate openings of many channels, are not observed under conditions of constitutive activity of the lightsensitive channels during whole cell recordings (62). It is
also not clear why the activity of TRPL channels, which is
observed under whole cell recordings in the trp mutant
(62), is absent in the dissociated rhabdomeral preparation.
The uncertainty as to the physiological significance
of the single-channel recordings emphasizes the importance of noise analysis, which has been used to estimate
single-channel properties from the so-called “rundown
current” (RDC). The RDC represents a spontaneous opening of light-sensitive channels, which develops after several minutes of whole cell recording accompanied by
illuminations (62) or under metabolic stress in the dark
(4). In the presence of 4 mM external Mg2⫹, TRPL channels have an apparent conductance of ⬃35 pS and TRP
channels ⬃4 pS. Both channels, however, are blocked by
divalent ions including Mg2⫹, and under divalent free
conditions, conductance values increase to ⬃70 pS
(TRPL) and ⬃35 pS (TRP), respectively (64, 68).
Power spectra have also been calculated from current noise, providing information on channel kinetics.
Power spectra of TRPL channels are consistent with a
channel with two open states with mean open times of 2
and 0.15 ms. Noise analysis data of heterologously expressed TRPL channels in Drosophila S2 cells were indistinguishable from that measured in Drosophila photoreceptors, and the single-channel conductance of the
heterologously expressed TRPL channel was directly confirmed by recordings of single channels (68). Power spectra of TRP channels (in the trpl mutant) are characterized
by higher frequencies with a calculated time constant of
0.4 ms.
2. TRP channels are highly permeable to Ca2⫹
The light-sensitive channels in invertebrate photoreceptors are essentially nonselective cation channels, and
the channels in Drosophila readily permeate a variety of
monovalent ions including Na, K, Cs, Li, and even large
organic cations such as Tris and TEA (55, 156). Moreover,
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the reversal potential of the LIC in WT and the trpl mutant
shows a marked dependence on extracellular Ca2⫹, indicating that TRP channels are permeable to Ca2⫹.
Our knowledge of the biophysical properties of the
TRP and TRPL channels has been facilitated by measurements of the in situ conductance in the trp and trpl
mutants. Permeability ratios to monovalent and divalent
ions in WT and trp flies have been calculated (60) using
the constant current equation and reversal potential data
obtained under various ionic conditions. With the assumption of Goldman-Hodgkin-Katz (GHK) constant field
theory, the quantitative dependence implies a relative
Ca2⫹ permeability (PCa:PNa) of ⬃40:1 (55, 60). More recently, the permeability ratios for a variety of divalent and
monovalent ions ratios have been determined under biionic conditions, where in these experiments Cs⫹ was the
only intracellular cation while the bath contained one of a
variety of monovalent or divalent cations. It was found
that TRPL represents a nonselective cation channel (PCa:
PCs ⫽ 4.3) and TRP, a channel highly selective for divalent
cations (PCa:PCs ⬎100:1). The WT values are explained by
approximately equal summation of TRP and TRPL conductance. On the assumption of independent mobility of
ions, these permeability data imply that in WT, 45% of the
LIC at resting potential is mediated by Ca2⫹, 25% by Mg2⫹,
and 30% by Na⫹ (using a physiological Ringer solution,
Ref. 158).
3. Magnesium conductance and block
Drosophila photoreceptors bathed in divalent free
medium respond to light in an outward current at membrane potentials equal to the resting potential (i.e., ⫺50
mV). Addition of Mg2⫹ to the bath resulted in light-induced inward current, indicating that Mg2⫹ permeates the
light-sensitive channels (60). Consistent with this observation, calculation of permeability ratios indicated that
the light-sensitive channels have a relatively high permeability to Mg2⫹ (60). This highly unusual property of the
TRP channels was also recently found in one of the TRPM
channels (123).
TRP and TRPL channels are also blocked by Mg2⫹ at
physiological concentrations. This property is common to
a variety of channels (64). For TRPL channels, the block
is moderate (maximally ⬃3-fold with an IC50 of 2–3 mM)
and has little, if any, dependence on either voltage or
extracellular Ca2⫹. In WT flies, where the current is dominated by the TRP channels, the block is much more
severe and is voltage dependent. The block is relatively
relieved at both hyperpolarized and depolarized potentials, thereby generating a characteristic inward and outward rectification, while the block is also stronger in the
absence of extracellular Ca2⫹. Under these conditions,
there is a maximum of ⬃20-fold block at resting potential,
with an IC50 of 200 ␮M Mg2⫹. In the presence of 1.5 mM
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extracellular Ca2⫹, the maximum block (in 20 mM Mg2⫹)
is ⬃10-fold. The block is reflected in a reduction in singlechannel conductance, which may be an important factor
in improving signal-to-noise ratio in a similar manner to
the conductance of the vertebrate cGMP-gated channels
(213).
The inward and outward rectification seen in the
current voltage (I-V) relationship of the WT conductance
is due in part to the voltage-dependent Mg2⫹ block. A
similar I-V function is found in trpl mutants, indicating
that this is a property of the trp-dependent channels.
Under divalent free conditions, the I-V relationship shows
a simple exponential outward rectification. The I-V relationship of TRPL channels shows outward rectification
both in the presence and absence of divalent ions, suggesting that it reflects an inherent property of the channels (64, 68, 158).
4. Ca2⫹ influx
Consistent with the electrophysiological determinations, the ability of the light-sensitive channels to permeate Ca2⫹ has been directly demonstrated by measurements of very large light-induced Ca2⫹ influx signals.
Ca2⫹-selective microelectrodes were applied in vivo
(139), and Ca2⫹ indicator dyes were used in both isolated
ommatidia of Drosophila (59, 140, 155) and in Calliphora,
in vivo (128). With the use of Ca2⫹ indicators of high and
low affinity (140) or ratiometric indicators (59), it was
found that during intense lights cellular Ca2⫹ reaches the
micromolar range. Ca2⫹ influx was much reduced in the
trp mutant (59, 139). The Ca2⫹ influx measurement in
Calliphora, in vivo, has shown that cellular Ca2⫹ is regulated in a graded fashion over a large range of adapting
light intensities. In dark-adapted photoreceptors, Ca2⫹
can reach a high level of 200 ␮M in ⬍20 ms (128). Moreover, Ca2⫹ imaging demonstrates colocalization of Ca2⫹
influx and extrusion. This colocalization ensures that after the cessation of light, removal of Ca2⫹ from the rhabdomere, where the phototransduction machinery resides,
is faster than in the cell body. This situation ensures a
rapid dark adaptation (127).
B. Activation of TRP and TRPL Channels
In Limulus ventral photoreceptors there is strong
evidence that light induces release of Ca2⫹ from intracellular stores via InsP3 receptors localized in the submicrovillar cisternae (SMC) and that Ca2⫹ can activate at
least one class of light-sensitive channel (reviewed in
Refs. 44, 124). The main evidence includes direct measurements of light-induced Ca2⫹ release coincident with,
or a few milliseconds before the light-induced current
(189), the ability of both InsP3 and Ca2⫹ to excite the
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light-sensitive channels (23, 135, 136) and the inhibition of
the light response by Ca2⫹ chelators (171). However, in
Drosophila, but not in the large flies (41), InsP3 and Ca2⫹
failed to excite the photoreceptor cells (57, 153).
Activation of PLC is necessary for Drosophila phototransduction since null PLC mutants (17) eliminate the
response to light (110). Therefore, extensive biochemical,
electrophysiological, and molecular genetic approaches
were applied to Drosophila photoreceptors to study the
link between activation of PLC and the opening of TRP
and TRPL channels. However, the signaling steps linking
between the products of PLC activation and the opening
of the light-sensitive channels in Drosophila remain unknown and controversial.
1. Possible roles of Ca2⫹ in excitation
There is strong evidence that the presence of Ca2⫹ in
the photoreceptor cell of Drosophila is necessary for
excitation, since prolonged Ca2⫹ deprivation reversibly
eliminates excitation (37, 60). Nevertheless, photolysis of
caged Ca2⫹ (DM-nitrophen) failed to activate the lightsensitive channels in null rhodopsin or PLC mutants,
which do not respond to light (57). The released Ca2⫹ did,
however, induce a substantial electrogenic Na⫹/Ca2⫹ exchange current in WT and the blind mutants. In WT flies,
there is an inherent problem since the Ca2⫹ uncaging light
also excites the photopigment. Indeed, in WT flies, when
the caged Ca2⫹ was released during the rising phase of the
light response, a large facilitation of the LIC was observed
while a profound inhibition was revealed when Ca2⫹ was
released during the steady-state phase. Interestingly, in
the trp mutant, upon the release of caged Ca2⫹, inhibition
of the LIC was observed during the rising phase and
facilitation at the falling phase. Both facilitation and inhibition occurred with a submillisecond latency, suggesting
it occurred at the level of the channels themselves and
indicating that the released Ca2⫹ had direct access to the
channels.
Alternative slower methods of application have been
used to raise cytosolic Ca2⫹, including perfusion of up to
0.5 mM Ca2⫹ from the whole cell patch pipette (56) and
application of the Ca2⫹ ionophore ionomycin (58). None
of these procedures appears to activate any light-sensitive
channels, although they could activate an electrogenic
Na⫹/Ca2⫹ exchange current and modulate the response to
light itself. These largely negative results do not rule out
the possibility that Ca2⫹ is necessary but not sufficient for
excitation. Also, because Ca2⫹ is known to exert both
positive and negative feedback on the response to light,
the exogenous application of Ca2⫹ in the above experiments might not be sufficiently refined to allow separation
between these opposing effects (37).
Physiol Rev • VOL
2. Light induces release of Ca2⫹
There is strong evidence for a massive light-induced
Ca2⫹ influx, which is associated with the LIC in the presence of normal extracellular Ca2⫹(see above). In contrast,
in Ca2⫹-free medium, most measurements have shown
no, or negligible, Ca2⫹ rise, suggesting there is very little
if any light-induced Ca2⫹ release from internal stores (59,
140, 155). These initial negative results probably arise
from the very small dimension of the putative InsP3sensitive Ca2⫹ stores, the SMC (61). More refined measurements, which took into account the small dimensions
of Drosophila SMC, indeed show light-induced release of
Ca2⫹ from internal stores that correlated with the ability
of the photoreceptors of the trp mutant to respond to light
(37). However, recent measurements by Hardie, Colley,
and colleagues (153) have shown that this light-induced
release of Ca2⫹ is similar in WT and in a mutant in which
the known InsP3 receptor was removed genetically. These
authors thus argue that this release of Ca2⫹ is not mediated via the InsP3 receptor, suggesting that this release is
from non-InsP3-sensitive stores and has no role in phototransduction (153).
3. Effects of Ca2⫹ chelators
The effects of Ca2⫹ chelators have been tested by
including the chelator in the patch pipette during whole
cell recordings of the LIC in Drosophila (56). As expected,
the slow buffer EGTA (as high as 18 mM) had only weak
effects on the light responses in normal Ringer solution
(1.5 mM Ca2⫹) and had no apparent effect in Ca2⫹-free
solutions. The fast chelator BAPTA, operating in the microsecond range, was much more potent than EGTA. A
high concentration of BAPTA (14 mM) reduced the sensitivity to light by ⬃5,000-fold in normal Ringer solution.
However, in Ca2⫹-free solution, the effects of BAPTA
were much lower, with only a 2 log unit reduction in
sensitivity (14 mM BAPTA), while no effect was observed
with concentrations below 3 mM. Therefore, Ca2⫹ regulates the light-activated channels with fast kinetics, while
cellular Ca2⫹ has to reach a threshold concentration to
manifest this effect.
Taken together, the available data suggest that Ca2⫹
has profound effects on excitation, but the specific roles
of Ca2⫹ are still unknown.
4. Involvement of InsP3 in phototransduction
Application of InsP3 by photoactivated caged InsP3 in
the Drosophila photoreceptors did not result in activation
of the light-sensitive channels (153). However, pipette
application to the cell during whole cell recordings of the
hydrolysis-resistant GTP analog guanosine 5⬘-O-(3-thiotriphosphate) (GTP␥S), which is expected to have an
excitatory effect, also failed to activate the transduction
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cascade. Therefore, there is probably a problem of accessibility of chemicals to the signaling membrane in the
preparation of isolated Drosophila ommatidia during
whole cell recordings. It is thus possible that these negative results may be attributed to the highly compartmentalized structure of the phototransduction machinery in
Drosophila.
An alternative method of application has been successfully attempted in the larger flies, Lucilia and Musca
(41, 180). These studies exploited the finding that Lucifer
yellow applied extracellularly is taken up by the photoreceptors following intense illumination, possibly via receptor-mediated endocytosis (201). Minke and Stephenson
(112) introduced extracellular GTP␥S into photoreceptors by application of intense light. The effect of GTP␥S in
Musca photoreceptors was manifested by a noisy depolarization in the dark, with properties of the response to
light. Similarly, extracellular application of InsP3 into
Musca or Lucilia retinae accompanied by intense white
light resulted in a large increase in baseline noise that
persisted in the dark long (⬎30 min) after the light. Application of 2,3-diphosphoglycerate (DPG), an inhibitor of
InsP3 hydrolysis, greatly enhanced the effect of exogenously applied InsP3 and also enhanced and prolonged
the physiological response to light, suggesting an endogenous production of InsP3 during illumination. Noise analysis suggested that the InsP3-induced noisy depolarization
is similar to the noisy depolarization induced by dim light
(41, 180).
