The TRP Superfamily of Cation Channels

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REVIEW

The TRP Superfamily of Cation Channels Craig Montell (Published 22 February 2005)

The transient receptor potential (TRP) protein superfamily consists of a diverse group of cation channels that bear structural similarities to Drosophila TRP. TRP channels play important roles in nonexcitable cells; however, an emerging theme is that many TRPrelated proteins are expressed predominantly in the nervous system and function in sensory physiology. The TRP superfamily is divided into seven subfamilies, the first of which is composed of the “classical” TRPs” (TRPC subfamily). Some TRPCs may be storeoperated channels, whereas others appear to be activated by production of diacylglycerol or regulated through an exocytotic mechanism. Many members of a second subfamily (TRPV) function in sensory physiology and respond to heat, changes in osmolarity, odorants, and mechanical stimuli. Two members of the TRPM family function in sensory perception and three TRPM proteins are chanzymes, which contain C-terminal enzyme domains. The fourth and fifth subfamilies, TRPN and TRPA, include proteins with many ankyrin repeats. TRPN proteins function in mechanotransduction, whereas TRPA1 is activated by noxious cold and is also required for the auditory response. In addition to these five closely related TRP subfamilies, which comprise the Group 1 TRPs, members of the two Group 2 TRP subfamilies, TRPP and TRPML, are distantly related to the group 1 TRPs. Mutations in the founding members of these latter subfamilies are responsible for human diseases. Each of the TRP subfamilies are represented by members in worms and flies, providing the potential for using genetic approaches to characterize the normal functions and activation mechanisms of these channels.

Introduction The transient receptor potential (TRP) superfamily is distinct from other groups of ion channels in displaying a daunting diversity in ion selectivities, modes of activation, and physiological functions. Nevertheless, they all share the common feature of six transmembrane domains, varying degrees of sequence similarity, and permeability to cations. A recurring theme in the TRP field is that these channels are of particular importance in sensory physiology. These include roles for TRP channels in each of the sensory modalities, ranging from vision to taste, smell, hearing, mechanosensation, and thermosensation. TRP channels also serve to allow individual cells to sense changes in the local environment, such as alterations in fluid flow and mechanical stress. The initial impetus to identify mammalian TRPs was to characterize the channel that might account for a highly Ca2+Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. E-mail: [email protected]

selective Ca2+ entry mechanism in nonexcitable cells, referred to as store-operated Ca2+ entry (SOCE) (1, 2). Stimulation of many nonexcitable cells with growth factors or hormones leads to activation of phospholipase C (PLC), production of inositol 1,4,5-trisphosphate (IP3) plus diacylglycerol (DAG), and release of Ca2+ from internal IP3-sensitive stores. This transient release of Ca2+ often results in a sustaining rise in Ca2+ through Ca2+ influx across the plasma membrane (3, 4). Interest in finding the channels responsible for these SOCE pathways garnered considerable interest, due to association of these modes of Ca2+ entry with processes ranging from T cell activation to apoptosis, cell proliferation, fluid secretion, and cell migration (3). Mammalian homologs of Drosophila TRP (5, 6) were candidate store-operated channels (SOCs), because the channel in flies functions in phototransduction, which is a PLC-mediated signaling pathway (7, 8). Moreover, Drosophila TRP was speculated to be a SOC. In the 10 years that have elapsed since the first reports of mammalian TRP channels (1, 2), there have been many surprises in the field. Unanticipated were the large size of the superfamily (Table 1), the unusual domain organization of certain members, the range of cation selectivities, and the panoply of activation mechanisms. An ironic twist is that some of the mammalian TRP channels appear to be SOCs, yet current evidence indicates that activation of Drosophila TRP is not dependent on release of Ca2+ from internal stores and is not a SOC. The critical importance of TRP channels is underscored not only by their roles in sensory physiology, but also by the demonstrations that mutations in TRP channels are associated with diseases ranging from hypomagnesemia to polycystic kidney disease and a neurodegenerative disease, mucolipidosis. Composition of TRP Superfamily The TRP superfamily is composed of seven subfamilies, all of which include six putative transmembrane domains (Fig. 1). The fourth transmembrane domain lacks the complete set of positively charged residues necessary for the voltage sensor in many voltage-gated ion channels (9). The structure of a TRP channel has not been solved; however, the channels appear to form tetrameric assemblies (10–13), consistent with the structures of voltage-dependent K+ channels. The initially identified members of the TRP superfamily are referred to as the “classical” or “canonical” TRPs and fall into the TRPC subfamily. The names of the remaining subfamilies are based on the original designation of the first recognized members of each subfamily (14). The Group 1 TRPs—TRPC, TRPV, TRPM, TRPN, and TRPA—share substantial sequence identity in the transmembrane domains. The TRPC, TRPM, and TRPN (except for NOMPC) proteins include a 23– to 25–amino acid “TRP domain” C-terminal to sixth transmembrane domain (Fig. 2). The two Group 2 subfamilies, TRPP and TRPML, are only distantly related to the Group 1 TRPs, owing to low sequence similarity and a large extracellular loop between the first and second transmembrane domains (Fig. 1). The TRPP

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REVIEW of light-induced Ca2+ influx (21). This phenotype, combined with the observation that fly vision requires PLC (7), raised the possibility that trp might encode the archetypal PLC-operated Ca2+ channel. This hypothesis was confirmed with the cloning and functional characterization of TRP. The gene trp encodes an

and TRPML subfamilies are both included in Group 2 because of similarities in their primary amino acid sequences and predicted topologies. Six of the subfamilies include members that are conserved in organisms as divergent as worms, flies, and humans. The remaining subfamily, TRPN, contains members in invertebrates (15) and in zebrafish (16), although no mammalian TRPN protein has been described. The TRP superfamily has ancient origins because distantly related proteins are expressed in fungi (17–19). These latter proteins belong to the TRPY subfamily, so named after the first reported member, the yeast vacuolar protein, Yvc1 (18). Owing to the weak relationship of the TRPY proteins to other TRPs, they are not classified along with either Group 1 or Group 2 TRPs.

Subfamily

Worms

Flies

Mice

Humans

TRPC

3

3

7

61

TRPV

5

2

6

6

TRPM

4

1

8

8

TRPA

2

4

1

1

TRPN

1

1

0

0

TRPP

1

3

1

3

3

TRPML

1

1

3

3

Total

17

13

28

27

2

Group 1 Subfamilies: TRPC Subfamily Invertebrate TRPC proteins. TRPC proteins contain three to four ankryin repeats and extensive amino acid similarity to Drosophila TRP, and are PLC-dependent nonselective cation channels. The founding member of the TRP family was discovered as a key component required for the light response in Drosophila photoreceptor cells. Mutations in trp cause the response to light to be transient (20) and result in a ~10-fold decrease in the level

Table 1. Composition of TRP superfamily in worms, flies, mice, and humans. 1Human TRPC2 is a pseudogene and is not counted. 2TRPP2-like and not TRPP1-like proteins are included. 3 Three very distantly related TRPP proteins (CG13762, CG16793, and CG9472) are not included (209).

Group 1 TRPs

TRPC

TRPV

TRPM

TRPA

TRPN

TRPC1

TRPV1

TRPM7

TRPA1

TRPN1

P

P

P

P

P

Extracellular

A A A A

N

Intracellular

TRP domain

cc

N

Kinase domain

N

N

N C

C

C

Group 2 TRPs

C

TRPP PKD2

C

TRPML MLN1

Extracellular

P

P

C

Intracellular

N

C

N

Fig. 1. The seven TRP subfamilies. Representatives of the five group 1 and two group 2 subfamilies are indicated at the top and bottom, respectively. Several domains are indicated: ankyrin repeats (A), coiled coil domain (cc), protein kinase domain (TRPM6/7 only), transmembrane segments, and the TRP domain (see Fig. 2).

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REVIEW eye-enriched protein with four N-terminal ankyrin repeats and an overall predicted topology similar to that of many members of the superfamily of voltage-gated and second messenger–gated ion channels (5, 6). Consistent with the structural similarities between TRP and known ion channels, in vitro studies have demonstrated that TRP is a cation channel with moderate selectivity for Ca2+ relative to Na+ (PCa:PNa ~ 10:1) (6, 22, 23). In addition to TRP, two other TRPC proteins are expressed in Drosophila, TRPL (24) and TRPγ (25), each of which shares ~45 to 50% amino acid identity with TRP over the N-terminal 800 to 900 amino acids. The sequence similarity encompasses all six transmembrane segments and decreases after the TRP domain (Figs. 1 and 2). The most highly conserved regions of the TRP domain include the TRP box 1 (Glu-Trp-Lys-Phe-Ala-Arg) and the TRP box 2, a proline-rich motif, which is one of two binding sites for a scaffold protein (Homer) in mammalian TRPC1 (26). The functional implications of the interaction between TRPC and Homer are intriguing and are discussed in the last section of this Review. As is the case with TRP (6), both TRPL (27) and TRPγ (25) are highly enriched in photoreceptor cells. Thus, all the TRPC members in Drosophila are expressed predominantly in the visual system. Analyses of loss-of-function and dominant-negative forms of the Drosophila TRPCs indicate that all three contribute to the light-dependent cation influx. Flies devoid of TRPL were originally reported to be indistinguishable from the wild type (27). However, the light response in trpl mutant flies displays several differences from that of the wild type, including changes in the permeability ratios for several cations (28) and a reduced response to a light stimulus of long duration (29). Double mutants lacking both TRP and TRPL are completely unresponsive to light (27), indicating that TRPγ cannot function independently of TRP and TRPL. TRPγ may function in combination with TRPL because the light response is nearly eliminated in trp mutant flies expressing a dominant-negative form of TRPγ (25). Caenorhabditis elegans encodes three TRPC proteins, one of which, TRP-3, has been subjected to genetic analysis (30). This protein is required in sperm for fertility in hermaphrodites and in males. The sperm are motile, but appear to be defective in a step following gamete contact, possibly gamete fusion. Activation of Drosophila TRP. Drosophila TRP is capable of functioning as a SOC, because it can be activated in tissue culture systems using drugs, such as thapsigargin, that cause release of Ca 2+ from the internal Ca 2+ stores (22, 23, 31). Thapsigargin treatment results in Ca 2+ release because it inhibits the smooth endoplasmic reticulum (ER) Ca2+-ATPase (adenosine triphosphatase) that normally counterbalances the constant leak of Ca2+ from the ER stores (32, 33). Despite the observation that TRP appears to function as a SOC in cultured cells (in vitro) (22, 23, 31), TRP is not activated through a store-operated mechanism in vivo. Introduction of either thapsigargin (34, 35) or IP3 (36) to photoreceptor cells does not activate cation influx. In addition, the Drosophila genome encodes a single relative of the mammalian IP3 receptor (IP3R) (37, 38), and mutations that eliminate this gene have no discernible effect on the photoresponse (39, 40). In addition, there exists a second Ca 2+-release channel, the ryanodine receptor, which is distantly related to the IP3R (37, 41). As is the case for the IP3R, there is only one ryanodine receptor homolog in Drosophila, and mutations in this locus have no effect on phototransduction (42). Thus, TRP function is not

dependent on either of the known Ca 2+ -release channels. Moreover, the lack of concordance between the in vitro and in vivo studies raises questions concerning the physiological relevance of using thapsigargin to address whether or not a given channel is a SOC.

Fig. 2. The TRP domain. This region is a highly conserved 23– to 25–amino acid region in TRPC, TRPN, and TRPM proteins, which is C-terminal to the transmembrane segments (see Fig. 1). TRP box 1 is invariant in the TRPC proteins. A variation of TRP box 1 is present in zTRPN1 and cNOMPC. A less conserved version of TRP box 1 is in each TRPM protein. The TRP box 2 is the prolinerich region. Sequences are from mammals, except for zTRPN1 (zebrafish), cNOMPC (C. elegans), and TRP, TRPL, TRPγ, and dTRPM (Drosophila).

Because IP3 is not sufficient to activate TRP, an alternative proposal is that activation of TRP is coupled to PLC activity through hydrolysis of the PLC substrate, phosphatidylinositol4,5-bisphosphate (PIP2) (43). Another proposal is that production of DAG rather than IP3 is essential for opening of the TRP channels. Consistent with this latter proposal, polyunsaturated fatty acids (PUFAs), which can be derived from DAG, lead to activation of TRP either in vitro or after application to isolated Drosophila photoreceptor cells (44). In addition, TRP is constitutively active in a mutant, rdgA, that disrupts an eye-enriched DAG kinase (45). Elimination of the DAG kinase also increases the small light response in strong norpA alleles, which disrupt the activity of PLC (46, 47). These results were interpreted as

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REVIEW additional evidence that PUFAs gate TRP, because elimination Activation mechanisms of mammalian TRPC channels. A of the DAG kinase should, in principle, result in higher levels common feature of the mammalian TRPC channels is that they of PUFAs. However, it has not been demonstrated that the levels are activated in cultured cells through pathways that engage of PUFAs are increased in rdgA, and it cannot be excluded that PLC [for example, see (52, 58–64)]. All of the TRPC-dependent the effects of PUFAs on TRP may be indirect (48). Thus, the conductances are nonselective cation channels, although there mechanism through which activation of PLC is coupled to are differences in the permeabilities of Ca2+ relative to Na+ and other cations (Table 3). As with the Drosophila TRPs, a activation of TRP remains controversial. controversial issue concerns the mechanism through which Given that Ca2+ release from internal stores does not function in Drosophila visual transduction, the transient response to stimulation of PLC and production of IP3 and DAG activates or potentiates TRPC-dependent conductance. Three models have light in trp mutant flies could be due to rapid Ca2+-dependent inactivation of the remaining influx channels in trp mutant been investigated, one of which is that TRPC channels are photoreceptor cells (49). Consistent with this proposal, mutation activated by production of IP3 and release of Ca2+ from internal stores. Another proposal is that a rise in DAG leads to opening of one of the calmodulin-binding sites in TRPL results in a of TRPC channels. Alternatively, TRPC channels may be activated sustained rather than a transient light response in trp mutant through an exocytotic mechanism. According to this latter model, flies (49). Furthermore, it was reported that the trp photoresponse PLC activation leads to a regulated translocation of the channels was similar to that of the wild type in the absence of extracellular from intracellular vesicles to the plasma membrane. Evidence Ca2+. However, this latter result has been challenged (50). An intriguing proposal that may account for the transient has been presented supporting each of these proposals, although light response in trp flies is that it results from depletion of the in only a few cases has the activation mechanisms underlying a substrate for PLC, PIP2, in trp photoreceptors (51). Using the mammalian TRPC channel been addressed in vivo. inwardly rectifying K+ channel Kir2.1 as a biosensor, it apSubset Members Length Percent ID Percent ID to Percent ID to pears that PIP2 concentrations (aa) within same set mTRPCs outside set fly TRPs4 are lower in trp cells than in 1 TRPC1 759–810 N/A 31–44 33–36 wild-type photoreceptor cells. 1 2 TRPC2 psuedo N/A 33–38 33–34 The decrease in PIP 2 is pro4322 posed to be a consequence of a 876–1172 requirement for Ca2+ influx to 37–38 33–38 3 TRPC3 828–848 71–793 down-regulate PLC activity TRPC6 815–931 70–72 35–37 32–37 and up-regulate PIP2 recycling 35–37 30–36 TRPC7 862 69–793 (51). However, direct evidence that the PIP2 concentration is 4 TRPC4 836–1077 65 35–45 42–47 reduced in trp photoreceptor TRPC5 966–975 65 34–43 40–43 cells and that this decrease results in the trp phenotype Table 2. Four sets of mammalian TRPC proteins. In many cases, the lengths of the TRPC proteins remains to be demonstrated. Mammalian TRPC proteins. differ owing to alternative RNA splicing. The percent identities apply to the N-terminal ~750 to 900 This portion of the proteins includes all six transmembrane segments and the TRP A total of seven TRPC proteins amino acids. domain. 1Human TRPC2 is a pseudogene (1). 2A bovine form of TRPC2 is predicted to encode have been described in mammals only four transmembrane segments (53). 3TRPC3 and TRPC7 share slightly greater amino acid (1, 2, 52–60). In contrast to mice identities to each other than to TRPC6. 4TRPγ is the most related to each mammalian TRPC and rats, humans express only protein and then to a lesser degree to TRPL and TRP. TRPγ shares 50% amino acid identity to six TRPCs, because TRPC2 is a either TRP or TRPL over the NH -terminal ~800 residues. TRP and TRPL are 45% identical over 2 pseudogene (1). The seven TRPC the N-terminal ~900 residues. mTRPC, mouse TRPC. proteins are subdivided into four At least four TRPC proteins (TRPC1, -2, -4, and -5) may be subsets on the basis of their primary amino acid sequences (Table 2 activated through a store-operated mechanism, because application and Fig. 3). As is the case for the three Drosophila TRPCs, the of IP3 or thapsigargin results in increases in cation influx in mammalian TRPC proteins include three to four ankyrin repeats, tissue culture cells expressing any one of these proteins (52, 56, six putative transmembrane domains, and amino acid sequence 57, 61, 65). The mechanism underlying SOCE is unresolved. identity (≥30%) over the N-terminal ~750 to 900 amino acids, One proposed mechanism is that a diffusible messenger (Ca2+ which extends to and includes the TRP domain. The sequences influx factor, CIF), which is generated upon release of Ca2+ of the mammalian TRPC proteins are quite variable in the region from the internal stores, leads to Ca2+ influx (66–68). Another C-terminal to the TRP domain. However, the lengths of the possibility is that SOCE depends on a conformational coupling C-terminal tails and the overall size of the mammalian TRPC between the IP3R and the influx channels (69). According to proteins (Table 1) are typically smaller than those of the Drosophila this second model, there is a direct interaction between the TRPCs (1124 to 1275 residues). Some of the TRPC proteins, such IP3R, situated in the intracellular Ca2+ stores, and the Ca2+ as TRPC1, are widely expressed, although each of the TRPCs is influx channels in the plasma membrane. Upon release of Ca2+ expressed in the nervous system and some are highly enriched in from the internal stores, there is a change in conformation in neurons. As is the case with the Drosophila TRPC proteins (23, the IP3R that induces a conformational shift in the SOCs, resulting 25), most of the mammalian TRPCs appear to form heteroin activation of Ca2+ influx. In support of the conformation multimers. The functional consequences of these heteromultimeric coupling model is the demonstration that the addition of assemblies are discussed later in this Review.

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REVIEW TRPC3 by IP3 in excised patches after the native IP3R was removed calyculin A, which causes condensation of cortical actin bundles at the plasma membrane, precludes SOCE and TRPC3 by extensive washing (79). Direct interactions between the IP3R and TRPC channels may be a common phenomenon, because activation in vitro (70, 71). This manipulation of the actin TRPC1 can coimmunoprecipitate with the type II IP3R from human cytoskeleton was interpreted as interfering with SOCE by platelets (82). Ca2+ release through another Ca2+-release channel, preventing access of the ER to the plasma membrane. the ryanodine receptor, can also lead to activation of TRPC3 (83). Alternatively, the reorganization of the cytoskeleton may Distinct TRPC3 channels appeared to be functionally coupled to disrupt Ca2+ entry by causing internalization of either TRPC1, TRPC3, or TRPC4 (72, 73). either the ryanodine receptor or the IP3R, but not both (83). In contrast to some TRPC channels that may be SOCs, other TRPC proteins, notably TRPC6 and -7, are activated in vitro by DAG (60, 74). These results are reminiscent of the report that PUFAs activate Drosophila TRP channels (60). However, it remains unclear whether DAG and PUFAs function directly or indirectly in gating TRP channels. Indirect activation of TRPC proteins by DAG could occur through production of longchain fatty acid metabolites, which can lead to mitochondrial uncoupling. Metabolic stress induced by mitochondrial uncoupling can activate TRPC proteins (75) by depletion of adenosine 5´triphosphate (ATP), as is the case with Drosophila TRP (48). The findings that some TRPC channels may be store-operated whereas others may be activated through production of DAG would suggest that different TRPC proteins are gated through distinct mechanisms. However, such a conclusion becomes murky with regard to TRPC2 and TRPC3. Evidence has been provided that mouse TRPC2 is activated in vomeronasal neurons through a rise in DAG (76). However, TRPC2 appears to be activated by Ca2+ release in mouse sperm (77). Potentially, the differences in these modes of activation could result either from heteromultimerization of TRPC2 with distinct subunits in sperm and vomeronasal neurons or could be due to differences in TRPC2 isoforms, which are generated by alternative mRNA splicing (61, 78). Different modes of activation have also been ascribed to TRPC3. According to one report, activation of TRPC3 depends on production of DAG (74), whereas other studies indicate that TRPC3 is storeoperated (52). Conformational coupling Fig. 3. Dendrogram showing the relatedness of the TRP proteins. The phylogenetic tree may activate TRPC3 because this channel was generated using MacVector 6.5.3 and includes all of the human TRPs, mouse TRPC2, interacts directly with the type I IP3R in and zebrafish TRPN1 (open box). In addition, one Drosophila and one C. elegans member vitro (79). The association between TRPC3 of each subfamily are included (indicated by filled boxes). and IP3R occurs through two regions in the The disparate observations that TRPC3 may be store-operated IP3R, which are situated between the N-terminal IP3 binding site and the transmembrane domains, and a small portion of TRPC3 in some studies and gated by DAG in others may reflect differences C-terminal to the transmembrane domains (80, 81). Additional in the cell types used for the expression studies. Different cell evidence consistent with the conformational coupling model is that lines may express distinct sets of endogenous proteins that interact introduction of IP3 and the IP3R appears to restore regulation of with TRPC3 and affect its mode of regulation. Thus, it is critical