Biochemical studies, which strongly support an essential role of PLC, also suggested indirectly that InsP3 is
involved in fly phototransduction. Evidence for operation
of a light-dependent PLC in fly photoreceptors was obtained from biochemical experiments in membrane preparations of Musca and Drosophila eyes. An eye membrane preparation was developed in which lightdependent phosphoinositide hydrolysis could be studied
under defined conditions, allowing the effects of activators and inhibitors to be analyzed. Musca eyes or Drosophila heads containing intact cells were preincubated
with [3H]inositol to label the phosphoinositides. The
membrane prepared from these cells was used to analyze
the hydrolysis of phosphoinositides. The Musca eye membrane preparation responded to illumination by an increase in the accumulation of InsP3 and inositol bisphosphate (InsP2), the products of polyphosphoinositide
hydrolysis by PLC. Addition of DPG substantially decreased the accumulation of InsP2 and concurrently increased the accumulation of InsP3, which thus becomes
the major product of the light-induced phosphoinositide
hydrolysis (41). It is apparent that fly membranes are
endowed with the enzymatic system necessary to produce InsP3 and to eliminate it after it has been produced.
Both the robust light-dependent preferential production
of InsP3 and the efficient turn-off mechanism to stop its
Physiol Rev • VOL
action are consistent with an internal messenger role of
InsP3 in fly phototransduction (for review, see Refs. 107,
167).
The Drosophila genomic sequence (2) identifies only
one InsP3 receptor gene in the Drosophila genome, and
mutations in this gene are lethal (1, 194). However, it is
possible to generate mutant photoreceptors in mosaic
patches by inducing mitotic recombination in heterozygotes. Intracellular recordings from photoreceptors in
such mosaic patches revealed no differences from WT,
leading the authors to conclude that the InsP3 receptor
played no role in phototransduction (1). A more detailed
study used mosaic eyes homozygous for a deficiency of
the InsP3 receptor of Drosophila, where elimination of
InsP3 receptor was confirmed by RT-PCR, Western blot
analysis, and immunocytochemistry (153). In this mutant,
all aspects of the photoresponse when examined in detail
by whole cell recordings were normal. These results indicate that the InsP3 receptor that was eliminated does
not participate in phototransduction, or that it is redundant to a function of another protein. The apparent conclusion from these experiments is that the InsP3 branch of
the inositol lipid signaling is not involved in phototransduction of Drosophila. However, this conclusion has been
recently questioned following the use of a novel antagonist of the InsP3 branch of the inositol lipid signaling.
The membrane-permeant compound 2-aminoethoxydiphenyl borate (2-APB) has proven effective as a probe
for assessing the involvement of InsP3 branch of the
inositol lipid signaling in intact cells. 2-APB at 75 ␮M
blocks receptor-mediated Ca2⫹ store emptying in intact
HEK293 cells as well as in several other cells tested (100).
In broken cells, 2-APB directly blocks InsP3-mediated
Ca2⫹ release from the ER. 2-APB has no effect on InsP3
binding, does not alter InsP3 production through agonistsensitive PLC, and does not modify the function of ryanodine receptors or voltage-gated Ca2⫹ channels (100,
102). 2-APB applied to the external medium reversibly
and efficiently blocks the endogenous and robust InsP3mediated signaling pathway of Xenopus oocytes by specifically inhibiting InsP3-induced release of Ca2⫹ from
internal stores. Thus 2-APB seems to operate at a stage
after production of InsP3 but before InsP3 action in mediating the rise in cellular Ca2⫹ (33). All the above features of 2-APB, together with very fast penetration into
the signaling region inside the cell, make 2-APB a useful
reagent for studies of the involvement of the InsP3 branch
in Drosophila phototransduction. Recent studies indeed
showed that 2-APB is highly effective at reversibly blocking the response to light of Drosophila flies in a lightdependent manner at a concentration range that coincided with its effectiveness in oocytes (33). Furthermore,
2-APB had no effect on the opening of the TRP and TRPL
channels themselves, indicating that it operates upstream
of the channels. Accordingly, because 2-APB seems to
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BARUCH MINKE AND BOAZ COOK
block InsP3-induced Ca2⫹ release in vertebrate cells, it
cannot be excluded that Drosophila photoreceptors use
the InsP3 branch of the inositol-lipid signaling pathway for
light excitation even if the mechanism and specificity of
2-APB action are not clear. The reconciliation of the
apparently conflicting knockout of the InsP3R and the
2-APB data is likely to shed important new light on the
mechanism of activation of TRP and TRPL (33). In addition, the same dilemma may apply to the activation mechanism of vertebrate homologs of the TRPC subfamily,
where similar apparently conflicting knockout of InsP3Rs
and the 2-APB data have been recently reported (101).
Thus, in the triple InsP3 receptor (InsP3-R) knockout
DT40 cells devoided of all three functional InsP3-Rs, SOC
activity was virtually identical to WT cells. However, in
both WT and mutant cells, 2-APB blocked SOC activity in
an identical manner (101).
5. Are DAG and polyunsaturated fatty acids second
messengers of excitation?
6. Constitutive activation of the TRP and TRPL
channels in the dark
The DAG branch of the inositol lipid signaling has not
been considered as a route of excitation in invertebrate
phototransduction because mutations in an eye-specific
PKC (inaC) lead to defects in response termination and
adaptation with no apparent effects on activation. This
notion has been recently supported by experiments in
Limulus showing that application of phorbol esters or the
alkaloid indolactam, which are PKC activators, largely
suppressed light-induced Ca2⫹ release and excitation at
an early stage of the cascade (39). Contrary to these
findings, application of phorbol esters and indolactam did
excite mollusk (Lima) photoreceptors in the dark. The
indolactam-induced excitation was also antagonized by
PKC inhibitors (40). Phorbol ester also mimicked the
effects of light in rdgB, a retinal degeneration mutant of
Drosophila in which degeneration is largely enhanced by
illumination (108).
Recently, a possibility has been considered that DAG
does not operate solely as activator of PKC but also in a
parallel pathway that leads to TRP or TRPL activation, as
well as of mammalian TRPC channels (75; see below).
DAG is a potential precursor for formation of polyunsaturated fatty acids (PUFAs) via DAG lipase, although the
activity of this enzyme has not been demonstrated in
Drosophila photoreceptors. Mutations in DAG kinase
(rdgA, Fig. 1) that inactivates DAG result in a severe form
of light-independent degeneration, although the reason
for this is unknown (103, 104). Recently, a possible mechanism has been suggested following the discovery of Hardie and colleagues (35, 154) that in the rdgA mutant TRP
and TRPL channels are constitutively open, while elimination of TRP by a mutation (in the double mutant rdgA;
trp) partially rescues both the degeneration and the ability to respond to light. The authors suggest that
Physiol Rev • VOL
elimination of DAG kinase by the rdgA mutation leads to
accumulation of DAG and hence of PUFAs, which constitutively activate the TRP and TRPL channels, leading to a
toxic increase in cellular Ca2⫹. Hardie and colleagues
further showed that application of PUFAs to isolated
Drosophila ommatidia reversibly activate the TRP and
TRPL channels as well as recombinant TRPL channels
expressed in Drosophila S2 cells. The latter were also
rapidly activated by application of PUFAs in inside-out
excised patches. Application of lipoxygenase inhibitors at
low concentrations induced production of bumplike unitary responses followed by activation of macroscopic
currents at higher concentrations. The authors suggest
that PUFAs may be messengers that directly mediate
activation of TRP and TRPL in Drosophila photoreceptors (35). It should be noted, however, that genes encoding for lipoxygenases have not been found in the Drosophila genome (2).
The ability to open TRP and TRPL channels in the
dark may provide some insight on their gating mechanism, since the mechanism of dark openings bypasses at
least some of the phototransduction stages that are activated by light.
RdgA is not the only Drosophila mutant in which
there is constitutive activation of the light-sensitive channels. Specific mutations in the trp gene also cause constitutive activation of the TRP channel, leading to extremely
rapid photoreceptor degeneration in the dark (214, Fig. 5).
A new mutant designated TrpP365 does not display the
transient receptor potential phenotype and is characterized by a near-normal level of the TRP protein and rapid,
semi-dominant degeneration of photoreceptors. Despite
its unusual phenotypes, TrpP365 is a trp allele because a
TrpP365 transgene induces the mutant phenotype in a
wild-type background, and a wild-type trp transgene in a
TrpP365 background suppresses the mutant phenotype.
Moreover, amino acid alterations that could cause the
TrpP365 phenotype are found in the transmembrane segment region of the mutant channel protein. These mutations are due to the following amino acid substitutions:
Pro(500)Thr, His(531)Asn, Phe(550)Ile, and Ser(867)Phe.
Whole cell recordings clarified the mechanism underlying
the retinal degeneration by showing that the TRP channels of TrpP365 are constitutively active, thus presumably
allowing a toxic increase in cellular Ca2⫹ (214, Fig. 6).
Constitutive activation of TRP and TRPL was also
obtained by metabolic stress, which turned out to be very
efficient in reversibly causing channels openings in the
dark in vivo. Accordingly, patch-clamp whole cell recordings and measurements of Ca2⫹ concentration by ionselective microelectrodes in intact eyes of normal and
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FIG. 5. Electron micrographs of
transverse sections through the layer of
Drosophila ommatidium TrpP365/trpCM
raised at 19°C and TrpP365/TrpP365. All
samples were obtained from newly
eclosed adult flies, and all flies were
marked with w. The TrpCM mutant is the
original, nearly null, Trp mutant. The
electron-dense structures are the rhabdomeres, which are composed of microvilli that contain the signaling molecules. The trpCM mutation eliminates most
of the TRP protein. The figure shows that
the degree of degeneration in a newly
eclosed fly depends on the amount of mutated TRP having the TrpP365 mutation.
[From Yoon et al. (214).]
mutant Drosophila revealed that anoxia rapidly and reversibly depolarizes the photoreceptors and induces Ca2⫹
influx into the photoreceptor cells in the dark (Fig. 7).
Furthermore, openings of the light-sensitive channels,
which mediate these effects, could be obtained by mitochondria uncouplers or by depletion of ATP in photoreceptor cells, while the effects of illumination and all forms
of metabolic stress were additive. Effects similar to those
found in WT flies were also found in mutants with strong
defects in rhodopsin, Gq protein, or phospholipase C, thus
indicating that the metabolic stress operates at a late
stage of the phototransduction cascade (Fig. 8). Genetic
elimination of both TRP and TRPL channels prevented
the effects of anoxia, mitochondria uncouplers, and depletion of ATP, thus demonstrating that the TRP and
TRPL channels are specific targets of metabolic stress.
These results suggest that a constitutive ATP-dependent
process is required to keep TRP and TRPL channels
closed in the dark, a requirement that would make them
sensitive to metabolic stress (4). The results also suggest
FIG. 6. Single-cell functional analysis
by whole cell recordings of the TrpP365
mutant. A: a typical light-induced current
(LIC) of a wild-type cell (left trace) in
response to an orange stimulus and the
absence of any responses in TrpP365/
trpCM and TrpP365/TrpP365 (middle and
right traces). The duration of the orange
light stimulus is indicated above each
trace. B and C compare families of current traces elicited by series of voltage
steps from photoreceptors of wild-type
(left column), the light-insensitive cells
of TrpP365/trpCM (middle column), and
TrpP365/TrpP365 homozygotes (right column). For each experiment, a series of
voltage steps was applied from a holding
potential of ⫺20 mV in 20-mV steps (bottom traces). B: membrane currents were
recorded 30 s after establishing the
whole cell configuration with physiological concentrations (1.5 mM) of Ca2⫹ in
the bath. C: application of 10 mM La3⫹ to
the bath suppressed the membrane currents. The figure shows that the trpP365
mutation eliminated the response to light
due to constitutive activity of the TRP
channels that could be blocked by La3⫹.
[From Yoon et al. (214).]
Physiol Rev • VOL
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BARUCH MINKE AND BOAZ COOK
redox sensor. A dominant negative form of TRPC3 abolished the oxidant-induced current in aortic cells, suggesting that TRPC3 is involved in this conductance (9). Recently it has been suggested that LTRPC7 are reporters of
the metabolic status of cells by being sensitive to Mg-ATP
(9). The latter finding is in accord with the findings in
Drosophila; however, because it is known that the role of
TRP channels in the fly is to respond to light stimuli, it is
conceivable that some of the reported sensitivities of TRP
channels are epiphenomena, while their main role is altogether different.
7. TRP and store-operated Ca2⫹ entry
FIG. 7. Anoxia activated the TRP and TRPL channels in both wildtype (WT) (A) and the PLC null mutant (norpAP24; B) as monitored by
Ca2⫹ influx. The figure shows extracellular voltage change (ERG, top
traces in A and B) and potentiometric measurements with Ca2⫹-selective microelectrode (ECa, bottom traces in A and B) in response to
orange lights and anoxia (N2) in WT Drosophila and norpAP24 mutant.
Note that there is no response to light (LM, light monitor) in the
norpAP24 mutant and the initial slow phase of the electrical response to
anoxia (before the arrow) is missing in the Ca2⫹ signals of both WT and
the mutant. *Movement artifact. [From Agam et al. (4).]
that the effects of PUFAs may be indirect, since these
compounds have been shown in many studies to function
as efficient uncouplers of mitochondria (7). Suppression
of TRP activity is not unique to Drosophila; the NADinduced activity of LTRPC2 is also suppressed by ATP
(see sect. IVD4, Ref. 160).
The constitutive activity of the TRP and TRPL channels may lead to cell death under metabolic stress, not
only in Drosophila but also in vertebrate cells, which
express TRPC and TRPM channels (9, 123). Aortic endothelial cells have been shown to express an oxidantactivated nonselective cation channel that functions as a
Physiol Rev • VOL
Because Drosophila TRP is the target of inositol lipid
signaling and TRP has a high Ca2⫹ selectivity, Hardie and
Minke (63) have proposed that excitation in Drosophila
might be analogous to the process of PI-regulated Ca2⫹
influx described in a wide variety of mammalian cell
types. Experiments designed to test whether a SOC-like
mechanism might operate in Drosophila phototransduction have been largely negative (58). Although both ionomycin and thapsigargin release Ca2⫹ from intracellular
compartments (58, 155), store depletion by these agents is
not associated with the activation of any currents, nor do
these agents block the response to light during several
minutes (but thapsigargin does block the LIC after long
exposure and confers a trp phenotype on WT flies; Ref.