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REVIEW to characterize the mechanisms regulating TRPC proteins in vivo. Unfortunately, there is a paucity of such studies because of the difficulties inherent in ascribing native conductances to specific TRPC channels. A native TRPC3-dependent conductance current has been characterized from the brains of neonatal rats, which is activated by a signaling pathway initiated by the neurotrophin BDNF (brain-derived neurotrophic factor), stimulation of its receptor (TrkB), and subsequent coupling of PLC-γ (84). This conductance, IBDNF, is not activated by DAG and is eliminated by inhibitors of the IP3R. Thus, at least one endogenous TRPC3 conductance requires activity of the IP3R and is not gated by DAG. TRPC3, as well as several other TRPC channels, appear to be situated primarily in intracellular compartments in native cells (84–86). These results raise the possibility that some TRPC channels are held in vesicular membranes and may be activated by regulated shuttling to the plasma membrane. If correct, such a proposal might

account for the observation that many TRPC channels are constitutively active in vitro. The possibility that activation of some SOCs might involve exocytosis is supported by the finding that SOCE is blocked by inhibitors of vesicular trafficking (87) and is prevented by inhibition of a protein, SNAP-25, that is required for the fusion of vesicles with their target membranes (88). Several in vitro studies provide evidence that exocytosis and endocytosis can alter the concentration of TRPC proteins in the plasma membrane and thereby modulate TRPC-dependent cation influx (72, 73, 89, 90). The reorganization of the cytoskeleton appears to participate in controlling the insertion and removal of several TRPC proteins from the plasma membrane (72, 73). Stimulation of receptors coupled to the activation of PLC can also increase the concentration of TRPC channels in the plasma membrane (89). Furthermore, proteins involved in the exocytotic machinery contribute to the movement of TRPC proteins to the plasma membrane (90–92).

TRPC subfamily Gene name

Chromosomal location

Selectivity PCa:PNa

TRPC1

3q22–q24

TRPC2

11p15.3–p15.4

Nonselective cation 2.7

TRPC3

4q27

1.6

TRPC4

13q13.1–q13.2

7 (>1003)

TRPC5

Xq23

9.5

TRPC6 TRPC7

11q21–q22 5q31.2

5 1.91; 52

Mode(s) of activation Store-operated? Store-operated?, DAG

Store-operated, DAG, exocytosis Store-operated? Store-operated?, exocytosis DAG Store-operated, DAG

Adult tissues with highest expression Heart, brain, testis, ovaries VNO, testis

Brain Brain, endothelia, adrenal gland, retina, testis Brain Lung, brain Eye, heart, lung

Functions and diseases Required for EPSC in Purkinje cells Acrosome reaction, male aggression, pheromone response (human TRPC2 is a pseudogene) – Vasorelaxation, neurotransmitter release Modulating neurite extension – –

TRPV subfamily Name

Chromosomal location

Selectivity PCa:PNa

Mode(s) of activation

Adult tissues with highest expression

TRPV1

17p13.3

Heat (43°C), vanilloids, anandamide, protons, PIP2, exocytosis

Trigeminal (TG) and dorsal root ganglia (DRG), urinary bladder

Hot pain sensor 43°C

TRPV2

17p11.2

9.6 (vanilloids) 3.8 (heat) 3

Heat (52°C), exocytosis, membrane stretch

Very hot pain sensor 52°C

TRPV3

17p13.3

2.6

TRPV4

12q24.1

6

TRPV5

7q35

>100

TRPV6

7q33–q34

>100

DRG, spinal cord (SC), brain, spleen, small and large intestine, vascular myocytes DRG, kerotinocytes, tongue, TG, SC, brain Kidney, lung, spleen, testis, endothelia, liver, heart, DRG, keratinocytes Kidney, duodenum, jejunum, placenta, pancreas Small intestine, pancreas, placenta, prostate4

Warm (30°–39°C) Osmotic cell swelling, phorbol esters, warm (27°C), 5´6´-EET, Low intracellullar Ca2+; hyperpolarization, exocytosis Similar to TRPV5, storeoperated (CRAC?), exocytosis

Functions and genetics

Warm temperature sensor (30°-39°C) Osmosensor warm temperature sensor (27°C) Ca2+ reabsorption in kidneys



Table 3. Properties of vertebrate TRPC and TRPV proteins. All of the chromosomal locations apply to the human genes. The biophysical features, expression patterns, and functions are based on studies in either murine or human cells. 1Spontaneous current. 2ATP-enhanced current. 3Applies to current absent in cells from TRPC4-knockout mice. 4Only in prostate cancer lesions.

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REVIEW Regulated translocation of TRPC proteins also occurs in vivo in both invertebrates and vertebrates. In Drosophila photoreceptor cells, TRPL shuttles from the microvillar membranes to the cell bodies in response to light, and this movement functions in long-term adaptation (93). The TRPC protein in worms, TRP-3, is expressed in spermatids in intracellular vesicles; upon sperm activation, TRP-3 is detected in the plasma membrane (30). This translocation is commensurate with an increase in SOCE in the sperm, suggesting that exocytosis is a mechanism regulating Ca2+ influx. In mammals, both TRPC3 and TRPC5 appear to undergo regulated insertion into the plasma membrane in hippocampal neurons (91, 92). In the case of TRPC5, exocytosis of the channel inhibits neurite outgrowth (92, 94). Group 1: TRPV Subfamily Invertebrate TRPVs. The first reported members of the TRPV subfamily include C. elegans OSM-9 (95) and the mammalian protein, TRPV1 (96). Mammals express six distinct TRPV proteins, whereas worms and flies encode five and two members, respectively (Table 1). The proteins that comprise the TRPV subfamily contain three to five ankyrin repeats and share ~25% amino acid identity to TRPC proteins over a span that includes transmembrane segments V and VI and the TRP box (Fig. 1). The osm-9 locus was originally shown to function in osmotic avoidance, olfaction, and mechanosensation (95, 97). In addition, osm-9 is required for “social feeding” (98), a phenomenon in which certain strains of worms feed in groups rather than individually, depending on a variation in a single amino acid in the putative G-protein–coupled neuropeptide receptor, NPR-1 (99). Mutation of osm-9 eliminates “social feeding” in the aggregation-positive npr-1 strain. OSM-9 has not been functionally expressed in vitro; however, both Drosophila TRPV proteins, Nanchung (Nan; hard-of-hearing) and inactive (Iav), are Ca2+-permeable, nonselective cation channels, which are activated in vitro by hypoosmolarity (100, 101). Nan and Iav are expressed in a sensory structure, the chordotonal organ, which participates in the auditory response, and mutations in either loci disrupt hearing. Mammalian TRPV1. The archetypal mammalian TRPV member, TRPV1, was identified through an expression cloning strategy for a channel activated by vanilloid compounds, such as capsaicin, which are present in spicy foods (for example, hot chili peppers) (96). Moderate heat (≥43°C) also activates TRPV1, demonstrating that thermal heat and the pungent ingredient in “hot” foods elicit their effects through the same channel. Anandamide, which is an endogenous compound structurally related to capsaicin, also is capable of activating TRPV1 (102). TRPV1 is a molecular integrator for multiple types of sensory input, particularly those that function in the sensation of pain. Protons (pH ≤5.9) reduce the heat threshold for activation of the TRPV1-dependent cation conductance (103, 104). A decrease in PIP2 concentration, which occurs through production of the pro-algesic agents bradykinin and nerve growth factor, potentiates the capsaicin-evoked response and decreases the temperature threshold for channel activation (105, 106). Analyses of TRPV1-deficient mice confirmed that the channel is activated in vivo through multiple mechanisms (107, 108). In wild-type animals, TRPV1 is enriched in small- to mediumdiameter dorsal root ganglia (DRG) neurons, which respond to moderate heat, pH, and capsaicin (96, 103, 109). However, DRG neurons isolated from TRPV1−/− mice show an impaired