37). Application of Ca-BAPTA to the cell via the recording
pipette, buffering Ca2⫹ at concentrations at a level of ⬃10
nM, activates an inward current following application of
ionomycin (58). However, it is not clear if this effect is
due to store depletion or other effects similar to those
activated by metabolic stress. The elimination of the
InsP3R by mutation without affecting phototransduction
has also been used as a strong argument against a role of
inositide-mediated store depletion in Drosophila TRP and
TRPL activation (1, 153). Thus, although conditions have
been found in Drosophila to activate TRP and TRPL
channels, which in some studies have been used to demonstrate SOC mechanism in vertebrate cells, it is not clear
whether they represent the mechanism operating normally and represent the physiological route of excitation.
C. Role of TRPC3 in Pontine Neurons
At present, TRPC2, TRPC3, TRPC4, and TRPC6 are
the TRPC channels that have been studied in detail in the
native tissue. A recent study by Montell and colleagues
(95) has shown that TRPC3 is highly enriched in neurons
of the brain, especially in the forebrain, midbrain, and
hindbrain of rat embryo. Because TRPC3 is predominantly expressed in the rat brain during a relatively narrow developmental period before and after birth (95), its
function may be related to developmental processes. Im-
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451
FIG. 8. Single cell functional analysis
by whole cell recordings from Drosophila photoreceptor showing that depletion
of ATP activates the TRP and TRPL
channels of WT cells in the dark. The
constitutive activity was demonstrated
by application of voltage steps in the
dark during whole cell recordings from
the photoreceptor cells during the slow
inward current. A: typical light-induced
currents (LIC) of a wild-type cell in response to 3 orange lights was followed
by appearance of slow inward current in
the dark when the pipette solution had
no ATP and NAD and the membrane voltage was held at ⫺50 mV. The top traces
in each pair indicate the duration of the
orange light stimuli. B: onset of the dark
inward current was accelerated by application of 0.1 mM DNP (arrow). Note that
no additional response to light was obtained after the dark inward current was
induced. C: comparison of families of
current traces elicited by series of voltage steps in the range of ⫺100 to ⫹80 mV
in steps of 20 mV (lower row), from photoreceptors of WT flies in the dark before
the inward current was induced, at the
peak of the inward current at 1.5 mM
external Ca2⫹, after Ca2⫹ was removed
from the external medium (0 Ca2⫹) and
after 10 ␮M La3⫹ was applied to the external medium (as indicated). D: effects
of DNP on the trp mutants A relatively
slow induction of a small and noisy inward current in the dark was observed in
null trp after application of 0.1 mM DNP
(arrow) during recordings without ATP
and NAD in the recording pipette. Inward
currents could not be induced without
application of DNP under similar recording conditions. The right traces show a
family of current traces elicited by series
of voltage steps as in C at 1.5 mM external Ca2⫹. E: recording from the trpl;trp
double mutant using pipettes in which
ATP and NAD were omitted and 0.1 mM
DNP was applied to the external medium
as indicated. No dark inward current was
observed even 16 min after the beginning
of whole cell recordings. The right traces
show a family of current traces elicited
by series of voltage steps as in C at 1.5
mM external Ca2⫹. Note that neither
light-induced currents nor inward currents in the dark could be induced in this
double mutant. [From Agam et al. (4).]
portantly, the temporal and spatial distribution of TRPC3
parallels that of the neurotrophin receptor TrkB. Activation of TrkB by brain-derived nerve growth factor (BDNF)
Physiol Rev • VOL
led to production of a PLC-␥-dependent nonselective cation current (IBNDF) in isolated pontine neurons. Evidence
that TRPC3 contributes to IBNDF in vivo is provided by the
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BARUCH MINKE AND BOAZ COOK
following observations: 1) IBNDF was activated by antiTRPC3 antibody both at the macroscopic current level
and in single-channel recordings. In these experiments
BNDF and the antibody acted synergistically. 2) Heterologous coexpression of TrkB receptor and TRPC3 in 293T
cells followed by current measurements indicated that
IBNDF was not observed in cells expressing either TrkB or
TRPC3 alone. 3) TRPC3 coimmunoprecipitated with TrkB
of rat brain, suggesting that the two proteins interact and
form a protein complex. 4) IBNDF was blocked by SK&F96365, a nonspecific inhibitor of TRPC3. Interestingly, the
properties of single-channel conductance that was activated by the anti-TRPC3 antibody in pontine neurons
showed smaller conductance (i.e., 27 pS, see Table 2) and
longer mean open time (⬃20 ms) relative to measurements in heterologously expressed TRPC3 channels (of
⬃60 pS and ⬍2 ms). In addition, unlike the heterologously
expressed TRPC3 channels (100), the native pontine
TRPC3 channels could not be activated by the 1-oleoyl-2acetyl-sn-glycerol (OAG) analog of DAG. Application of
InsP3 activated the pontine TRPC3 channels, and inhibition of either PLC by its antagonist U73122 or the InsP3
receptor by xestospongin (100) inhibited the IBNDF current. These results are consistent with activation of
TRPC3 via the conformation coupling hypothesis (see
below), not only in heterologous system (89, 100) but also
in vivo (95).
D. Control of Ca2ⴙ Influx in Vascular Smooth
Muscle Cells
There are several studies reporting that TRP homolog channels are involved in the control of vascular
smooth muscle contraction and relaxation (for review,
see Ref. 163). Thus TRPC1 has been recently reported to
be involved in SOC activity of resistance arterioles, arteries, and veins from human mouse or rabbit (206). TRPC1specific antibody targeted to the outer vestibule of TRPC1
channel identified TRPC1 in native vascular smooth muscles. Interestingly, peptide-specific binding of the antibody selectively blocked SOC activity in these cells, suggesting that TRPC1 underlies SOC activity and hence
Ca2⫹ influx to these cells (206).
In perhaps the most detailed study to date, Mori and
colleagues (81) provided compelling evidence that TRPC6
is a requisite component of the ␣1-adrenoreceptor activator (␣1-AR) Ca2⫹-permeable nonselective cation channel
(NSCC) (reviewed in Ref. 163).
1. TRPC6 is the essential component of ␣1-AR
Ca2⫹-permeable NSCC channel
The ␣1-AR is distributed widely in the vascular system and played a central role in control of systemic blood
pressure via sympathetic innervations. Mori and colPhysiol Rev • VOL
leagues (81) have found that heterologous expression of
murine TRP6 in HEK293 cells reproduced almost exactly
the essential biophysical and pharmacological properties
of ␣1-AR-NSCC, previously identified in smooth muscles
of rabbit portal vein. These properties include activation
by DAG, S-shaped I-V relationship, high divalent cation
permeability, single-channel conductance of 25–30 pS,
augmentation by flufenamate, and blockade by Ca2⫹,
La3⫹, Gd3⫹, SK&F-96365, and amiloride. The level of
TRPC6 mRNA expression was much higher in smooth
muscles of both murine and rabbit portal vein than of
other TRPC members such as TRPC1 or TRPC3. Furthermore, treatment of primary cultured portal vein myocytes
with TRPC6 antisense oligonucleotides resulted in
marked inhibition of TRP6 immunoreactivity and selective suppression of ␣1-AR-activated store depletion-independent cation current and Ba2⫹ influx.
2. TRPC4 mediates vasorelaxation of blood vessels
A relatively small fraction of TRPC4 channels is expressed in vascular endothelial cells. However, TRPC4 of
endothelial cells is potentially of high functional importance, since Ca2⫹ influx to these cells, presumably
through TRPC4 channels, may contribute to increase in
cellular Ca2⫹, which is necessary for synthesis and release of vasoactive compounds (nitric oxide and prostaglandins) (49). A strong support for this notion has been
recently provided by production of knockout mice designated TRP4⫺/⫺ (49).
Nilius, Flockerzi, and colleagues (49) have studied
primary cultured mouse aortic endothelial cells (MAECs)
of normal mice, which express TRPC4, and compared
them with similar MAECs of TRP4⫺/⫺ lacking TRPC4
(but not other TRPC channels, as tested in brain tissue)
using a protocol that activates SOC channels. To deplete
the InsP3-sensitive Ca2⫹ stores they used either InsP3
applied to the patch-clamp pipette or the Ca2⫹ pump
inhibitor tert-butyl-benzohydroquinone (tBHQ). Ca2⫹
store depletion quickly led to a constitutive inward current with inward rectification, which was effectively
blocked by La3⫹ or Mg2⫹ (in Ca2⫹-free medium) in a
similar manner to the TRP channels of Drosophila. The
constitutively active channels revealed a high Ca2⫹ permeability, PCa/PNa ⫽ 159.7 (calculated using the constant
field theory). Strikingly, the constitutive SOC current was
missing in TRP4⫺/⫺ knockout mice. Furthermore, vasoactive agonists (ACh, ATP) induced an increase of free
cellular Ca2⫹, which was markedly reduced in the knockout mice. Importantly, stimulation of endothelial cells of
the knockout mice with the vasoactive agonist revealed
that vasorelaxation of aorta rings was impaired.
The above experiments constitute the first direct evidence for the physiological relevance in mature cells of
mammalian TRPC channels and for SOC in endothelial
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TABLE
2. Functional properties of “TRP-homolog” and “TRP-related” channels
Channel
Single-Channel
Conductance, pS
Relative
Permeability
PCa/PNa
4 (in divalent),
35 (divalent
free), in
Drosophila
35 (in divalent)
in Drosophila,
70 (in divalent
free) in
Drosophila
110 (PCa/PCs in
trpl mutant),
40 (PCa/PNa in
WT)
4.3 (PCa/PCs) in
Drosophila trp
mutant,
3 (PCa/PNa) in
Sf9
Heterologous
Expression in
the Following
Cells
Mechanism
of Activation
Species Used
for Studies in
the Native
Tissue
Possible
Function
Reference Nos.
TRP-homolog channels
TRP
TRPL
TRP␥
TRPC1
PUFAs?
Ca2⫹?
Sf9, 293T,
Xenopus
oocytes
Drosophila
Light-activated
channel
PUFAs?
DAG (in Sf9)?
293T, S2, Sf9,
CHO, Xenopus
oocytes
Drosophila
Light-activated
channel
293T
Drosophila
Light-activated
channel
subunit
SOC
CHO, Sf9, T293
Xenopus oocytes
SOC?, InsP3
COS-M6
Rat, snake,
mouse sperm
1.6
DAG, SOC?
InsP3 (conformational
coupling)
HEK293, COS,
CPAE, 293T,
CHO
Rat
5
DAG
CHO, HEK, COS
Portal vein
myocytes
␣1-AR activated
5.4
1, 7, 160 in
native
endothelial
cells
1, 14
DAG
SOC? PLC
HEK
CHO, HEK
Mouse
Vasorelaxation
SOC? PLC
CHO, HEK293
16 (from noise
analysis)
TRPC2
TRPC3
TRPC6
23 (noise
analysis),
66 (CHO cells),
27 (pontine
neurons)
35, 28
TRPC7
TRPC4
42
TRPC5
66, 48
Pheromone,
acrosomal
reaction
Neurotrophinactivated
channel
35, 37, 52, 53, 55,
57–64, 68, 126,
140, 153, 156,
158, 190, 209
35, 37, 52, 66, 68,
69, 76, 77, 90,
91, 129, 130,
174, 186, 198,
210, 211, 219
207
18, 97, 98, 99,
173, 178, 206,
218, 221
84, 96, 184, 192
9, 74, 85, 88, 89,
95, 114, 217,
218, 220
16, 19, 74, 81,
114
131
49, 144, 161, 183,
185, 197
132, 145, 161,
178
TRP-related channels
OSM-9
VR1
C. elegans
Embryonic
dorsal root
ganglia from
rat
3.8 (heat)
9.6 (capsaicin)
PKC, anadamide
Xenopus
oocytes,
HEK293
VRL-1
2.9
High
temperature
Xenopus oocyte,
HEK293
GRC
PCa/PCs ⫽ 0.89
Growth factor
CHO
6.3
Osmolarity
HEK293
OTRPC4
35
⬃110
88 (at ⫹60 mV)
30 (at ⫺60 mV)
ECaC
CaT2
CaT1
PCL
Polycystin-2
LTRPC2
LTRPC7,
TRP-PLIK
107
0.5;
42 in divalent
free
137 (at ⫾50 mV)
60
105
130
⬃5
⬃6
Nonselective
2.8
Ca2⫹
ADP-ribose
Reduction in
Mg-ATP
Xenopus
oocytes,
HEK293
Xenopus
oocytes, CHO
Rabbit kidney
Xenopus
oocytes,
CHO
HEK-293
CHO, HEK-293
C. elegans; renal
tubular cells,
U937 cells
Human cells
DT-40 B cells
Osmoreception,
olfaction
Thermal and
inflammatory
pain
High
temperature
pain
Growth factor
regulated
channel
Osmosensory
transduction
Ca2⫹ transport
in epithel
Ca2⫹ absorption
in the intestine
Male mating
(LOV-1)
Channel linked
to cellular
metabolism
36
25, 34, 222
26
86
177
73, 195
137
138, 215
11, 31
54
141, 160
123, 159
CHO, Chinese hamster ovary cells; DAG, diacylglycerol; InsP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; WT, wild type; PUFA,
polyunsaturated fatty acid; PKC, protein kinase C.
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BARUCH MINKE AND BOAZ COOK
cells, which seems to be specific to TRPC4 in these cells
(49). It is interesting to note that heterologous expression
of mouse TRPC4 and TRPC5 in HEK cells resulted in
markedly different results (161). In the heterologous HEK
cells, TRPC4 could not be activated by store depletion; it
shows relatively large single-channel conductance and
much smaller selectivity to Ca2⫹ relative to Na⫹ (see sect.
VC2). Further experiments are required to determine if
the above differences arise from the different cellular
environment of the heterologously expressed TRPC4
channels.
E. TRPC2 Regulates Ca2ⴙ Entry Into Mouse Sperm
Triggered by Egg ZP3
An important function of a TRP homolog channel,
TRPC2, in the native cell has been recently demonstrated
in the fertilization process of the mammalian egg. This
multistep process begins by association of the sperm with
a glycoprotein constituent of the zona pellucida, ZP3, of
the egg leading to release of hydrolytic enzymes from the
sperm acrosome and remodeling of the sperm surface.
This so-called acrosomal reaction is critical for penetration of the sperm into the egg and involves a G protein and
PLC-mediated transduction cascade that eventually leads
to activation of SOC channels and sustained increase in
intracellular Ca2⫹. It has been recently shown that TRPC2
is essential for activation of the sustained Ca2⫹ influx into
sperm by ZP3 (84). The essential role of TRPC2 in the
acrosomal reaction was demonstrated by inhibition of
ZP3-induced Ca2⫹ influx and acrosomal reaction by antiTRPC2 antibody (84). The abundant expression of TRPC2
isoform in the sperm is also consistent with the role of
TRPC2 in the acrosomal reaction. The same argument is
valid for the localization of TRPC2 to the VNO, which
suggests that it may function in the specific sensory system of pheromone detection. Because TRPC2 is a pseudo
gene in humans, pheromone detection seems to be lost in
humans. However, with regard to fertilization, a protein
different from TRPC2 should function in the acrosomal
reaction of humans.
F. The TRP-Related Group
Technical difficulties preclude the analysis of several
members of this subfamily, such as VRL1, the C. elegans
osm-9, ECaC1, and other channels in their native tissue.
An important approach to overcome this difficulty is to
produce knockout mice, in which the specific TRP-related
channel has been eliminated, and compare specific function, which is mediated via TRP-related channels. Their
specific role can thus be studied from the behavior to the
cellular level. This approach has been used for TRPC4
(see sect. IVD2) and for VR1 channels (see below). In
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addition, the cellular localization of these channels to
specific tissue or sensory neurons allows some predictions on the function of these channels and comparison of
these predictions to the actual measured properties in
heterologous systems.
1. VR1 channels mediate specific pain
Pain is initiated when peripheral terminals of a subgroup of sensory neurons are activated by noxious chemicals, pH, and mechanical or thermal stimuli. Nociceptors
are characterized, in part, by their sensitivity to capsaicin
(27). Capsaicin, the active ingredient in hot pepper, is an
excitatory neurotoxin that selectively destroys primary
afferent nociceptors in vivo and in vitro. Heterologous
expression of VR1 in HEK293 cells induced necrotic cell
death within several hours of continuous exposure to
capsaicin while control cells were not affected (27).
These results are reminiscent of the effect of the trpP365
and the rdgA mutations in Drosophila; they produce similar effects in vivo presumably via constitutive activity of
the TRP channels (see above). The natural stimulus that
activates VR1 channels (also designated TRPV1) seems to
be noxious heat, since the temperature-response profile
of VR1 matches very closely those reported for heatevoked pain response in mammals and in cultured neurons. Also, ruthenium red, which blocks VR1 activity in
mammalian cells or in oocytes, blocks heat-evoked nociceptive response in the rabbit ear (27). In addition, the
heat-evoked currents observed in both VR1-expressing
cells and cultured sensory neurons are carried through
outwardly rectifying, nonselective cation channels. Reduction in tissue pH, which usually results from infection,
inflammation, or ischemia, seems also to be endogenous
modulators of VR1. Interestingly, the endogenous cannabinoid receptor agonist anandamide was found to induce
vasodilatation by activating vanilloid receptor on perivascular sensory neurons. Heterologous expression of VR1 in
HEK293 cells and Xenopus oocytes (222) validates that
anandamide is an agonist of this receptor.
An important step toward the establishment of the in
vivo function of VR1 has been made by the creation of
knockout mice lacking VR1. Sensory neurons from mice
lacking VR1 are severely deficient in their response to the
noxious stimuli heat (⬎43°C), protons, or vanilloid compounds but exhibited normal responses to noxious mechanical stimuli (25).
Recently, a functional study of the mechanism underlying activation of VR1 channels in both the native neuronal cells and a heterologous system has been reported.
(150). Because PKC is a prominent participant in pain
signaling (28, 29, 87), the role of PKC in VR1 activation
was studied. Application of 12-tetradecanoylphorbol 13acetate (TPA), which activated endogenous PKC, to Xenopus oocytes expressing VR1 markedly increased the
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amplitude of capsaicin, anandamide, or proton-activated
currents, while TPA induced a current by itself. Specific
PKC inhibitors antagonized the above effects. Excised
outside-out membrane patches of VR1 expressing oocytes
showed pronounced activation of single channels during
application of capsaicin or TPA, with a conductance of
⬃110 pS. This single-channel conductance is much larger
than previously reported for the same channel (i.e., 35 pS)
(27) (see Table 2). Replacing ATP with the hydrolysis-resistant analog adenosine 5⬘-O-(3-thiotriphosphate)
(ATP␥S) activated the channels in a similar manner to
TPA, suggesting that phosphorylation of proteins by PKC
underlies channels activation. Importantly, similar singlechannel activation by capsaicin, TPA, or the algestic peptide bradykinin was observed in patch-clamp cell-attached recordings from sensory neurons, isolated from
the dorsal root ganglia of rat embryos. Recently, it has
been demonstrated that bradykinin and nerve growth factor (NGF) release VR1 from PIP2-mediated inhibition.
Accordingly, diminution of plasma membrane PIP2 by
antibody sequestration of PLC hydrolysis mimics the potentiating effects of bradykinin and NGF (34). Modulation
of PIP2, which may regulate VR1 function, has been suggested also to regulate TRPL activity in heterologous
system (47). Such a mechanism is unlikely to function in
vivo, since depletion of PIP2 in the trp mutant that has
only TRPL channels suppresses, rather than facilitates,
TRPL channel activity (67). A modulatory role of anadamide in PKC-VR1 signaling was examined by application
of anandamide to excised patches of Xenopus oocytes
expressing VR1. Application of anandamide (10 ␮M) produced a progressive increase in single-channel activity
and in the macroscopic current (150). This and the above
studies demonstrate again the complex nature of TRP
gating in different cells.
2. C. elegans osm-9 may regulate olfaction
and osmoregulation
G. NOMPC Is a Channel That Mediates
Mechanosensory Transduction
The mechanotransduction channel NOMPC belongs
to a unique subfamily of the TRP-related subfamilies
(196). The evidence that NOMPC mediates mechanosensory transduction came from a highly (10% of control)
reduced mechanoreceptor current in mechanosensory
neurons located at the mechanoreceptor organ of the
Drosophila nompC mutant (196). Mechanosensory neurons reveal a pronounced degree of adaptation to mechanical stimuli. Thus the intensity-response relationship
between the amplitude of the mechanoreceptor current
and displacement of the hair bristle shows a very steep
dependence (range of 10 ␮m) during constant background displacement. The adaptation is manifested by a
shift of the intensity response curve with increasing the
intensity of the background displacement. Interestingly,
one allele of nompC (nompC4) shows an abnormally
rapid adaptation, whereas the amplitude of the mechanoreceptor current is normal. This mutant may prove valuable to study the regulation mechanism of the channel. At
present there are no data on the ionic mechanism of the
mechanoreceptor current and on properties of the
NOMPC channels.
H. Summary
Functional analysis of C. elegans OSM-9 was based
on behavioral tests in mutant animals. These tests indicate an osm-9 gene function in a specific subset (AWA) of
neurons that mediate olfactory transduction, response to
osmotic and nose-touch stimuli. It is also involved in slow
adaptation to olfaction stimuli (36). No functional studies
at the cellular level are available.
3. The epithelial Ca2⫹ channels ECaC1, CaT1, CaT2,
and OTRPC4
Although the functional properties of ECaC1, CaT1,
CaT2, and OTRPC4 (see Fig. 4) have been derived from
heterologous expression studies (see below), their rather
specific localization to epithelial cells of specific organs
points to the possible function in the native tissue as the
gate keeper of 1,25-dihydroxyvitamin D3-dependent active
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transepithelial Ca2⫹ transporter in the kidney (73, 137) or
a highly Ca2⫹-selective SOC channel in the duodenum
(CaT1) (129). OTRPC4, which functions as an osmoreceptor and is expressed predominantly in the distal nephron
of the kidney, a region exposed to hypotonic solution, is
activated by a decrease in osmolarity, and thus its activation mechanism fits with its localization (177). Their functional expression in heterologous cells supports this conclusion (see below).
The expression pattern of various members of the
TRP family to specific cells and tissues may give us a clue
regarding their specific functions, which seem to be diverse. Unfortunately, the absence of selective antagonists
and the difficulty in analyzing many members of the TRP
family in the native tissue impose a great difficulty in
understanding the function of these TRP channels and in
resolving their mechanism of activation and properties
under physiological conditions. The detailed studies in
Drosophila that combine genetic dissection with powerful electrophysiological and single-cell Ca2⫹-measuring
techniques may also provide important clues regarding
the activation mechanism of at least the mammalian
TRPC channels that have striking structural similarity to
Drosophila TRP and TRPL. There is a steadily growing
research on mammalian members of the TRP family that
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have been conducted in the native tissue. New studies
show a crucial role of TRPC2 in the process of acrosomal
reaction in sperm, while TRPC3 has a role in developing
pontine neurons. Similarly, studies of VR1, TRPC1,
TRPC4, and TRPC6 in primary afferent neurons, salivary
glands, and vascular smooth muscles have revealed an
essential role in nociceptors, in control of fluid secretion
in salivary glands (172), and in control of smooth muscle
tone, respectively. It thus seems that studies in the native
tissue are indispensable. In the following section, recent
studies on coexpression of two different TRPC channels,
which probably form heteromultimeric channels, are reviewed. The striking differences in channel properties of
homo- and heteromultimeric channels described below,
the possible sensitivity of TRPs to the cellular environment, and the probable protein-protein interactions in
most TRPs emphasize the need to study TRP channels in
the native cellular environment.
V. FUNCTIONAL ANALYSIS
IN HETEROLOGOUS SYSTEMS
A. Heterologous Expression of trpl, But Not trp,
Leads to Channels With Similar Properties
of the Native Channel
Expression of cDNA of trpl and possibly of trp in a
variety of expression systems has provided important
support that the above gene products function as channels. The expression cells include two insect cell lines
(Spodoptera Sf9 cells and Drosophila S2 cells), mammalian cell lines (HEK293T and CHO cells), and Xenopus
oocytes (52, 69, 90, 129, 143, 174, 209). Schilling and
colleagues (190) first reported heterologous expression of
TRP. These investigators found that expression of trp
cDNA in Sf9 cells led to the appearance of a novel conductance that could be activated by thapsigargin. However, there is only limited similarity between the properties of the heterologously expressed TRP-dependent
conductance and the light-induced conductance of Drosophila. The TRP-dependent currents in the Sf9 cells
markedly differed from the light-activated current of Drosophila in several main aspects. The I-V relationship is
approximately linear in Sf9 cells expressing TRP, while
showing inward and outward rectification in Drosophila.
When the holding voltage is stepped, there is instantaneous current with no time-dependent kinetics in the cell
expressing TRP unlike the LIC. The TRP-dependent current of Drosophila shows positive and negative feedback
effects of Ca2⫹ on the LIC, which are completely absent in
the Sf9 cells. The TRP-dependent conductance in Sf9 cells
has low permeability to Ba2⫹ and is not blocked by Mg2⫹
in concentrations that have a strong blocking effect in
Drosophila (158). Xu et al. (209) functionally expressed
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TRP and found a novel conductance that could be activated by thapsigargin in HEK293T cells. However, the
properties of this current like those of the Sf9 cells
showed several quantitative discrepancies from the in situ
current (e.g., less permeable to Ca2⫹, less sensitive to
block by either La3⫹ or Mg2⫹ , and linear I-V curve without rectification).
Expression of TRP did show appearance of the TRP
protein in the heterologous system in the few cases where
it was investigated (52, 174, 209). Nevertheless, in several
unpublished studies no effect of transient TRP expression
was found in either HEK293 cells (Minke, unpublished
observations) or in Drosophila S2 cells. Expression of
TRP in Xenopus oocytes has provided inconsistent data,
whereas one study showed enhancement of the endogenous Ca2⫹-activated Cl⫺ current in TRP expressing oocytes (143), and another study failed to see any significant
effect when TRP was expressed alone without TRPL (52).
Taken together, it seems doubtful whether the heterologously expressed TRP reaches the surface membrane and
functions as a channel; all reported conductance can be
explained by enhancement of endogenous activity of
channels of the host cells.
Indeed, the GTP binding protein Rab6, which is required for vesicular trafficking within the Golgi and postGolgi compartments, turned out to be essential for TRP
expression in normal amounts in the native tissue (168).
Thus overexpression of mutant Rab6 protein shows reduction in both rhodopsin (Rh1) and TRP proteins. Rab6
is not the only protein required for TRP expression. Pak
and colleagues (93) found a novel protein called INAF,
which appears to be required for normal level of TRP
expression and perhaps also for TRP function in the fly
eye. Accordingly, a null mutant of INAF specifically reduced the amount of TRP to a very low level and seems to
specifically affect the TRP activity (93).