response to each of these sensory stimuli (107, 108). Furthermore, the mutant mice showed a reduced behavioral response to vanilloid compounds and a reduced withdrawal response after tail immersion in a hot waterbath (107). However, using a hotplate to perform the assay, there was no reported difference between wild-type and TRPV1−/− mice (108). Nevertheless, the knockout mice exhibited a clear and pronounced deficit in thermal hyperalgesia resulting from prior inflammation (107, 108). It is possible that regulated insertion of TRPV1 into the plasma membrane may contribute to hyperalgesia. In support of this, TRPV1 binds to the vesicular SNARE proteins snapsin and synaptotagmin IX, which participate in exocytosis (110), and TRPV1 colocalizes in vesicles in the cell bodies and processes of DRG neurons along with synaptotagmin IX and VAMP2. These may correspond to cytoplasmic transport packets similar to those reported for TRPC5. Phosphorylation of TRPV1 by protein kinase C (PKC) promotes the insertion of the channel into the plasma membrane, and this translocation appears to be regulated by SNARE proteins. Mammalian TRPV2, -3, and -4. At least three additional TRPV channels (TRPV2, TRPV3, and TRPV4) are activated by thermal heat, but with thresholds distinct from that of TRPV1 (111–116). The four thermally activated channels form the first of two TRPV subsets [TRPV1-4 (Fig. 3)] on the basis of their primary amino acid similarity, modes of activation, and ion selectivities. TRPV2 requires a noxious heat threshold (≥52°C) (111), whereas TRPV3 (112–114) and TRPV4 (115, 116) are activated by warm temperatures, with thresholds of 30°C and 27°C, respectively. TRPV1, -2, -3, and -4 channels are expressed in neurons in the DRG and elsewhere (Table 3), although TRPV3 and TRPV4 are enriched in keratinocytes (112, 114, 117, 118). Other than TRPV1, none of the other TRPV thermosensors is activated by capsaicin; however, at least two of these channels respond to additional stimuli. Mouse TRPV2 has been reported to participate in cation influx after translocating from intracellular pools to the plasma membrane in response to stimulation with either insulin growth factor I or neuropeptide head activator (119, 120). However, these studies were performed using an in vitro expression system, and it remains to be determined whether TRPV2 displays similar growth factor–induced translocation in vivo. Decreases in osmolarity and cell swelling can also lead to TRPV2 activation (121). TRPV4 is another polymodally activated channel that was initially shown to be activated by hypertonic cell swelling (122–124). TRPV4 can also be activated by phorbol esters and by chemicals, such as anandamide and arachidonic acid, which lead to the formation of the cytochrome P450 epoxygenase, 5´6´-epoxyeicosatrienoic acid (5´6´-EET) (125, 126). The various modes of TRPV4 activation do not appear to occur through a single mechanism because a point mutation in the third transmembrane domain inhibits activation by heat and phorbol esters, but not by cell swelling or arachidonic acid (127). Conversely, blockers of 5´6´-EET and phospholipase A2 (PLA2) decrease activation by cell swelling, but not by phorbol esters or heat. TRPV4 is activated by a range of mechanisms, which overlap with those that stimulate the nematode OSM-9. Introduction of TRPV4 into osm-9 mutant worms restores behavioral responses to hypertonicity and light mechanical stimuli to the nose, but not avoidance to noxious odorants (128).

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REVIEW These results suggest that there is substantial evolutionary conservation in the mechanisms leading to activation of these channels. Mammalian TRPV5 and -6. The remaining two TRPV channels, TRPV5 and TRPV6, form a second TRPV subset [TRPV5-6 (Fig. 3)]. These two channels are distinct from all other mammalian TRPs in displaying very high selectivity for Ca2+ (PCa:PNa>100) (129–131). As with other Ca2+-selective channels, the TRPV5-6 channels become permeable to monovalent cations in the absence of divalent cations. It has been proposed that TRPV6 may be the highly Ca2+-selective, store-operated channel, referred as CRAC (132), which was originally described in mast cells and T lymphocytes. However, several biophysical properties of the TRPV6-dependent current appear to be distinct from those of ICRAC (133). Nevertheless, it remains possible that TRPV5 or TRPV6 may be one subunit of a CRAC channel. As appears to be the case for several TRPC proteins, exocytosis of TRPV5 and TRPV6 may also contribute to activation of these channels (11). Group 1: TRPM Subfamily The TRPM subfamily is composed of eight mammalian members (Table 4), three of which are highly unusual “chanzymes” consisting of C-terminal enzyme domains. TRPM proteins share ~20% amino acid identity to TRPCs over a ~325-residue region that includes the C-terminal f ive transmembrane segments and the TRP domain (Fig. 2). The N-terminal domain of TRPM proteins is devoid of ankyrin repeats and is considerably longer (~750 residues) than the corresponding regions in TRPC and TRPV proteins (~325 to 450 residues). The total length of TRPM proteins (~1000 to 2000 amino acids) varies primarily because of diversity in the regions C-terminal to the transmembrane segments. Six of the TRPM members fall into three subsets, each containing two members: TRPM1/3, TRPM4-5, TRPM6-7. Members within the latter two subsets exhibit similar biophysical properties, in addition to high levels of amino acid similarity. TRPM2 and TRPM8 do not fall into multimember subsets, although they are more related to each other than to members of the other subsets. TRPM proteins are encoded in the Drosophila and C. elegans genomes, and one such protein, GON-2, is required for mitotic cell divisions of the gonadal precursor cells in worms (134). TRPM1 and –3. The designation of the TRPM subfamily is based on the former name of the founding member, melastatin (TRPM1), which is a putative tumor suppressor protein. TRPM1 was isolated in a screen for genes whose level of expression correlated with the severity of metastatic potential of mouse melanoma cell lines (135, 136)). TRPM1 expression in the cell lines and in melanocytic neoplasms is inversely correlated with melanoma aggressiveness (135, 137). An alternatively spliced isoform of TRPM1 encodes a short protein (~500 residues; TRPM1-S), which consists exclusively of the N-terminal region of the full-length form (TRPM1-L) and is devoid of any predicted transmembrane segments (135, 138). TRPM1-S associates with TRPM1-L and suppresses the activity of TRPM1-L by inhibiting its translocation to the plasma membrane (139). TRPM1-L activity was assayed by Ca2+ imaging because patch-clamp recordings of either TRPM1 or its close relative, TRPM3, have been difficult to perform. However, there is a report indicating that the constitutive cation currents resulting from expression of TRPM3 are augmented by hypotonic conditions (140). Alternatively, it has been suggested that TRPM3 may be a SOC (141).

TRPM4 and -5 are VCAMs. The biophysical features of the currents mediated by the TRPM4 and TRPM5 channels are distinct from those of other TRPs and may account for the Ca2+activated monovalent currents identified in various excitable and nonexcitable cells. TRPM4 is expressed as at least two isoforms, TRPM4a and TRPM4b (139, 142), which consist of shorter and longer N-terminal regions, respectively. Whereas TRPM4a displays little activity (139, 143), TRPM4b and TRPM5 appear to be directly activated by an increase in intracellular Ca2+ concentration (142–145). Continuous exposure to Ca2+ desensitizes TRPM5, and this effect is partially reversed by PIP2 (144). TRPM4b and TRPM5 are not Ca2+ permeable, but are selective for monovalent cations (142–145). In addition, both the TRPM4a- and TRPM5-dependent currents activate more slowly at positive potentials and deactivate at negative potentials (143–146). The voltage dependence, Ca2+ activation, and monovalent cation selectivities of these currents (IVCAM) are all features distinct from those of other TRP-dependent currents. Ca2+-activated monovalent cation currents (ICAM) have been described in a diversity of cell types, such as pancreatic acinar cells, cardiac myocytes, and neurons of the central nervous system and peripheral nervous system (147, 148). Moreover, these currents have been implicated in processes ranging from controlling the firing properties of pyramidal cells to mediating the slow oscillations in thalamocortical neurons during sleep (149, 150). Whether these endogenous Ca2+-activated monovalent cation conductances display voltage dependencies similar to that of ICAM remains to be addressed; however, it is intriguing to speculate that they are due to TRPM4b and TRPM5. Among the many cell types expressing TRPM5 are taste receptor cells, and TRPM5 functions in the chemosensory responses to sugars, glutamate, and quinine (151, 152). TRPM6 and -7 chanzymes. The domain organization of the TRPM6/7 proteins is striking, owing to the presence of a C-ter minal atypical protein kinase domain (153, 154). The protein kinase was identified in one report in a screen for PLC-interacting proteins (153). This domain is also expressed as a separate 347–amino acid enzyme independently from the TRPM domain. The protein kinase contains a FYVE (Fab1, YOTB, Vac1, and EEA1) domain zinc finger motif (155) and is most related to the atypical α-kinase family (156), which includes myosin heavy chain kinase A (157) and elongation factor–2 kinase (158). Despite a lack of apparent sequence similarity with conventional protein kinases, the threedimensional structure of α-kinases bears similarities with that of conventional protein kinases (159). The specific function of the TRPM protein kinase has been controversial, but it appears that the enzymatic activity is not a strict requirement for channel activity. According to one study, TRPM7 channel function was augmented by the addition of ATP and inhibited by an inactivating mutation in the enzyme (75). Another study concluded that TRPM7 is suppressed by intracellular Mg2+ and that the addition of ATP promotes activation of TRPM7 by lowering the concentration of free Mg2+ (154). Point mutations or deletions in the protein kinase domain did not substantially impair channel activation, but affected the sensitivity of the channel to Mg2+ suppression (160). However, for unknown reasons, the point mutations resulted in an increase in Mg2+ sensitivity, whereas the deletion had the opposite effect. Nevertheless, the findings that TRPM7 conducts Mg2+ and is

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REVIEW suppressed by increasing concentrations of Mg2+ provide for an efficient mechanism of negative-feedback regulation. The C-terminal protein kinase domain mediates the effects of adenosine 3´,5´-monophosphate (cAMP) on channel activity (161). An increase in cAMP levels elevates TRPM7-dependent currents, and this effect is eliminated by mutations that abolish the TRPM7 kinase activity. In addition to Mg 2+, channel activity of TRPM7 is also suppressed by hydrolysis of PIP2 (162). Whether this latter regulatory mechanism is related to the finding that TRPM7

binds to PLC remains to be resolved (153). It is intriguing to speculate on whether Mg2+ activates the PLC associated with the chanzyme, leading to diminution of PIP2 concentration. In fact, Mg2+-activated PLCs have been described in certain cell types, such as B lymphocytes (163). The highly related chanzyme, TRPM6, was identified as the mutated gene in patients with hypomagnesemia (164, 165). The biophysical features of the current resulting from in vitro expression of TRPM6 are quite similar to those of TRPM7. As with TRPM7, the TRPM6-dependent current is carried mostly

TRPM subfamily Name

Chromosomal location

Selectivity PCa:PNa

TRPM1

15q13–q14

TRPM2 TRPM3

21q22.3 9q21.11

Nonselective cation ~0.3 1.6

TRPM4

19q13.33

TRPM5

11p15.5

TRPM6

9q21.13

TRPM7

15q21

TRPM8

2q37.2

Monovalent selective cation Monovalent selective cation Primarily divalent, e.g., Mg2+ and Ca2+ Divalentspecific, e.g., Mg2+ and Ca2+ 3.3

Mode(s) of activation

Adult tissues with highest expression

Translocation?