The situation is very different for heterologous expression of TRPL, in which the appearance of a nonselective cation conductance has been reported after expression in Sf9 cells (69, 77), S2 cells (68), HEK293 cells (209),
CHO cells (69), and Xenopus oocytes (52, 91). In several
cases, a constitutive activity of the TRPL channels was
largely enhanced by coexpressed receptors, which activate endogenous G protein-coupled PI pathways. A common feature of the TRPL expression studies is a constitutive spontaneous activity, which increases with time
during whole cell recording, although Minke and colleagues (52) and Montell and colleagues (209) reported
that this was prevented when coexpressed with a large
quantity of TRP. The channel properties of TRPL when
expressed in Drosophila S2 cells were compared with the
TRPL-dependent light-sensitive conductance as determined in the native cells of trp mutants during whole cell
recordings (158). A variety of properties including singlechannel conductance and open times, ionic selectivity for
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monovalent and divalent ions, block by Mg2⫹, and I-V
relationship were found to be indistinguishable between
native and heterologously expressed TRPL. The properties of TRPL channels expressed in other expression systems appear similar (90, 130). Thus, in contrast to the
apparent failure to heterologously express functional
TRP, heterologous expression of TRPL appears to form a
conductance similar to that found in the native tissue,
except that some factor is required to prevent constitutive
activity, which does not occur under normal conditions in
vivo. The recent cloning of TRP␥ indeed solved this latter
issue (207). Thus heterologous expression of a TRP␥TRPL construct in 293T cells displayed an agonist (ATP)activated current, which was not observed in control
cells. The TRP␥-TRPL expressing cells do not show constitutive activity, and the I-V relationships show the outward rectification typical for the TRPL channels in situ,
strongly suggesting that TRP␥-TRPL forms a heteromultimer (207).
B. Mechanism of Activation of TRPL
1. Receptor-mediated activation
Coexpression of TRPL and the M5 muscarinic receptor in Sf9 cells using the baculovirus expression system
reveals an increase in basal Ba2⫹ influx typical of the
constitutive activity of TRPL channels in these cells. Addition of carbachol to these cells produced a large increase in cellular Ca2⫹, which could be blocked by atropine and by gadolinium (Gd3⫹, 0.1–1.0 mM) but was
insensitive to La3⫹ (10 ␮M). Depletion of the Ca2⫹ stores
by thapsigargin had no effect, suggesting that TRPL is a
store-independent channel (76, 77). Similar results were
also obtained by coexpression of TRPL with other hormone receptors in Sf9 cells such as the histamine, thrombin, and thromboxane A2 receptors (129, 130). The typical
outwardly rectifying TRPL current was observed after
hormonal stimulations following the TRPL expression in
these cells.
2. Activation by InsP3
TRPL channels expressed in Sf9 cells were activated
by inclusion of InsP3 in the recording pipette (43). Although heparin blocked activation by InsP3, it could only
partially counteract activation by a coexpressed bradykinin receptor, suggesting an additional alternative route of
excitation. The authors concluded that the effect of InsP3
was not mediated via a rise in Ca2⫹, since it was unaffected by inclusion of 1 mM EGTA in the pipette. The
authors suggested that InsP3 might directly gate TRPL via
conformational coupling (see below). Zimmer et al. (219)
also found an excitatory effect of InsP3 but reported that
since this was abolished by prior depletion of stores with
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thapsigargin it was an indirect action via a rise in Ca2⫹.
Schultz and colleagues (129) using Sf9 cells did not find
any effect of InsP3 but reported that activated Gq/11 subunits could activate TRPL channels either when overexpressed or, in a few cases, when applied to inside-out
patches. Although they initially suggested that TRPL
channels were directly gated by the G protein in similar
manner to some K⫹ channels, more recently they concluded that their findings are best explained by G proteinmediated activation of PLC (70).
3. Effects of Ca2⫹-CaM
TRPL was initially isolated on the basis that the
expressed protein binds CaM via two putative CaM binding sites (147). Subsequently, the two CaM binding sites
(designated CBS1 and CBS2) have been more accurately
identified and studied in vitro and in vivo (32, 164). CBS1
binds CaM in a Ca2⫹-dependent fashion (300 –500 nM
Ca2⫹). In contrast, CBS2 binds CaM in the absence of
Ca2⫹ with dissociation occurring at levels above 5 ␮M.
The authors suggest that dissociation of CaM from CBS2
and Ca2⫹-dependent CaM binding to CBS1 are the mechanism of TRPL activation (198). Similar inhibition of
TRPC channels by CaM has been recently demonstrated
(182). In general, similar dependence of TRPL activation
on CaM binding was obtained by expression of TRPL in
CHO cells. However, these experiments showed Ca2⫹
dependence of both CaM binding sites CBS1 and CBS2,
although they differ in their affinity to CaM with Ki values
of 12.3 nM and 1.7 nM for CBS1 and CBS2, respectively.
Both sites bind CaM only in the presence of Ca2⫹, and the
CaM antagonist calmidazolium inhibits the Ca2⫹-dependent activation of TRPL current, suggesting modulation of
TRP currents by cellular Ca2⫹ via CaM (186). Lan et al.
(91) reported that heterologous expression of TRPL in
Xenopus oocytes leads to an elevated free Ca2⫹, which
was increased by low concentrations of CaM and inhibited at high. Zimmer et al. (219) reported that TRPL
activity in CHO cells was enhanced by application of Ca2⫹
with an EC50 of 500 nM and that this could be reduced by
the CaM antagonist calmidazolium.
Schultz and colleagues (130) have obtained different
results in Sf9 cells that express TRPL channels together
with the histamine receptor. The histamine-induced
TRPL-dependent currents revealed reversible inhibition
by Ca2⫹ (IC50 of 2.3 ␮M). Consistent with these observations, inside-out patches revealed TRPL channel activity
that was reversibly inhibited by Ca2⫹ (10 –50 ␮M) in the
cytosolic side. Interestingly, the CaM inhibitor calmidiazolium had no inhibitory effect on channel activity, thus
suggesting that CaM was not involved in the inhibition
(130). Consistent with inhibition of the TRPL channels by
Ca2⫹ in Sf9 cells, experiments in the native tissue of
Drosophila have shown pronounced inhibition of the LIC
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in the trp mutant, which has only TRPL channels. TRPL
channels appear to be negatively regulated by Ca2⫹, since
in the presence of extracellular Ca2⫹ the response to light
is reduced by approximately threefold (158). Similarly
caged Ca2⫹ released during the rising phase of the light
response inhibited the light response in trp mutants (59).
To examine if CaM involves TRPL inhibition by Ca2⫹,
Zuker and colleagues (164) generated transgenic flies in
which TRP channels were absent and the native TRPL
channels were replaced with constructs with CBS1 or
CBS2 deleted. Activation by light in these flies was essentially unaltered, whereas there were major defects in
response termination.
Hardie and Raghu (66) examined the effect of Ca2⫹ in
S2 cells coexpressing the TRPL channels with a muscarinic receptor. Releasing caged Ca2⫹ resulted in a pronounced facilitation of the spontaneous TRPL activity.
Releasing caged InsP3 released Ca2⫹ from internal stores
but had at most a small effect on TRPL activity and none
at all when BAPTA was included in the pipette solution. In
contrast, excitation of TRPL via the coexpressed muscarinic receptor resulted in a rapid excitation of TRPL channels, which was not blocked by prior depletion of stores
by InsP3 or thapsigargin or by inclusion of BAPTA in the
pipette. The results suggest that a process requiring G
protein and PLC, but not InsP3, underlies activation of
heterologously expressed TRPL channels while channel
activity can be modulated by Ca2⫹-CaM. Taken together,
the bulk of data suggest that in heterologous systems
TRPL is constitutively open and that the channel can be
negatively and positively regulated by Ca2⫹-CaM.
4. Activation by PUFAs and DAG
PUFAs have been shown to activate recombinant TRPL
expressed in S2 cells (35). This preparation has an advantage over the photoreceptor cells, because it allows measurements of single-channel activity. Importantly, PUFAs
elicited TRPL channel activity in excised patches, suggesting
direct activation of the channel by PUFAs. The fact that
activation of TRPL by PUFAs was observed in the photoreceptors of the null norpA mutant, which lacks the photoreceptor specific PLC (17) and does not respond to light, was
used as a strong argument in favor of direct activation of the
channels without a need for PLC or other upstream proteins.
Schilling and colleagues (47), who measured the effects of
DAG analogs and PUFAs on TRPL expressed in Sf9 cells,
reported apparently different results. In contrast to the lack
of any effect of DAG or DAG analogs on Drosophila photoreceptors, the DAG analog 1-stearoyl-2-arachidonyl-glycerol
(SAG) (but not OAG) induced Ca2⫹ influx and elicited single-channel activity in TRPL-expressing Sf9 cells. Consistent
with the data reported by Hardie and colleagues (35),
PUFAs activated both Ca2⫹ influx in intact Sf9 cells expressing TRPL and elicited single-channel activity in excised
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patches. However, in contrast to previous claims that PLC is
not directly involved in single-channel activation of excised
patches, single-channel activation was elicited by application of three different PLCs. Furthermore, activation of
TRPL channels in Sf9 cells by PUFAs was markedly attenuated after inhibition of PLC by the U73122 antagonist (but
not by the control compound U73343) (47). The authors
concluded that both the hydrolysis of PIP2 and the generation of DAG are required to rapidly activate TRPL channels
after receptor stimulation and that the effect of PUFAs is
mediated at least in part via PLC. The reports are, therefore,
contradictory, supporting either direct or PLC-mediated activation of TRPL by PUFAs. These results may arise from
the multiple effects that PUFAs have while the activation
of photoreceptors may arise indirectly from the effects of
PUFAs as mitochondria uncouplers causing metabolic
stress (4).
5. Store-operated Ca2⫹ entry
Almost all studies have found that TRPL is not activated by store depletion. One study has reported activation of TRPL by thapsigargin (211), but the possibility that
the regulation was due to the associated increase in Ca2⫹
was not rigorously excluded.
In contrast, as mentioned above, heterologously expressed TRP is presumably activated by store depletion.
6. Coexpression of TRP and TRPL channels
Coexpressing Drosophila TRP and TRPL in Xenopus
oocytes in cRNA ratios of 10:1 reveals a novel inward
current showing inward and outward rectification that
was activated by Ca2⫹ store depletion. This current was
neither observed in native oocytes nor in oocytes expressing either TRP or TRPL alone by the same amounts of
cRNA, implicating a cooperative action of TRP and TRPL
in the depletion-activated current (52). Heterologous coexpression of TRP and TRPL in 293T cells produced an
outwardly rectifying novel current, which could be activated by store depletion. Immunoprecipitation studies
revealed physical interaction between TRP and TRPL as
found in the native Drosophila retina (209). Evidence that
TRP and TRPL form heteromultimeric channels was obtained by suppression of the TRP-dependent current by
dominant negative form of TRP, and vice versa. It was
also shown that the NH2 terminal and transmembrane
domains contributed to subunit assembly. Because each
of the channels by itself can be opened by light, the
relevance of coexpression of TRP and TRPL to the native
system is still not clear.
C. Heterologous Expression
of “TRP-Homolog” Channels
Functional analysis of the TRPC group has been confined mainly to heterologous systems (but see TRPC2,
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TRPC3, TRPC4, and TRPC6). Not surprisingly, the activation mechanism and biophysical properties remain unclear and controversial. Thus conflicting results have
been reported for the same channel for reasons that remain unclear. A possible explanation may be the use of
different expression system and expression levels of the
TRPC homolog and possible interaction with endogenous
proteins of the expressing cells.
TRPC1, TRPC4, and TRPC5 homologs seem to form
one functional group. However, several studies suggest
that they are activated by store depletion and therefore
consider them as SOCs (15), while other studies show
store-independent activation (70).
1. TRPC1
The TRPC1 isoform of human was transiently coexpressed with the M5 muscarinic receptor in COS cells.
Activation of the endogenous inositol lipid signaling of
these cells through the M5 receptor was obtained by
application of carbachol (CCh). The depletion of the Ca2⫹
stores leads to the opening of Ca2⫹-permeable surface
membrane channels. This entry of Ca2⫹ is the manifestation of the endogenous SOC in control cells. In the cotransfected COS cells with TRPC1 and M5 receptor, a
significantly larger Ca2⫹ influx relative to control cells
was observed (218). Additional studies on a spliced variant of TRPC1 (221) expressed in CHO cells investigated
some biophysical properties of the expressed TRPC1 homolog. Transient expression of TRPC1A induced a constitutive inward cationic current in Ca2⫹-free medium,
which was significantly larger than the control. Biophysical analysis of this current revealed a nonselective cation
current with similar permeability for Na⫹, Ca2⫹, or Cs⫹.
The current was enhanced in stably expressing cells perfused with Ca2⫹ buffer after application of InsP3 or thapsigargin, which presumably depletes the Ca2⫹ stores and
was inhibited by the lanthanide Gd3⫹. Based on these
results, TRPC1A was characterized as a SOC channel.
Expression of the human TRPC1 isoform in the insect Sf9 cells using the baculovirus expression system
(173) provided a different conclusion. Expression of
TRPC1 caused an increase in basal cytosolic free Ca2⫹ as
well as Ba2⫹ concentration as a function of posttransfection time. The latter indicated activation of an influx
pathway. Whole cell recordings indicate that the inward
current was nonselective with respect to Ca2⫹, Ba2⫹, or
Na⫹ and was blocked by La3⫹. Both Ba2⫹ influx and the
cationic current were unaffected by thapsigargin, InsP3,
or internal Ca2⫹ buffers. Thus, according to this study,
TRPC1 is a nonselective cationic channel, which is store
independent. The authors propose (without further evidence) that it requires additional subunits to become a
SOC. Similar failure to activate human TRPC1 expressed
in HEK cells by store depletion was observed by GrosPhysiol Rev • VOL
chner and colleagues (97). Interestingly, TRPC1 could be
activated (apparently directly and not via PKC) by the
DAG analog OAG (Table 2). Previous studies by Schultz
and colleagues (74, 100) first showed that DAG and its
analogs activate TRPC3 and TRPC6 channels, but could
not activate TRPC1 under the same experimental condition. The difference in the results of the two groups can
be explained by the suppression of the OAG-induced response in TRPC1 by Ca2⫹, which was present in the
external medium of the initial experiments (97).