Eye

ADP-ribose, NAD, H2O2 Decrease in osmolarity, store-operated? Ca2+-activated, voltagemodulated (VCAM) Ca2+-activated, voltagemodulated (VCAM) Mg2+ inhibited, translocation

Brain Human kidney, mouse brain Prostate, colon, heart, kidney, Small Intestine, liver, lung, taste receptor cells Kidney, small intestine

Phosphorylation, Mg2+-ATP

Kidney, heart

Menthol, icilin, cool (23°–28°C)

Prostate, TG, DRG

Functions and genetics – Redox sensor – – Taste1 Mg2+ absorption hypomagnesemia, hypocalcemia Cell viability

Cool temperature sensor (23°–28°C)

TRPA and TRPN Name

Chromosomal location

Selectivity PCa:PNa

TRPA1

8q13

0.8

zTRPN1



?

Mode(s) of activation Cold (17°C), icilin, mustard oil, wasabi, cannabinoids (THC), cinnaldehyde, bradykinin, mechanical deflection? Mechanical stimuli?

Adult tissues with highest expression

Functions and genetics

DRG, hair cells

Cold pain sensor (17°C), hearing1,2

Ear, eye

Hearing2

TRPP and TRPML Name

Chromosomal location

Selectivity PCa:PNa

Mode(s) of activation

Adult tissues with highest expression

Functions and genetics

TRPP2

4q21-q23

Nonspecific cation

Kidney, widely expressed

Polycystic kidney disease

TRPP3

10q24

4.3

5q31 19p13.2– p13.3 1p22 1p22.3

– Nonspecific cation – –

Modulated by Ca2+, translocation?, interaction with polycystin-1, protons Modulated by Ca2+, inhibited by troponin I – Inhibited by reduction in pH, modulated by Ca2+? – –

TRPP5 TRPML1 TRPML2 TRPML3

Kidney, heart Testis, heart Brain, heart, skeletal muscle – Cochlea hair cells1

– – Lysosomal trafficking?, mucolipidosis type IV – Hearing1

Table 4. Properties of vertebrate TRPM, TRPA, TRPN, TRPP, and TRPML proteins. All of the chromosomal locations apply to the human genes, with the exception of TRPN1, for which a chromosomal location is not indicated because this gene is present in zebrafish, but not humans or rodents. With the exception of zTRPN1, the biophysical features, expression patterns, and functions are based on studies in either murine or human cells. 1Based on studies in mice. 2Based on studies in zebrafish.

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REVIEW by divalent cations, such as Mg2+ and Ca2+, and is inhibited by there is controversy concerning the activation mechanism of Mg2+ (166). TRPM6 is capable of efficiently heteromultimerizing mammalian TRPA1. According to one group, TRPA1 is activated with TRPM7, and this interaction appears to promote translocation by cold temperatures below 17°C (182). Although this thermoof TRPM6 from an intracellular compartment to the plasma activation has not been reproduced in another study (183), membrane (167). there is agreement that various chemical agonists lead to The TRPM6 and TRPM7 chanzymes represent the first activation of TRPA1 (182, 183) (Table 5). These include channels shown to be highly permeable to Mg2+ (154, 166). pungent compounds present in mustard oil and other natural The TRPM7-dependent Mg2+ influx contributes to cell survival sources and the psychoactive compound in marijuana ∆ 9because TRPM7 knockdown cells are not viable unless they tetrahydrocannabinol (THC). In addition, TRPA1 is activated are bathed in a medium rich in Mg2+ (160). TRPM7 is also by an agonist (bradykinin) that initiates a PLC-coupled signaling permeable to trace and toxic metals, such as Zn2+ and Ni2+ pathway (184). (168). Thus, entry of Zn 2+ and Ni 2+ through TRPM7 may The mouse and zebrafish TRPA1 proteins are required for contribute to the accumulation and toxicity of these metals. hearing and may be mechanically gated channels (185). A TRPM6 or TRPM7 may correspond to a Mg2+-inhibited divalent mechanically gated channel is thought to be activated by a current, referred to as MIC or MagNuM, that has been identified gating spring, and it has been proposed that the large number of in T lymphocytes and rat basophilic leukemia (RBL) cells (163, ankyrin repeats in near the N terminus may serve this purpose 169–171). (185, 186). TRPM2 and -8. TRPM2 is a second type of chanzyme Two Drosophila TRPAs appear to be thermosensors. The represented by members of the TRPM family. TRPM2 is Drosophila homolog of TRPA1 is also thermally activated; composed of a C-terminal ADP-ribose pyrophosphatase however, it is activated in vitro by warm rather than cold (NUDT9) domain (172, 173). TRPM2 is activated by either temperatures (187). A role for dTRPA1 in vivo has not been ADP-ribose or agents, such as H2O2, which affect the redox described. A phenotypic analysis of another Drosophila TRPA, state (172, 173). The alteration in redox state appears to activate referred to as Painless, demonstrates that it functions in larvae TRPM2 through binding of β-NAD+ (nicotinamide adenine in the avoidance of noxious heat above ~38°C (188). Currently, dinucleotide) to the NUDT9 domain (174). Such a mode of there is no evidence that the Drosophila TRPA channels activation has important implications in terms of the mechanism function in mechanotransduction. underlying hypoxia-induced cell death (see below: TRPs and hypoxia-induced cell death). A short form of TRPM2, which is Group 1: TRPN Subfamily composed of the N terminus and the first two transmembrane The TRPN subfamily includes a single member in worms, flies, segments, binds to and suppresses the activity but not the localand zebrafish (15, 16, 189), although no TRPN protein has been ization of the long form of TRPM2 (175). These data are consistent reported in mammals. Each of the TRPN proteins include 29 with the f indings that the N-terminal fragments of the ankyrin repeats N-terminal to the six transmembrane segments Drosophila and mammalian TRPCs can bind to and suppress (15, 16, 189) (Fig. 1). TRPN proteins share ~20% amino acid the activities of full-length TRPC proteins (23, 25, 75, 176). identity to TRPC proteins over a ~400–amino acid segment that TRPM8 is most related to TRPM2, although it is not a spans the six transmembrane domains. However, TRPN proteins chanzyme. TRPM8 is a “thermoTRP,” which is activated by differ from TRPC, TRPV, and TRPM proteins in that they do moderately cool temperatures and by chemicals, such as menthol not include a TRP domain. and icilin, that evoke the sensation of thermal coolness (177, Several lines of evidence indicate that TRPN channels may 178). Interestingly, the molecular bases for cold activation of be mechanically gated. The founding member of the TRPN TRPM8 and heat activation of TRPV1 are conceptually similar. subfamily, Drosophila NOMPC, is most likely a subunit for a Activation of both of these channels by temperature involves mechanically gated channel because it is expressed in changes in the voltage-dependent activation curves by temperature (179, 180). In addition, Mammalian thermal-sensitive channels menthol and capsaicin, ligands that activate Channel Threshold Chemicals that evoke thermal-like response TRPM8 and TRPV1, respectively, do so by TRPV1 Capsaicin ≥43 shifting the activation curves. Whether the other thermoTRPs are activated through a TRPV2 – ≥52 similar mechanism remains to be addressed. TRPV3 – ≥30-39 Group 1: TRPA Subfamily The TRPA proteins are characterized by the presence of large numbers of ankyrin repeats in the N-terminal domain (8-18) (Fig. 1). There is only one TRPA member in mammalian organisms, although there are four in Drosophila (Table 1). The founding TRPA protein, TRPA1, was isolated in a screen for genes down-regulated in fibroblasts that have undergone oncogenic transformation (181). Several TRPA proteins have been reported to be thermosensors (Tables 4 and 5); however,

TRPV4 TRPM8 TRPA1

≥27 23-28 17

– Menthol, icilin Icilin, mustard oil

Drosophila thermal-sensitive channels Painless (TRPA subfamily) TRPA1

≥39



≥27



Table 5. Mammalian and fly thermosensitive TRPs. Those that respond to warm temperatures are red and those that respond to cold are blue.

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REVIEW mechanosensory organs and the mechanosensory response is greatly reduced in loss-of-function mutants (15). In addition, a C. elegans TRPN protein appears to be expressed in mechanosensory neurons (15). The zebrafish homolog, TRPN1, is expressed in hair cells of the inner ear and is required in larvae for hearing (16). However, these TRPN proteins have yet to be characterized in vitro, and it is not yet clear whether either of these proteins is capable of functioning independently as a channel. Nevertheless, the large number of ankyrin repeats in TRPN has been proposed to function as a gating spring (186). Group 2 Subfamilies Overview. The archetypal TRPP and TRPML proteins, TRPP2 (polycystin-2, PKD2, PC2) and TRPML1 (mucolipin, MCOLN1, ML1), respectively, were identified in screens for the genes disrupted in human diseases. The TRPP and TRPML proteins are designated as Group 2 TRPs, owing to their relatively high similarity to each other but low primary sequence similarity to Group 1 TRPs. Nevertheless, as is the case with the Group 1 TRPs, the Group 2 members contain six predicted transmembrane domains and appear to be cation-permeable channels. Group 2: TRPP subfamily. TRPP2 was discovered as one of the gene products mutated in many cases of autosomal dominant polycystic kidney disease (ADPKD) (190). ADPKD results in kidney failure in ~1 in 1000 individuals, due to the formation of benign, fluid-filled cysts (191–193). Human TRPP2 interacts with TRPP1 (polycystin-1, PKD1, PC1) (194, 195), and mutations in one or the other of these two proteins account for ~95% of autosomal-dominant PKD (191). Both proteins are widely expressed (190, 196–198), and cyst formation may arise in other tissues, as well. Mice with targeted mutations in TRPP2 die in utero and display cyst formation in the maturing nephrons and pancreatic ducts (199) and defects in the cardiac septum (200). Thus, the mouse model recapitulates many of the features of human ADPKD. In addition, TRPP2-deficient mice display a laterality phenotype (201, 202), which is described in more detail below. TRPP proteins appear to be present throughout the animal kingdom and, in addition to TRPP2, include two other mammalian proteins predicted to contain six transmembrane domains, referred to as TRPP3 (PKD2L1, polycystin-L, PCL) (203, 204) and TRPP5 (PKD2L2) (205, 206), and proteins in C. elegans (LOV-2) (207, 208), flies (AMO) (209, 210), and sea urchins (suPC2) (211). TRPP proteins share ~25% amino acid identity to the most closely related TRPC proteins, TRPC3 and TRPC6, over a region spanning transmembrane segments IV, V, and the pore-loop (H5 segment), which is a hydrophobic domain between segments V and VI that contributes to ion selectivity (212) (Fig. 1). Mammalian TRPP2 contains a Ca2+binding motif (EF-hand) and a coiled-coil domain near the C terminus, but does not include any ankyrin repeats or a TRP domain. In addition, TRPP proteins include a large extracellular loop between the first and second presumed transmembrane segments (Fig. 1). TRPP1 is a very large protein consisting of 11 predicted transmembrane domains, with amino acid sequence similarity to the transmembrane domains in TRPP2. In addition, TRPP1 includes an ~2500–amino acid extracellular domain and an intracellular C-terminal domain (196) that interacts with the C terminus of TRPP2 (194, 195). The interaction between the two