Expression of TRPC1 in human mandibular gland
cell line (HSG) resulted in a three- to fivefold increase in
SOC activity measured by thapsigargin-stimulated Ca2⫹
influx, while similar transfection with TRPC3 did not
show enhancement of SOC activity. Interestingly, transfection of HSG cells with antisense TRPC␣1 cDNA significantly attenuated the endogenous SOC activity, while
changes in SOC were associated with corresponding changes in the levels of TRPC1 in the plasma membrane (98).
2. TRP4 and TRP5
The bovine TRPC4 homolog was studied electrophysiologically after overexpression in HEK cells (144).
Depletion of the Ca2⫹ stores in low Ca2⫹-buffered cells by
GTP␥S, InsP3, or thapsigargin induced a large inwardly
rectifying current that was not observed in the control
cells. The relative permeability of the expressed channel
to cations was PCs:PNa:PCa:PBa ⫽ 1:1.1:7.7:12.3. Inability to
resolve single-channel activity was interpreted as an indication of very low single-channel conductance. Thus, according to this study, TRPC4 is a SOC with high permeability to divalent ions (144, 197). Results similar to those
obtained in the TRPC4 isoforms were also obtained after
expression of TRPC5 in HEK cells (144). The authors
concluded that these channels have properties reminiscent of CRAC channels.
Two other studies on mouse isoforms of TRPC4 and
TRPC5 (161) and on TRPC5 (131) expressed in HEK cells
reported conflicting results. In TRPC5-expressing cells,
activation of the endogenous purinergic receptor by application of ATP induced a large transient increase in
cellular Ca2⫹ and inward current only in the presence of
external Ca2⫹ (131). Omission of Ca2⫹ from the recording
pipette abolished the ATP-induced current, suggesting
that increase in cellular Ca2⫹ is required for TRPC5 activation. The permeability ratio of the current was PCa:PNa:
PCs ⫽ 14.3:1.5:1.0, showing Ca2⫹ selectivity. La3⫹ and
Gd3⫹ and SK&F-6365, an inhibitor of nonselective cation
and some Cl⫺ channels, inhibited the Ca2⫹ transients.
Single-channel activity during patch-clamp recordings at
low external Ca2⫹ show single-channel conductance of
47.6 pS. Ca2⫹ store depletion by thapsigargin had no
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effect larger than the control, thus indicating that this
channel is not SOC (see Table 2).
Detailed functional studies of mouse TRPC4 and
TRPC5 expressed in HEK cells have shown that these two
channel types are not only structurally related but also
display similarities in their mechanism of regulation and
biophysical properties (161). Contrary to the experiments
on bovine and rat orthologs, activation of the mouse
isoforms was independent of the filling state of the internal Ca2⫹ stores. Importantly, these channels did not respond to InsP3, in contrast to TRPC3 channels (see below) (161). Activation via a G protein-coupled PLC was
demonstrated by activation with CCh or histamine (after
coexpression with the histamine receptor) by application
of GTP␥S, which activated single channels, and by inhibition of the agonist-induced current by the PLC inhibitor
U73122. In contrast to the proposed similarity to CRAC
channels, the studies on mouse TRPC4 and TRPC5 reveal
large single-channel conductance of 41 and 63 pS in the
presence of Mg2⫹ and similar conductance to both monovalent and divalent cations. Similarly, single-channel conductance of 47.6 pS was measured in another study of
heterologously expressed TRPC5 in HEK293 cells (212).
The conductance and the inward and outward rectification of the I-V relation were markedly dependent on
Mg2⫹. In the absence of Mg2⫹, the rectification was lost,
reminiscent of the effect of Mg2⫹ on Drosophila TRP.
Surprisingly, La3⫹ at concentrations up to 1 mM facilitated TRPC4- and TRPC5-dependent currents (161). The
reasons for the marked differences in regulatory and biophysical properties between species and studies in different laboratories remain unknown. Interestingly, the rat
ortholog of TRPC4 lacks the entire putative S2 region. A
rat ortholog expressed in Xenopus oocytes was functionally characterized as SOC (185). It is still not clear if the
missing S2 segment is responsible for this phenomenon.
Thus the bulk of the most recent studies on TRPC4 and
TRPC5 in heterologous systems suggest that these channels are receptor-activated and store-independent nonspecific cation channels, in contrast to studies in native
endothelial cells (49). Importantly, DAG or DAG analogs
do not activate these channels.
3. TRPC2
Only a few studies of the heterologously expressed
mouse TRPC2 channel are available. Nevertheless, the
specific expression of rat TRPC2 isoform to the VNO
organ of rat and sperm of mice have already provided a
very significant clue as to the functions of TRPC2. Accordingly, TRPC2 may be the target channel of a G protein-coupled receptor that mediates detection of pheromones.
Heterologous expression of mouse TRPC2 in COS
cells expressing the muscarinic receptor M6 revealed a
Physiol Rev • VOL
typical SOC. The expressed TRPC2 channels were tested
by the same paradigm described above for TRPC1 and
show a significant enhancement of Ca2⫹ influx after store
depletion by thapsigargin (192). These findings seemingly
contradict the expectation of Liman et al. (96) that expect
TRPC2 to be store-independent channels, since the VNO
organ where TRPC2 is localized is composed of microvilli
and has no Ca2⫹ stores.
4. TRPC3, TRPC6, and TRPC7
The members of this group of TRP channels are
closely related in molecular structure and function. The
human TRPC3 has been extensively characterized by a
number of investigators. It was stably expressed in HEK
cells (217) and bovine pulmonary artery endothelial cells
(85) and transiently expressed in CHO cells (220). The
heterologously expressed TRPC3 channel has been studied by various techniques including whole cell recordings
of macroscopic current, excised patch-clamp recordings
of single channels, as well as fluorescence Ca2⫹ measurements using Ca2⫹ indicators. Essentially all investigators
reported similar properties of the TRPC3 channel (193).
TRPC3 forms a nonselective cation channel that is activated by a G protein-coupled receptor, G protein, and
PLC. La3⫹, Gd3⫹, and SK&F-96365 are potent blockers of
the channel that has a single-channel conductance of
60 – 66 pS. The I-V relation shows inward and outward
rectification. Most studies also reported that TRPC3 is a
store-independent channel and that thapsigargin has no
effect. Nevertheless, several investigators have reported
some enhancement of the receptor-activated current in
TRPC3-expressing cells by thapsigargin treatment (89,
218). A different line of evidence supporting the notion
that TRPC3 function as SOC channels was provided by
stable transfection of HEK293 cells with a fragment of
human TRPC3 in antisense orientation. In the antisense
transfected cells, activity of SOC channels monitored by
Ba2⫹ influx after store depletion with thapsigargin was
reduced by 32% compared with cells expressing sense
TRPC3 (205).
Interestingly, in addition to activation by several agonists via a receptor and in few cases by store depletion,
TRPC3 could be activated by DAG or its analogs (74, 100).
Different studies have reported conflicting data with regard to the effects of InsP3. Although Hoffman et al. (74)
reported that application of InsP3 has no effect on human
TRPC3 or TRPC6 expressed in CHO cells, other studies
on human TRPC3 expressed in either bovine pulmonary
artery endothelial cells or in HEK293 cells show clear
activation by InsP3 (89, 145). DAG and its analogs seem to
activate the TRPC channels directly and not via the
InsP3R because application of the InsP3R antagonist
2-APB does not affect the activation by DAG analogs
(100).
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The most interesting studies on TRPC3, which open
new avenues toward understanding TRP gating, have described the interactions between TRPC3 and the InsP3R
(88, 100). The study by Muallem and colleagues (88) has
provided for the first time experimental evidence supporting the conformational coupling hypothesis of inositidemediated Ca2⫹ entry. This hypothesis proposes that information is transferred through direct coupling between the
InsP3R in the ER and a surface membrane Ca2⫹-permeable channel to control Ca2⫹ entry (13, 82). Drosophila
TRP has been previously proposed to constitute the surface membrane channel, which interacts with the InsP3R
(109), but no experimental evidence has been provided to
support this hypothesis. Muallem and colleagues (88),
using stably expressed TRPC3 in HEK293 cells, have
shown that excised membrane patches containing TRPC3
channels but no InsP3R because of extreme washing can
be activated by addition of vesicles containing native or
recombinant InsP3R bound to its ligand. The physical
interaction of TRP and InsP3R was also demonstrated
by coimmunoprecipitation and glutathione-S-transferase
(GST)-pulldown experiments and identifies two regions
of InsP3R NH2 terminal that interact with one region of
the COOH terminal of TRPC3. The interacting peptide
regions were overexpressed in HEK293 cells and showed
modulation of endogenous Ca2⫹ entry (19). Activation of
the InsP3R could be blocked by two known inhibitors,
heparin and xestospongin (88). Interestingly, overexpression of the TRPC3 channel induced increased expression
of the InsP3R (19, 88). The conformational coupling hypothesis was supported by experiments of Gill and colleagues (100), who compare the dependence of divalent
ions entry on the InsP3R in native SOC system and the
TRPC3-expressing HEK293 cells. These investigators
show that that displacement of actin by the phosphatase
inhibitor calyculin in HEK293 cells inhibits activation of
both native SOC by store depletion and activation of
TRPC3 channels by an agonist, presumably by preventing
interaction between the InsP3R and TRPC3. Furthermore,
inhibition of the InsP3R by xestospongin and by 2-APB
blocked both SOC and TRPC3 activities (100). The association with InsP3R has also been reported for TRPC1 and
TRPC6 by coimmunoprecipitation (19, 99).
A recent study has shown that interaction of the
TRPC channel and the InsP3R is not confined to a few but
to all TRPC channels (182). Interestingly, the InsP3R binding domain also interacts with CaM in a Ca2⫹-dependent
manner with affinities ranging from 10 nM for TRPC2 to
290 nM for TRPC6. In the presence of Ca2⫹, the TRPCInsP3R interaction is inhibited by CaM. In addition, other
binding sites for CaM and InsP3Rs are present in the ␣but not the ␤-isoform of TRPC4, while TRPC4 is activated
strongly by an antagonist of CaM and high (50 ␮M) concentration of the TRPC binding peptide of the InsP3R in
Physiol Rev • VOL
inside-out membrane patches. Thus both the InsP3R and
CaM are involved in the control of TRPC gating (182).
A detailed study on TRP6 heterologously expressed
in HEK293 cells has been described above (81). Although
TRPC7 has not been studied as extensively as TRPC3, the
limited studies show that essentially it has very similar
functional properties to those of TRPC3 (20). DAG also
activates TRPC7 (131).
5. Coexpression of TRPC1 and TRPC3
The ability of TRPC homologs to coassemble has
been established by studies demonstrating that heterologously expressed TRPC1 and TRPC3 can be coimmunoprecipitated (209). Coexpression of human TRPC1 and
TRPC3 in HEK cells resulted in formation of channel
activity with properties different from those of cells expressing either TRPC1 or TRPC3 alone, which were studied under the same experimental conditions (97). Accordingly, in coexpressing cells, receptor activation by
carbachol suppressed Ca2⫹ entry to a level smaller than
that observed in TRPC3-expressing cells. Furthermore,
OAG-induced Sr2⫹ entry was reduced relative to that
measured in cells expressing TRPC3. Importantly, coexpression of TRPC1 together with TRPC3 generated constitutively active conductance that was strongly inhibited
by extracellular Ca2⫹. These observations suggest that
coassembly of TRPC1 and TRPC3 resulted in formation of
heteromeric channel with properties different from the
homomultimeric channels with regard to modulation by
Ca2⫹ and possibly other characteristics (97). Additional
evidence for functional consequences of coexpressing
TRPC1 and TRPC3 came from experiments in which HEK
cells, which stably expressed human TRPC3, were transiently transfected with human TRPC1 segment in antisense orientation. In these cells, SOC activity as measured
by Ba2⫹ influx after store depletion was suppressed by
55% relative to cells coexpressing sense TRPC3 plus sense
TRPC1 (205).
6. Coexpression of TRPC1 and TRPC5
The ability of TRPC homologs to coassemble has
been also demonstrated in the mouse brain tissue where
TRPC1 and TRPC5 were coimmunoprecipitated (178). Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted
in novel nonselective cation channel with a voltage dependence similar to NMDA receptor channel (but lacking
the Mg2⫹ dependence), but unlike that of any reported
TRPC channel including TRPC1 or TRPC5 alone. TRPC1/
TRPC5 heteromultimers were activated by Gq-coupled
receptors but not by depletion of intracellular Ca2⫹
stores. Interestingly, single-channel conductance of
TRPC1/TRPC5 heteromultimere was approximately eightfold smaller than that of TRPC5 (slope conductance of 5
pS relative to 38 pS) (178).
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D. Heterologous Expression
of “TRP-Related” Channels
vated currents. Store depletion by thapsigargin had no
effect (31).