polycystins appears to be critical for function. According to one study, introduction of TRPP2 into Chinese hamster ovary (CHO) cells does not result in any discernible channel activity (213). However, coexpression of TRPP1 along with TRPP2 induces translocation of TRPP2 to the plasma membrane and production of a Ca2+-permeable nonselective cation conductance (213). According to another study analyzing TRPP2 in lipid bilayers, the cytoplasmic tail of TRPP1 increased the channel activity of TRPP2. Nevertheless, controversy exists as to whether TRPP2 is a cation influx channel (213–215) or a new type of Ca2+-release channel (216). TRPP3 has also been functionally expressed and shown to be a nonselective cation influx channel that is positively regulated by intracellular Ca2+ (217). Currently, there is no evidence that TRPP3 Ca2+ influx activity requires TRPP1. Evidence has been presented raising the possibility that TRPP1 may also be a cation influx channel (218). However, it was not excluded that the currents induced by expression of TRPP1 were due to indirect effects on an endogenous channel. Nevertheless, this possibility is consistent with the substantial similarity between TRPP2 and the C-terminal six transmembrane domains of TRPP1. ADPKD primarily affects the kidneys; however, it is also frequently associated with clinical manifestations in various other tissues (219). TRPP1 and TRPP2 are concentrated in mono- or primary cilia (201, 220–222). In mammals, monocilia are present on the surface of most cells and consist of nine doublets of microtubules distributed near the cell periphery (9+0 structure). Motile cilia are similar in structure and include two central pairs of microtubules, in addition to the nine peripheral doublets (9+2 structure). Analyses of the mammalian TRPPs and their invertebrate homologs suggest a conserved role for TRPPs in both motile and nonmotile axonemal structures. The C. elegans homologs of both the TRPP1 and TRPP2 homologs (LOV-1 and PKD2, respectively) are required for male sensory behavior and are enriched in sensory cilia (207, 208). In the sea urchin, TRPP1 and TRPP2 homologs are present in the acrosomal region of sperm (211, 223), and in Drosophila the TRPP2 homolog, AMO, is concentrated in the sperm tail (209). Mutation of amo does not affect sperm development, mating, or entry of the sperm into the females (209, 210). Rather, there is a defect in transfer of the sperm from the uterus to sperm storage organs. In female flies, sperm is subsequently released from the sperm storage organs so that eggs can be fertilized over an extended period following mating. The localization of TRPPs to both motile and primary cilia suggest that TRP channels may play a role not only in the sensory modalities, but in allowing individual cells to sense their local environment. TRPPs may be situated on primary cilium, which protrude from the surface of most vertebrate cells, to facilitate the detection of extracellular signaling molecules or to sense changes in fluid flow, osmolarity, or other mechanical stress. Consistent with this proposal, cultured kidney cells from wildtype, but not TRPP1 mutant, cells display an increase in Ca2+ influx in response to fluid flow (222). Similarly, the flowinduced Ca2+ influx was disrupted in wild-type cells bathed in antibodies that recognize the extracellular domains of either TRPP2 or TRPP1 (222). Interestingly, a role for TRPPs in detecting fluid flow is consistent with the findings that TRPP2 knockout mice display

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REVIEW a defect in left-right asymmetry (201, 202). Wild-type vertebrates, such as mice and humans, display lateral asymmetry in the heart and several other organs. This left-right asymmetry is established during embryogenesis and requires leftward flow of fluid over the node, which is generated by motile cilia. TRPP2 is normally expressed in node monocilia, and TRPP2deficient embyros do not display the flow-induced rise in intracellular Ca2+ (201, 202). The combination of studies on TRPP2 indicates that it may be a mechanosensor that responds to fluid flow. Group 2: TRPML subfamily. The TRPML subfamily is defined by a human protein, TRPML1 (mucolipidin1; ML1), encoded by the MCOLN1 gene (224–226). Mutations in MCOLN1 are responsible for a lysosomal storage disorder, mucolipidosis type IV (MLIV), which leads to severe neurodegenerative defects. Although the disease primarily affects the nervous system, MCOLN1 RNA is expressed in most tissues. The three mammalian TRPML proteins are small (538 to 580 residues), relative to other TRP-related proteins, and the level of primary amino acid sequence identity to TRPC proteins is quite limited. Analysis of TRPML1 using Prof ileScan (http://myhits.isb-sib.ch/cgi-bin/motif_scan), an algorithm that compares proteins to known motifs and patterns (227), reveals a stronger relation to TRPCs than to other proteins in the databases. The similarity of TRPML1 (amino acids 331 to 521) to TRP spans the region that includes transmembrane segments three to six and the putative pore-loop region. TRPML1 has stronger sequence identity to members of the TRPP subfamily (≥20% amino acid identity over a region that includes transmembrane segments four to six) and includes a large extracellular loop between transmembrane domains one and two similar to that of TRPP proteins (Fig. 1). Notable sequence motifs in TRPML1 include a lipase serine active-site domain, a bipartite nuclear localization signal, and a putative late endosomal-lysosomal targeting signal. The subcellular distribution of the TRPML proteins is unresolved, but one hypothesis is that TRPML1 is an endosomaland lysosomal-associated cation channel, which is required for the re-formation of lysosomes (228). According to one expression study in Xenopus oocytes, TRPML1 is a cation channel, which translocates to the plasma membrane upon treatment with the Ca2+ ionophore, ionomycin (229). TRPML1 has also been reported to be inhibited by a decrease in pH (230). Two mutations associated with weak or moderate forms of MLIV do not eliminate channel activity, but prevent the pH-mediated inhibition of the current (230). The roles of TRPML2 and TRPML3 in humans are not known; however, mutations in mouse Mcoln3 are responsible for the hearing loss and pigmentation defects associated with varitint-waddler mice (231). Consistent with a role in hearing, TRPML3 is expressed in hair cells, both in a vesicular compartment and in the stereocilia. Currently, the specific role of TRPML3 in hearing and pigmentation is not known. Single representatives of the TRPML subfamily are encoded in worms and flies and are between 40 and 44% identitical to mammalian TRPMLs over most of the proteins (224–226). Within a domain encompassing transmembrane segments three to six, the percent identity between TRPML1 and these invertebrate homologs rises to nearly 60%. The C. elegans TRPML gene, cup-5, has been subjected to genetic analyses (232, 233). As appears to be the case in MLIV

(232), cup-5 mutant worms accumulate large vacuoles and display defects in endocytosis and lysosome biogenesis (234). In addition, introduction of either the human MCOLN1 or MCOLN3 genes into worms rescues the cup-5 phenotype to a great extent (233, 235). The ability of MCOLN3 to rescue cup-5 suggests that the ML1 and ML3 proteins may be serving similar functions at the molecular level, even though they are not redundant in mice or humans. Functions of TRP Proteins TRP proteins in vascular endothelial cells and myocytes. Sustained Ca2+ entry in vascular endothelial cells leads to changes in cell shape (236) and affects vessel tone and permeability (237, 238), angiogenesis (239), and leukocyte trafficking (240). TRPC channels may mediate these Ca 2+ entry pathways because different TRPCs are expressed in various endothelial cells (158, 176, 236, 241, 242). Furthermore, a dominant-negative form of TRPC3 inhibits SOCE in umbilical vein endothelial cells (176) and oxidant-induced cation influx in aortic endothelial cells (242). The first mouse knockout of a TRPC protein, TRPC4, provides evidence for a TRPC protein in endothelial cell function (242). TRPC4 -−/−− mice are viable and reach maturity, but they display impaired vasorelaxation of the aortic rings (242). This defect may be a consequence of a perturbation in SOCE because agonist-induced Ca2+ influx is almost eliminated in aorta endothelial cells isolated from the TRPC4−/− mice. TRPC proteins are also expressed in other nonexcitable cells that are proposed to be regulated by SOCE. These include pancreatic β cells (243), human platelets (82), rabbit portal vein smooth muscle (244), and salivary gland cells (245). TRPC1 is a candidate for modulating the secretion of fluids and electrolytes in salivary glands, because SOCE is reduced in salivary gland cells transfected with antisense TRPC1 RNA (245). TRP proteins may also function in vascular smooth muscle cells. TRPC6 is expressed in rabbit portal vein myocytes, and introduction of TRPC6 antisense oligonucleotides to such primary cells inhibits the nonselective cation channel activated by α1-adrenoreceptor agonists (244). The α1-adrenoreceptor functions in the control of systemic blood pressure (246), and it is possible that it may do so through activation of TRPC6. TRPV2 is also expressed in vascular myocytes, where it appears to be activated by membrane stretch (121). TRPC proteins in fertilization. Fertilization of a mammalian egg is a multistep process that begins with association of the sperm with a glycoprotein, ZP3, in the egg’s extracellular matrix (247). The sperm-ZP3 interaction triggers the release of hydrolytic enzymes from the sperm acrosome and remodeling of the sperm surface. These events, referred to as the acrosomal reaction, are critical for penetration of the egg by the sperm, ultimately leading to zygotic development. Association of the sperm with ZP3 initiates the acrosomal reaction through a signaling cascade that involves G proteins (heterotrimeric GTPbinding proteins) (248), PLC-δ4 (249), transient Ca2+ influx through voltage-gated channels, and a more sustained influx through a SOC (250). The identity of the SOC has been elusive, although mouse TRPC2 has been suggested to be a subunit of the ZP3-triggered channel. A TRPC2 isoform is highly enriched in the sperm, and antibodies to an extracellular domain of TRPC2 inhibit the ZP3-induced Ca2+ influx and acrosomal reaction (77). However, a knockout of mouse TRPC2 does not show defects in fertilization (251). TRPC2 cannot participate in

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REVIEW fertilization in humans, because the human TRPC2 is a pseudogene (1). At least four TRPC proteins have been localized in human sperm (252), raising the possibility that one or more TRPCs functions in fertilization. Consistent with a possible role for mammalian TRPCs in fertilization, a TRPC-related protein in C. elegans, TRP-3, is expressed in sperm and is required for fertility (30). TRP proteins in the kidneys, urinary bladder, and small intestine. In humans, TRPM6 is present at the highest levels in the kidneys and small intestine, and mutations in this chanzyme result in hypomagnesemia with secondary hypocalcemia (164–166). The defect in Mg2+ absorption results in a variety of symptoms, including seizure and muscle spasms during infancy. TRPM7 is also permeable to Mg2+, and interactions between TRPM6 and TRPM7 may also contribute to the disease (see below). Several members of the TRPV subfamily are present in the kidneys, one of which, TRPV4, is predominantly in the distal nephron of the kidneys (122–124, 253). TRPV4 is activated in vitro by decreases in osmolarity and is present in a region of the kidneys that may be exposed to hypotonic fluid (122–124). TRPV4-mutant mice display an impairment in osmotic sensation; however, this phenotype is attributed to a role of TRPV4 in the brain, which leads to an increased level of serum arginine vasopressin in response to hyperosmolarity (254). Whether TRPV4 has a role in the regulation of electrolyte or fluid transport in distal nephron has not been demonstrated. TRPV5 and TRPV6 are present primarily in the kidneys and small intestine, respectively, and may play important roles in Ca2+ absorption (130, 255). A definitive demonstration that TRPV5 functions in Ca2+ reabsorption in the kidney has been provided by an analysis of TRPV5-deficient mice (256). The defect was observed along the kidney’s distal convolution, where TRPV5 normally is present. In addition, the mutant mice excreted about six times more Ca2+ in the urine than did wildtype animals. Interestingly, the expression of genes encoding Ca 2+ transport proteins, such as the Na + /Ca 2+ exchanger (NCX1), was reduced in the kidneys. The dysfunction in Ca2+ homeostasis in the TRPV5−/− animals also resulted in defects in bone structure. In addition to a requirement for TRPV protein in the kidneys, at least one member of this subfamily, TRPV1, is required for normal urinary bladder function (257). TRPV1-knockout mice display normal bladder morphology and excrete similar volumes of urine as wild-type animals do. However, the mutant animals show a higher frequency of small volume eliminations and an increase in bladder capacity and nonvoiding bladder contractions (257). The bases of these phenotypes are unresolved, but it is noteworthy that ATP and purinergic signaling has been linked to bladder function (258–261) and stretch-evoked ATP release is greatly reduced in urothelial cells from TRPV1−/− mice (257). Currently, there is no evidence that TRPV1 is activated by membrane stretch or hypotonicity, although it is plausible that heteromultimers composed of TRPV1 are stretch-activated channels. TRPC proteins in neuronal development and plasticity. Mammalian TRPC RNAs and proteins are each expressed in the brain (262) and several, such as TRPC3 (52, 84, 263), TRPC4 (263), and TRPC5 (57, 58), are highly enriched in the brain. Others, such as TRPC1, are expressed in various tissues in addition to the central nervous system (1, 2). TRPC1 is required for the

excitatory postsynaptic conductance (EPSC) in cerebellar Purkinje cells, through a pathway initiated by glutamate stimulation and involving activation of a group I metabotropic glutamate receptors (mGluR1), Gαq, and PLC-β (264). Although the function of this postsynaptic current is unresolved, these data raise the possibility that TRPC1 functions in neuronal plasticity. TRPC3 may participate in activity-dependent changes that occur in the mammalian brain around the time of birth. The TRPC3 protein is detected primarily in the brain immediately before and after birth and is activated in vivo by a pathway that is initiated by BDNF (84). BDNF functions in neuronal differentiation and survival (265, 266), which occurs through changes in transcription many hours after exposure to the neurotrophin. However, there is evidence that BDNF is involved in synaptic plasticity and can cause rapid effects, such as morphological changes at the growth cone and modulation of neurotransmitter release (267–269). Because these effects are too rapid to occur through transcriptional induction, they may be mediated by BDNF-stimulated Ca2+ influx through TRPC3. Direct evidence that TRPC channels function in neurotransmitter release and growth cone extension has been obtained for TRPC4 and TRPC5, respectively. Many dendrites contribute to synaptic transmission through release of the neurotransmitter GABA (γ-aminobutyric acid). The release of GABA is reduced in TRPC4-mutant mice, indicating that this channel promotes this process (270). Another exciting finding is the observation that TRPC5 channels appear to function in neurite extension and affect growth cone morphology (94). Growth cones possess TRPC5 and display a current reminiscent of TRPC5. A dominant-negative form of TRPC5 induces longer growth cones, suggesting that TRPC5 activity inhibits neurite extension. The pathway leading to TRPC5 activation involves stimulation of a receptor tyrosine kinase, which in turn engages phosphoinositide 3-kinase (PI3K), the guanosine triphosphatase Rac1, and PI5K (92). Vertebrate TRP proteins in sensory physiology. An emerging theme is that many vertebrate members of the TRP superfamily function in sensory perception, as is the case for invertebrate TRPs. These include chemosensation, hearing, osmosensation, and thermosensation. At least two TRP channels function in chemosensation, one of which (TRPM5) is present in taste receptor cells and is required in the mouse for the bitter, sweet, and umami responses (151, 152). TRPM5 is also present in other tissues (271) and presumably has additional roles independent of taste transduction. Features that distinguish TRPM5, and the highly related channel, TRPM4b, from other TRP channels is that they are voltage-modulated, Ca2+-activated monovalent selective cation channels (142–146). These are properties of currents that have been implicated in diverse processes ranging from slow oscillations in the thalamocortical neurons during sleep (149) to the firing properties of pyramidal cells in the cortex (150) and signaling in cardiomyocytes (272, 273). Whether TRPM4b or TRPM5 function in these latter processes remains to be determined. One TRPC protein, TRPC2, plays a role in the pheromone response. TRPC2 is expressed as at least two alternatively spliced isoforms, one of which (TRPC2α) is enriched in the vomeronasal organ (VNO) of rodents (55, 61, 78). In the VNO, pheromone presentation activates TRPC2 through a DAGdependent pathway (76). Surgical ablation of the VNO indicates that this organ functions in the initiation of male sexual behavior

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REVIEW in rodents (274). However, mating in TRPC2−/− mice is normal (251, 275). Rather, TRPC2-deficient mice fail to display the pheromone-induced male-male intraspecies aggression (251, 275). TRPC2-knockout males exhibit sexual behavior, without preference, toward castrated intruder males or females. Thus, TRPC2 function is required for sex discrimination. It is noteworthy that the VNO may not be functional in humans (276), and that human TRPC2 is a pseudogene (1). Therefore, TRPC2 cannot contribute to sexual discrimination in humans as it does in mice. Three vertebrate TRPs function in hearing, one of which, TRPN1, is the zebrafish homolog of the Drosophila mechanotransduction channel, NOMPC (16). RNA encoding TRPN1 is present in embryonic and larval hair cells and in multiple tissues in the adult, including the ear. Reduction in TRPN1 expression, using morpholino antisense oligonucleotides, does not affect larval hair cell number or morphology, but inhibits auditory and vestibular function. The subcellular distribution of the TRPN1 protein is not known, and it is unclear whether it is the mechanotransduction channel. Nevertheless, a TRPN protein cannot serve as a mechanosensory channel in humans because members of the TRPN subfamily are not encoded in the genomes of mammalian organisms. A third TRP protein that participates in hearing is a member of the TRPML subfamily. Mutations that disrupt mouse TRPML3 result in hearing loss, vestibular defects, and other abnormalities (231). TRPML3 is present in hair cells, although it is not a strong candidate mechanotransduction channel because it does not appear to be localized to the tips of stereocilia where the transduction machinery is situated. In addition to hair cells, TRPML3 is present in other cell types and tissues and has been proposed to function generally in intracellular ion homeostasis. TRPA1 has emerged as the elusive mechanically gated transduction channel necessary for the auditory response in mammals (185). Disruption of TRPA1 expression causes defects in hearing, and the protein is present in the tips of stereocilia where the transduction machinery is situated. Furthermore, there is an increase in TRPA1 expression coincident with the initiation of transduction in hair cells. In addition to facilitating an animal’s ability to sense stimuli in the external milieu, some TRP channels enable individual cells to detect changes in the local cell environment. These include alterations in fluid flow and osmolarity. Both TRPV4 (122–124) and TRPP2 (277, 278) appear to participate in detection of such localized stimuli. Among the most exciting findings in the TRP field are the demonstrations that six mammalian members of the TRP superfamily respond to thermal temperature [Table 5 (also see above)] (96, 111–115, 126, 177, 178, 182). These six TRP channels belong to three different subfamilies (TRPV, TRPM, and TRPA) and together account for the responses to a wide range of temperatures extending from noxious heat (≥43°C) to uncomfortable cold (≤17°C). Furthermore, at least three of the thermoTRPs also respond to chemicals that elicit the sensation of thermal heat or thermal cold (96, 177, 178, 182–184), providing at least part of the molecular mechanisms for these phenomena. Many questions remain concerning the thermoTRPs. Are there one or more additional TRPs, which respond to temperatures in the ultracold range? What are the identities of other proteins that interact with and modulate the activities of thermoTRPs?