1. VR1 and VRL-1
3. Coassembly of polycystin-1 and polycystin-2
produces a nonselective cation channel
Heterologous expression of the vanilloid receptor 1
(VR1), the heat-gated ion channel, in HEK293 cells and in
Xenopus oocytes has indicated that VR1 is a Ca2⫹-permeable, nonselective cation channel. Measurements of I-V
relations of a capsaisin-induced current show outward
rectification with reversal potential of ⬃0 and no significant preference for monovalent cations with PK:PNa
⫽ 0.94 and PCs:PNa ⫽ 0.85 (27). The capsaisin-induced
current exhibits a notable preference for divalent cations
with PCa:PNa ⫽ 9.6 and PMg:PNa ⫽ 4.99 while the permeability sequence is Ca2⫹ ⬎ Mg2⫹ ⬎ Na⫹ (see Table 2). The
capsaicin-induced inward current is rapidly (within ⬃200
ms) inactivated in the presence of external Ca2⫹ while the
inactivation is abolished in Ca2⫹-free medium. Capsaicininduced current was also examined at the single-channel
level using excised patches and showed unitary conductance of 76.7 pS at positive potentials and 35.4 pS at
negative potentials with Na⫹ as a sole charge carrier (27).
The capsaicin activates the channels when applied on
either side of the membrane, most likely because of the
hydrophobic nature of the compound. Activation of heterologously expressed VR1 by the endogenous cannabinoid anandamide revealed biophysical properties similar
to those measured after activation by capsaicin (222).
VRL-1, which is activated by high temperature but
not by capsaicin and pH, has biophysical properties somewhat different from those of VR1. The I-V relation shows
both inward and outward rectifications and reduced selectivity to divalent cations, PCa:PNa ⫽ 2.94 and PMg:PNa
⫽ 2.40 (26) (Table 2). Similar properties of I-V relations
were obtained in CHO cells expressing the VRL-1 homolog GRC, which has ⬃80% amino acids sequence identity with VRL-1 (86).
2. Polycystin-L is a Ca2⫹-regulated cation channel
Polycystin-L (PCL) shares significant homology to
the polycystin-2 channel (50% identity and 71% similarity).
Heterologous expression of PCL in Xenopus oocytes has
provided strong evidence that this protein is a calciummodulated nonselective cation channel that is permeable
to Na⫹, K⫹, and Ca2⫹; PNa:PK:PRb:PLi ⫽ 1:0.98:0.97:0.87.
The channel is about five times more permeable to Ca2⫹
than to Na⫹ at ⫺50 mV. Single-channel recordings
showed that the PCL channels are activated directly by
Ca2⫹ and that the channel conductance is 120 –135 pS at
80 mM Ba2⫹, Sr2⫹, or Ca2⫹, with relatively long mean
open times. Divalent ions were more permeable than
monovalent except for Mg2⫹ that efficiently blocked the
channel at negative holding potentials. A relatively large
concentration of La3⫹ (0.1 mM) inhibited the Ca2⫹-actiPhysiol Rev • VOL
Attempts at demonstrating channel activity similar to
PCL by in heterologous expression of polycystin-2 have
failed. However, the indistinguishable phenotype resulting from mutation in either PKD1 or PKD2 genes suggested that their gene products interact to form a functional unit. Indeed, the COOH termini of polycystin-1 and
polycystin-2 interact through their coiled-coil domains.
Coexpression of PKD1 and PKD2 in CHO cells generated
time-dependent outwardly rectifying current that was
⬃20-fold larger than in mock transfected cells (54). The
expressed channel is a nonselective cation channel (PNa:
PCs:PNMDG ⫽ 1.1:1.0:0.6). The channel is about sixfold
more permeable to Ca2⫹ than to monovalent cations.
Extracellular Ca2⫹ also inhibited the inward current as
found in other TRP channels.
4. The Ca2⫹ transport epithelial channel proteins
ECaC, CaT1, CaT2, and CaT-L
Both putative Ca2⫹ transport channels have been
initially heterologously expressed in Xenopus oocytes,
and ECaC was also expressed in HEK293 cells. Functional
data on the ECaC channel includes 45Ca2⫹ uptake, electrophysiology, and fluorimetric measurements. 45Ca2⫹ uptake revealed linear uptake over 2 h, showing increased
influx when extracellular Ca2⫹ was increased up to ⬃1
mM. The Ca2⫹ influx was inhibited by La3⫹ ⬎ Cd2⫹
⬎ Mn2⫹, whereas Mg2⫹ had no effect. Permeability to
Na⫹ was negligible in double labeling experiments (in
which both Ca2⫹ and Na⫹ were labeled), whereas Ba2⫹ or
Sr2⫹ did not affected the Ca2⫹ influx. Acidification (to pH
5.9) had a significant inhibitory effect on Ca2⫹ influx
reminiscent of the marked effect of acidosis on calciuresis in vivo. These experiments show that apical Ca2⫹
influx, possibly via ECaC, which is highly selective for
Ca2⫹, is the rate-limiting step of transcellular Ca2⫹ transport (73). Patch-clamp whole cell recordings and fluorimetric measurements of Ca2⫹ influx in heterologously
expressed ECaC in HEK293 cells showed a distinctive
Ca2⫹ permeability and constitutive activity of the ECaC
channels with PCa:PNa ⫽ 107 and divalent cation selectivity profile of Ca2⫹ ⬎ Mn2⫹ ⬎ Ba2⫹ ⫽ Sr2⫹. The I-V
relationship showed pronounced inward rectification
(195).
A close relative of ECaC designated CaT2 has been
recently cloned (137). CaT2 has 84.2 and 73.4% amino acid
identity to ECaC and CaT1, respectively. However, unlike
ECaC, CaT2 is kidney specific in rat. Heterologous expression of CaT2 in Xenopus oocytes produced Ca2⫹-activated novel inward current that was also induced by Sr2⫹
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and Ba2⫹ but not Mg2⫹, whereas Cd2⫹ was a potent
inhibitor that also permeated the channel. Antagonists of
the L-type voltage-activated Ca2⫹ channels have only little
effect even at high concentrations. Thus CaT2 like ECaC
seems to participate in Ca2⫹ entry into cells of the distal
convoluted tube and connecting segment of the nephron
(137).
45
Ca2⫹ uptake experiments in Xenopus oocytes expressing CaT1 showed properties similar to those described for ECaC channel (138). Electrophysiological
studies in oocytes expressing CaT1 channels using twoelectrode voltage clamp revealed a large inward current
that was initially activated by extracellular application of
Ca2⫹ and then rapidly inactivated. The I-V relationship of
the current shows inward rectification. Intracellular injection of EGTA reduced the amplitude of the Ca2⫹-activated
current and abolished the inactivation phase. The CaT1expressed channels are permeable to Ca2⫹ but also to
Na⫹, K⫹, and Rb⫹ in contrast to the ECaC channel. La3⫹,
as well as inorganic ions, which block voltage-gated Ca2⫹
channels (e.g., Ni2⫹, Co2⫹, Cd2⫹), also block the inward
current as well as Ca2⫹ influx in CaT1-expressing cells.
Like the ECaC channel, the CaT1 channel was also
blocked by acidic pH and did not produce current enhancement after store depletion (138).
Additional highly Ca2⫹-selective CaT1-related channel protein has been recently cloned and designated CaTL (202). Heterologous expression of CaT-L in HEK293
cells resulted in a large inwardly rectifying current, indicating that CaT-L forms a constitutively active ion channel. Detailed electrophysiological tests clearly established
that CaT-L has the hallmarks of Ca2⫹-selective channel
that can also be partially blocked by Ca2⫹. Measurements
of intracellular Ca2⫹ using a fluorescent Ca2⫹ indicator
revealed that the constitutive permeability to Ca2⫹ also
largely elevated cellular Ca2⫹, thus making CaT-L a suitable channel for Ca2⫹ transport in the endogenous tissue
(202).
5. CaT1 manifests pore properties similar to ICRAC
The molecular identity of ICRAC was an enigma for a
long time. Because ICRAC has been the classical SOC and
members of the TRP family have been activated by SOC
mechanism, members of the TRP family have been implicated as potential candidates of ICRAC. The Ca2⫹ transport
epithelial channel proteins are the only members of the
TRP family with the high selectivity to Ca2⫹ characteristic
of ICRAC. A recent study by Clapham and colleagues (215)
has provided evidence that CaT1 is a promising candidate
of ICRAC. Heterologous expression of CaT1 in CHO-K1
cells formed a large inwardly rectifying current when
intracellular Ca2⫹ was buffered to by 10 mM EGTA in the
pipette. The reversal potential of the current was ⫹49 mV
as expected from a highly selective Ca2⫹ channel. The
Physiol Rev • VOL
relative conductance of the expressed CaT1 was Ca2⫹ ⬎
Ba2⫹ ⬎ Sr2⫹ ⬎ Mn2⫹, with a permeability ratio PCa/
PNa ⫽ 130, similar to ICRAC. Extracellular Ca2⫹, but not
Ba2⫹ or Sr2⫹, inactivated the CaT1 current. CaT1 current
also revealed the anomalous mole fraction (the capacity
to hold two or more divalent cations in the pore simultaneously) a typical property of a highly Ca2⫹-selective
channels. One of the hallmarks of ICRAC is its increased
permeability to monovalent ions in divalent free medium.
This property was also found in CaT1-dependent current.
The single-channel conductance of ICRAC has been reported to increase from 0.5 to 40 pS in the absence of
divalent ions (134); the CaT1-dependent single-channel
conductance to Na⫹ in divalent free condition was ⬃42
pS. However, no data in physiological solution were reported. In addition to the above properties, CaT1-dependent current was also similar to ICRAC in the kinetics of
the whole cell current and block by La3⫹. Importantly, the
CaT1-dependent current was activated by Ca2⫹ store depletion obtained by application of InsP3 or thapsigargin in
the presence of 10 mM EGTA. Thus CaT1 seems to fulfill
several criteria associated with a channel producing ICRAC
(215).
6. OTRPC4 confers sensitivity
to extracellular osmolarity
Transient transfection of HEK293 or CHO or BBL (rat
basophilic leukemia) cells with OTRPC4 resulted in significant elevation of cellular Ca2⫹. Reduction in extracellular osmolarity resulted in a large and reversible increase
in cellular Ca2⫹. Conversely, reduction in extracellular
osmolarity led to reduction in cellular Ca2⫹. All other
members of this subfamily with similar structure (i.e.,
VR1, VRL-1, and ECaC) do not respond to similar changes
in osmolarity. The response to change in osmolarity was
rapid (within 30 s). A response to osmolarity is observed
at changes of 30 mosM (i.e., ⬃10% change), whereas Ca2⫹
concentration increases linearly with the increase in osmolarity in a range larger than 100 mosM. Store depletion
or activators of VR1, VRL-1, PLC, and mechanical stretch
had no effect, whereas La3⫹ (100 ␮M) and ruthenium red
(10 ␮M) blocked the Ca2⫹ influx. Electrophysiological
characterization of the OTRPC4-expressing cells revealed
constitutively active channels, with I-V relation showing
outward rectification with a zero reversal potential at
physiological solutions. Measurements of the ionic selectivity show that OTRPC4 is a nonselective cationic channel with PCa/PNa of ⬃6. Single-channel recordings confirmed the spontaneous openings of the channels and the
I-V relation of the macroscopic current, while the probability of openings depended on the osmolarity with relatively long delay (⬃70 s) after increase in osmolarity.
Regulation of cell volume has been shown to rely on Ca2⫹
influx, and OTRPC4 properties are consistent with these
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studies (177). Its high sensitivity to change in extracellular osmolarity and its specific localization to the region of
the kidney where cells are exposed to hypotonic solutions
made OTRPC4 a promising candidate for an osmoreceptor channel (177).
7. LTRPC7 (TRP-PLIK or TRPM7) is a MgATPregulated channel required for cell viability
TRPM7 was cloned independently by Scharenberg
and colleagues (123) and by Clapham and colleagues
(159) and was characterized as an ion channel with a
kinase domain. Its channel properties were characterized
by heterologous expression in CHO-K1 cells using patchclamp recordings. TRPM7-dependent current showed a
large outward rectification. The relative permeability to
cations was 1.1, 0.97, and 0.34 for K⫹, Na⫹, and Ca2⫹,
respectively, and therefore, it was considered as a nonselective cation channel. The TRPM7-dependent current
was weakly inhibited by La3⫹ and required a high (2 mM)
concentration for a significant block. The slope conductance of single-channel current was 105 pS (159). The
effects of mutations that alter the kinase activity on channel function revealed a marked decrease in current, suggesting that the kinase activity is required for channel
function. This conclusion was supported by experiments
in which current amplitudes in cells dialyzed with an
ATP-containing pipette (5 mM NaATP, 1 mM Mg2⫹) initially increased, followed by a slow decrease over several
minutes. Currents in cells that were dialyzed with 0 mM
ATP were significantly smaller (159).
TRPM7 was also designated LTRPC7 by Scharenberg
and colleagues (123). Targeted deletion of LTRPC7 in
DT-40 B cells was lethal, indicating that LTRPC7 has a
fundamental and nonredundant role in cellular physiology. Heterologous expression of LTRPC7 in HEK-293 cells
revealed a large outwardly rectifying current. Substitution
of monovalent ions with choline in the bath had no effect,
suggesting that the inward current was carried by divalent
ions when they are present in the medium. However, in
divalent free medium, the channel is highly permeable to
monovalent ions. Further studies showed that the pore of
the channel had a high affinity for and is permeant to both
Ca2⫹ and Mg2⫹, and the channel shows Mg2⫹-dependent
anomalous mole fraction behavior. At the same time outward currents are inhibited with increasing Mg2⫹ concentration. The permeability of the channel to Mg2⫹ is reminiscent of a similar property of the Drosophila TRP
channel (60, 64). Interestingly, MgATP at millimolar concentrations suppressed channel activity of LTRPC7. It
turned out that MgATP acted as a physiological regulator
of LTRPC7. This conclusion was supported by application
of hydrolysis-resistant ATP analog. Thus Mg-ATP␥S (but
not Na-ATP␥S) effectively and reversibly suppressed
LTRPC7-dependent current, suggesting that phosphorylaPhysiol Rev • VOL
tion of the channel in MgATP-dependent manner directly
regulates the channel activity. In this respect, LTRPC7
belongs to of Mg-nucleotide-regulated metal ion currents
(MagNuM). The author suggested that the effects of
NaATP on TRP-PLIK reported by Clapham and colleagues
(159) might be explained by a drop in cellular Mg2⫹ and
not due to regulation of channel function by the kinase
domain.
The studies on LTRPC7 indicate that this channel is
an intracellular ligand-gated ion channel whose activation
may be linked to cellular energy metabolism through its
sensitivity to cytosolic MgATP levels and that it effectively permeates both Ca2⫹ and Mg2⫹. It is thus striking
that similar properties characterize the Drosophila TRP
channel, which is also permeable (and partially blocked)
by Mg2⫹ and shows extreme sensitivity to metabolic
stress in vivo in a manner that depletion of ATP opens the
TRP channel in the dark while ATP suppresses channel
opening (4).
8. LTRPC2 (TRPM2) gating involves ADP-ribose
and ␤-NAD
Heterologous expression of LTRPC2 in HEK293 cells
forms a current that was induced by application of 100
␮M ADP-ribose (ADPR), showing a linear I-V relationship
and reversing at 0 mV, thus suggesting that this is a
nonselective cation channel activated by ADPR. No current was formed either by application of NAD⫹, cyclic
ADPR, ATP and other nucleotides, or by store depletion
(141). The channel was permeable to both monovalent
and divalent cations. Excised inside-out patch-clamp recordings revealed that the channel is most likely directly
opened by ADPR at concentration of 30 ␮M or higher
showing no desensitization. The slope conductance of the
single channel was 60 pS. On the basis of the heterologous
expression of LTRPC2, a similar analysis was performed
on native U937 cells, which express LTRPC2. Essentially
similar properties were found including activation by
ADPR (141). These studies show that ADPR can regulate
directly the permeability of one member of the TRP family
raising the possibility that the NUD9-H domain (see sect.
IIID) of the channel is involved in channel gating. A more
recent study has shown that both ADPR and ␤-NAD activate the LTRPC2 expressed in monocyte cell lines. Biophysical studies confirm LTRPC2 as Ca2⫹-permeable nonselective cation channel. As mentioned above, ATP
strongly suppressed NAD- and ADP-activated LTRPC2
channels (160).
9. Summary
The first TRPs to be studied in heterologous systems
are the original Drosophila TRP and TRPL, which are
activated in response to light stimuli. Although Drosophila phototransduction is very well characterized, heterol-
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ogous expression of TRP and TRPL had limited success. It
was virtually impossible to activate TRP, and TRPL was
constitutively active spontaneously. Relevance to the native process of phototransduction was only shown unambiguously in the properties of the TRPL channels. If researchers had to elucidate the role of TRP and TRPL using
heterologous expression experiments, we would still be
very far from that goal.
In the new era of genomic advancement, reverse
genetics has provided a valuable tool in finding new proteins and using them to elucidate biological processes.
However, in contrast to forward genetics where the starting point is a defective function, finding a gene and its
expression pattern is far away from finding a functional
role in a biological process. Heterologous expression of
TRPs has provided a myriad of effectors involving many
cellular processes. This variability could result from the
characteristics of the TRP channel family; however, some
of it most probably results from features of the channels
that are not directly related to their main role but become
prominent in the foreign cellular environment. The large
variability, the sensitivity to cellular conditions, and the
lack of a common activator or antagonist make it difficult
to study these channels in isolation from their natural
cellular environment. Only in conjunction with other
strategies will the heterologous expression experiments
be able to realize the advantage of allowing analysis of
isolated components.
VI. TRP LOCALIZES AND ANCHORS THE
TRANSDUCTION SIGNALING COMPLEX
OF DROSOPHILA
Recently, it has become apparent that TRP plays an
important role in organization of multiprotein signaling
complexes. An important step toward understanding Drosophila phototransduction has been the finding that some
of the key elements of the phototransduction cascade are
incorporated into supramolecular signaling complexes
via a scaffold protein, INAD. The INAD protein was discovered because of a Drosophila mutant designated inactivation but no afterpotential D (InaD). The first discovered inaD mutant, InaDP215, is a dominant mutant
isolated by Pak (133) on the basis of an abnormal prolonged depolarizing afterpotential (PDA). The InaD gene
was cloned and sequenced by Shieh and Zhu (169) and
found to bind TRP by overlay assays and coimmunoprecipitation in the original InaD mutant, InaDP215, and WT
(32). Studies in Calliphora by Paulsen and colleagues (79)
have shown that INAD binds not only TRP but also PLC
(NORPA) and eye-specific PKC (INAC). The interaction of
INAD with NORPA and INAC was confirmed in Drosophila (32, 187). It was further found that inaD is a scaffold
protein, which consists of five ⬃90-amino acid protein
Physiol Rev • VOL
interaction motifs called PDZ (PSD95, DLG, ZO1) domains (187). These domains are recognized as protein
modules that bind to a diversity of signaling, cell adhesion, and cytoskeletal proteins (5, 42, 162) by specific
binding to target sequences typically, although not always, in the final three residues of the COOH terminal.
The PDZ domains of INAD bind to the signaling molecules
as follows: PDZ1 and PDZ5 bind PLC (170, 187, 191), PDZ2
and PDZ4 bind eye-PKC (3, 187), and PDZ3 binds TRP (32,
169, 187). TRPL appears not to be a member of the
complex, since unlike PKC, NORPA, and TRP it remains
strictly localized to the microvilli in the inaD1 null mutant
(187). However, coimmunoprecipitation studies have indicated that TRP and TRPL interact directly when heterologously expressed in 293T cells as well as in Drosophila
head extracts (209). Further studies are required to establish if the observed TRP-TRPL interactions have clear
functional consequences in vivo.
Several studies by Montell and colleagues have
shown that in addition to PLC, PKC, and TRP other signaling molecules such as CaM, rhodopsin (32), and
NINAC (200) bind to the INAD signaling complex. Such
binding, however, must be a dynamic process (117). It
should be noted, however, that rhodopsin is at least 10fold more abundant than INAD, and it is an immobile
protein. This fact thus suggests that there are two populations of rhodopsin molecules: a minor functional fraction, which is bound to INAD, and a large fraction of
nonfunctional unbound rhodopsin molecules. Studies of
bump properties in Drosophila are inconsistent with such
hypothesis, since every absorbed photon has the same
probability of inducing a quantum bump regardless of its
site of absorption (71).
Biochemical studies in Calliphora have revealed one
important aspect of associating signaling protein together
by showing that both INAD and TRP are targets for phosphorylation by the nearby eye PKC (78, 79). The association of TRP into transduction complexes may also be
related to increasing speed and efficiency of transduction
events as reflected by the immediate vicinity of TRP to its
upstream activator, PLC, and its possible regulator, PKC
(78, 80).
An important aspect of the formation of the supramolecular complex has been recently established by showing
that TRP plays a major role in localizing the entire INAD
multimolecular complex. It was found that the association between TRP and INAD is essential for correct localization of the complex in the rhabdomeres as found in
other signaling systems (e.g., Ref. 5). This conclusion was
arrived at by using Drosophila mutants in which the
signaling proteins, which constitute the INAD complex,
were removed genetically and by deletions of the specific
binding domains, which bind TRP to INAD (94). These
experiments show that INAD is correctly localized to the
rhabdomeres in inaC mutants (where eye PKC is missing)
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BARUCH MINKE AND BOAZ COOK
and in norpA mutants (where PLC is missing), but severely mislocalized in null trp mutants (94, 188), thus
indicating that TRP (but not PLC or PKC) is essential for
localization of the signaling complex to the rhabdomere.
To demonstrate that specific interaction of INAD with
TRP is required for the rhabdomeric localization of the
complex, the binding site at the COOH terminal of TRP
was removed (94, 188) or three conserved residues in
PDZ3 which are expected to disrupt the interaction between PDZ domains and their targets (45) were modified
(188). As predicted, both TRP and INAD were mislocalized in these mutants (188). Interestingly, in a null trp
mutant, ⬃25% of the level of rhabdomeric INAD was still
present, most likely due to a second site of INAD binding
to a still unidentified protein, via PDZ1 (188). The study of
the above mutants was also used to show that TRP and
INAD do not depend on each other to be targeted to the
rhabdomeres; thus INAD-TRP interaction is not required
for targeting but for anchoring the signaling complex (94,
188). TRP thus serves as an anchor that localizes the
signaling complex to its subcellular site. Because almost
all members of the TRP family contain multiple putative
protein-protein interaction sites, it is most probable that
they have an important functional facet as anchors that
localize multiple signal transduction elements. Additional
experiments on TRP and INAD further show that INAD
has other functions in addition to anchoring the signaling
complex. One important function is to preassemble the
proteins of the signaling complex (188). Another important function, at least for the case of PLC, is to prevent
degradation of the unbound signaling protein by a still
unknown mechanism (187).
The mammalian scaffold protein NHERF binds
TRPC4, TRPC5 and PLCs. Recent coimmunoprecipitation
studies have shown, for the first time, that mammals may
organize their TRPC channels in supramolecular complexes similar to the INAD signaling complex. Murine
TRPC4 and TRPC5, as well as PLC-␤1 and PLC-␤2 interact
with the first PDZ domain of the Na⫹/H⫹ exchanger regulatory factor (NHERF). This interaction was demonstrated in vivo (TRPC4 and PLC-␤2), in HEK293 cells
expressing TRPC4, and in adult mouse brain by coimmunoprecipitation. Because NHERF has two PDZ domains
and it binds also to the cytoskeleton via other molecules,
the cytoskeleton seems to be a part of this supramolecular organization (183). Functional studies are required to
determine the functional consequences of this organization of signaling and structural proteins.
VII. CONCLUDING REMARKS
Phosphoinositide-mediated signaling is present in almost every eukaryotic cell and plays a central role in
many aspects of cell signaling, yet many aspects related to
Physiol Rev • VOL
the surface membrane channel, which is the target of this
transduction cascade, are elusive. The main unclear aspects are the identity, the mechanism of activation (including gating), and the specific cellular function of the
channel. The family of TRP channel proteins constitutes
the only surface membrane channels with known molecular identity that are activated by the phosphoinositide
cascade. By now, there is sufficient data to indicate that
phosphoinositide-mediated channels form a heterogeneous family with diverse properties and functions. Molecular identification as “TRP-homolog” or “TRP-related”
of key molecules that mediate specific disease or specific
sensory function constitutes an important clue as to the
general function of this molecule and strongly suggests
that PLC is involved in its activation. With such an important role in signaling, it is not surprising that there is much
interest in trying to unravel the mechanism of activation
of TRP channels.
Several mechanisms have been proposed to account
for activation of TRP channels. The two main mechanisms are 1) store operation, via conformational coupling
between TRP and the InsP3 receptor, and 2) store independent, via activation of PLC that leads to production of
second messenger such as DAG or PUFAs. Although both
mechanisms have been formulated in very general terms,
there are more questions than answers regarding the underlying molecular details, and different investigators
have reported conflicting results regarding the basic classification of various TRP channels as store operated or
store independent. A possible explanation of this confusion is that the mechanism of activation and regulation of
TRP gating involves interactions among several key proteins. Such protein-protein interactions have been reported for voltage- and ligand-gated channels, although
the basic mechanism of gating is known for these channels. For TRP channels in which the basic gating mechanism is obscure, the knowledge about other interacting
proteins seems to be crucial for understanding TRP activation and gating mechanism. We already know that Drosophila TRP is attached to a supramolecular complex via
the scaffold protein INAD, which includes several signaling proteins, and similar mechanisms exist in vertebrate
cells (e.g., via the scaffold protein NHERF). There are
indications that TRP channels form heteromultimers and
that all TRPC channels interact with the InsP3R and the
ryanodine receptors. If such interactions are crucial for
TRP activation in the native system, it is expected that
overexpression of TRPs in heterologous systems leads to
confusing results. This is because of possible absence of
crucial interacting signaling proteins or disruption of the
normal stoichiometry among the signaling molecules that
lead to abnormal and variable signaling complexes according to the expression level of TRP and the specific
cellular environment of the host cell. Even heterologous
expression of TRPL, which seems to produce channel
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467
activity with properties similar to that observed in the
native system, results in constitutive activity of the TRPL
channels unlike the situation in the native system.
It is, therefore, important to direct future efforts
toward investigating the activation mechanism of the various TRP channels in the native cells. Genetic tools seem
to be especially suitable for such an approach. It is not
clear if all members of the TRP family share similar
mechanism of activation, but the similar structural features of most members and the involvement of PLC in
their activation suggest that in general they share a common mechanism of activation, although the details may be
different in the various members of this diverse family of
channel proteins.
We thank many colleagues especially from Charles S. Zuker’s lab for comments on the manuscript and Hagit Cohen
Ben-Ami for preparation of the figures. We thank Drs. Richard
Payne, Donald L. Gill, and Zvi Selinger for critical reading of the
manuscript. We also thank Roger C. Hardie and Zvi Selinger for
permission to use their unpublished observations.
Experiments described in this review were supported by
National Eye Institute Grant EY-03529, the Israel Science Foundation, the German Israeli Foundation, the United States-Israel
Binational Science Foundation, the Minerva Foundation, and the
Moscona Foundation (to B. Minke).
Address for reprint requests and other correspondence: B.
Minke, Dept. of Physiology, The Hebrew University-Hadassah
Medical School, Jerusalem 91120, Israel (E-mail: Minke@md2.
huji.ac.il).
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