TRPs and hypoxia-induced cell death. Anoxic conditions, such as occur during metabolic stress, can lead to massive death of neurons and other cell types. Consequently, understanding the molecular mechanisms underlying this phenomenon has important implications for treating ischemic cell death. A series of studies indicate that uncontrolled activity of TRP channels may be a major factor leading to anoxic cell death. The first evidence linking metabolic stress to cell death was obtained in studies of TRPC channels. Drosophila TRP and TRPL are constitutively active in vivo under anoxic conditions or as a result of application of mitochondrial uncouplers or depletion of ATP (48). Furthermore, mutations that cause constitutive activation of TRP result in neurodegeneration in Drosophila photoreceptor cells (279). Oxidative stress may also result in activation of mammalian TRPC proteins. Endothelial cells express an oxidant-activated nonselective cation channel that functions as a redox sensor in the vascular endothelium, and a dominant-negative form of TRPC3 abolishes the oxidantinduced current (75). These experiments suggest that either TRPC3 or a channel capable of heteromultimerizing with TRPC3 contributes to this conductance. On the basis of these studies, oxidative stress in the mammalian brain could potentially result in constitutive activation of TRP proteins, which in turn could result in cell death due to uncontrolled influx of Ca2+. The potential contribution of TRP channel to hypoxia-induced cell death has received considerable support from analyses of two TRPM channels: TRPM2 and TRPM7. TRPM2 can be activated by H2O2, which leads to elevated levels of β-NAD+ (174, 280). Disruption of native TRPM2 expression in a rat insulinoma cell line suppressed the H2O2-induced cell death (174). Thus, TRPM2 may lead to Ca2+ overload and neuronal cell death as a result of a change in redox status. Prolonged oxygen and glucose deprivation appears to lead to activation of TRPM7 in neurons, and blocking TRPM7 expression in primary neurons inhibited the cell death resulting from anoxia (281). Thus, drugs that inhibit TRP proteins would offer a new therapy for minimizing the neurodegeneration associated with strokes and other traumas that induce oxidative stress. Heteromultimeric Interactions Among TRP Family Members A recurring theme is that many TRPs in native systems form heteromultimeric channels composed of two or more TRP subunits. In Drosophila photoreceptor cells, the three TRPC proteins that mediate the light-activated conductance assemble to form TRP homomultimers and heteromultimers of TRPL and TRP or TRPL and TRPγ (23, 25). TRP is ~10-fold more abundant than TRPL and TRPγ and forms a regulated channel in the absence of other subunits. Thus, TRP may be expressed in vivo primarily as a homomultimer. By contrast, in vitro expression of just TRPL or TRPγ results in constitutive activity of these channels. However, TRP/TRPL and TRPL/TRPγ heteromultimers are regulated channels with biophysical properties distinct from those of the homomultimers. The homo- and heteromultimeric interactions between the TRP subunits occur through the coiled-coiled region in the N-terminal segments (25). In addition, the transmembrane segments also contribute to subunit interactions. Heteromultimeric interactions also occur among mammalian TRPC channels. Based on analyses in human embryonic kidney 293 (HEK293) cells (282) and in the brain (283), there is a

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REVIEW strong preference for interactions between those channels that belong to the same subset: TRPC4/5 and TRPC3/6/7. TRPC1 displays a propensity to interact with TRPC4 and TRPC5, whereas TRPC2 does not appear to heteromultimerize with other channels. An important question concerns the functions of the heteromultimeric interactions. Possibilities include effects on the channel localization or stability and alterations in the biophysical properties of the currents. Each of the TRPC proteins has been expressed individually in tissue culture cells, and in many cases expression of these proteins results in the appearance of constitutive cation influx [for example, see (60, 62–65, 284-286)]. This property of many TRPC-dependent currents is similar to the constitutive influx resulting from in vitro expression of either Drosophila TRPL or TRPγ. TRPL/TRPγ coassemble to produce a PLC-regulated channel (25). Thus, it is plausible that some TRPC proteins are channel subunits that depend on interactions with other TRPC proteins for regulated activity. Consistent with this proposal, a TRPC3-dependent conductance endogenous to pontine neurons is not constitutive; rather, it is activated through a signaling pathway involving TrkB and PLC-γ (84). Whether TRPC3 interacts with another TRPC protein in vivo has not been addressed, although TRPC3 does interact in vitro with TRPC1 (23). However, in contrast to TRPL/TRPγ heteromultimers, coexpression of TRPC1 and TRPC3 in tissue culture cells generates a larger constitutively active conductance than do either of the individual proteins (287). Thus, if TRPC1 and TRPC3 form heteromultimers in vivo, they may include additional subunits to form regulated channels. TRPC1 forms functional heteromultimers with either TRPC4 or TRPC5 in the mammalian brain. TRPC4 and TRPC5 coimmunoprecipitate with TRPC1 from rat brains (85). Moreover, coexpression of either TRPC4 or TRPC5 with TRPC1 in tissue culture cells results in the production of nonselective cation conductances distinct from those generated by expression of the individual proteins (85). The conductances arising from TRPC1/TRPC4 and TRPC1/TRPC5 heteromultimers are augmented by activation of receptors that engage Gq proteins [the G protein family of α subunits that controls phosphoinositide (PI)-specific PLCs], but not by release of Ca2+ from internal stores. However, constitutive activity occurs in the absence of receptor activation. Thus, as is the case with TRPC1/TRPC3 heteromultimers, it is likely that additional subunits interact with and participate in the regulation of TRPC1/TRPC4 and TRPC1/TRPC5 heteromultimers in vivo. Indeed, there is evidence that TRPC heteromultimers consist of three TRP subunits (288). These include TRPC1/3/5, as well as other combinations of subunits: TRPC1 with TRPC4 or -5 and TRPC3 or -6. Thus, differential TRPC subunit interactions may give rise to a daunting diversity of channels and conductances. Heteromultimerization is not limited to TRPC, but appears to typify members of other TRP subfamilies, such as TRPV and TRPM. The C. elegans TRPV proteins, OSM-9 and OCR-2, may form heteromultimers in vivo, because the two proteins mutually influence their subcellular localizaton in those sensory cilia that normally coexpress both proteins (289). In a similar fashion, it has been proposed that the Drosophila TRPV channels, Iav and Nan, interact directly, because elimination of one protein results in instability of the other in cilia required for the auditory response (101). Several lines of evidence indicate that the mammalian TRPV5 and TRPV6 form functional heteromultimers

(11). Both channels are coexpressed in kidneys, and tagged versions of the proteins coimmunoprecipitate from Xenopus oocytes. Moreover, coassembly of TRPV5/6 heteromultimers alters several biophysical properties, including Ba2+ selectivity and Ca 2+-dependent inactivation, from those observed for homomultimeric channels. The two chanzymes with atypical protein kinase domains, TRPM6 and TRPM7, are also coexpressed in several tissues, such as kidneys, and interact directly (167). The TRPM6/7 heteromultimer display a larger conductance than either homomultimer. In HEK293 cells, the heteromultimeric interactions are required for insertion of TRPM6 in the plasma membrane. Interestingly, a mutation in TRPM6 that causes hypomagnesemia and hypocalcemia disrupts association with TRPM7 and insertion in the plasma membrane. It remains to be determined whether members of different TRP subfamilies associate in vivo. However, TRPC1, but not TRPC3, interacts directly with the TRPP2 in vitro (290). The functional implications of this interaction have not been described. Association of TRP Channels into Macromolecular Assemblies The Drosophila signalplex. It now appears that many members of the TRP superfamily exist in macromolecular assemblies composed of multiple signaling components. The existence of a TRP-containing supramolecular signaling complex (signalplex) was first demonstrated in Drosophila photoreceptor cells (7). The molecular scaffold for the signalplex is INAD (inactivationno-afterpotential D), a protein that consists of five ~90–amino acid protein domains referred to as PDZ (PSD-95, DLG, zona occludens–1) domains. INAD binds directly to a minimum of seven proteins that function in phototransduction (Fig. 4A). These include TRP (291, 292), TRPL (293, 294), PLC-β (292, 295), rhodopsin (293, 295), protein kinase C (PKC) (293, 296), calmodulin (293, 295), and the NINAC (neither-inactivationnor-afterpotential C) myosin III (297). INAD also binds in vitro to the Drosophila homolog of the FK506-binding protein, dFKBP59 (294). This latter protein also interacts with TRPL and down-regulates the channel, although a role for dFKBP59 in the photoresponse has not been described. The number of proteins that bind to INAD exceeds the number of PDZ domains. INAD is capable of forming homomeric interactions (293), thus providing the binding capacity to simultaneously nucleate a large array of target proteins. Of primary importance is the identification of the functions of the signalplex. Because light-dependent cation influx occurs within milliseconds of activation, it would seem that coupling of the signaling components into a macromolecular assembly would serve to facilitate rapid activation. However, deletion of the INAD binding site in TRP has no effect on the kinetics of activation (298). Thus, a direct association of TRP with INAD is not required for the light response. Nevertheless, it has not been excluded that the mutated TRP still associates indirectly with the signalplex and that such an interaction could contribute to activation. One role of the signalplex is to retain signaling proteins in the microvillar portion of the photoreceptor cells, the rhabdomeres. In wild-type photoreceptor cells, the proteins that participate in phototransduction are highly enriched in the rhabdomeres (7, 299), which are the functional equivalent of the

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REVIEW outer segments in mammalian photoreceptor cells. In inaD of INAD with NINAC has a direct role in signaling. Mutation mutant flies, the localizations of at least three INAD targets, of the INAD-binding site in NINAC has no effect on protein TRP, PKC, and PLC, are severely disrupted (295, 299). INAD stability or rhabdomeral localization, but causes a profound functions in retention rather than targeting of these proteins delay in termination (297). The basis for the requirement for to the rhabdomeres (298, 300). In addition, elimination of the NINAC/INAD interaction for response termination is not INAD or the INAD-binding sites in TRP, PKC, or PLC results known, although the observations that NINAC binds actin in instability of these INAD-binding proteins (298, 299, 301). and that INAD associates with both NINAC and TRP raises the The requirement for the TRP-INAD interaction for retention possibility that actin or myosin force generation functions in in the rhabdomeres is reciprocal. Mutation of the INAD-binding turning off the light-sensitive cation channels. site in TRP results in mislocalization of INAD and, as a conseMammalian TRP supramolecular complexes. Mammalian quence, mislocalization of PLC, PKC, and TRP (298). Elimination TRPC proteins also appear to be organized into macromolecuof any other known INAD-binding protein has no effect on the lar assemblies. For example, TRPC3 is activated through a pathlocalization of INAD. Thus, it appears that TRP and INAD form way initiated by the BDNF receptor TrkB, and TRPC3 immunothe core complex required for retention of the signalplex in the precipitates with the TrkB from rat brains (84). This interaction rhabdomeres. Decreases in the concentration of signaling proteins in the rhabdomeres, due to disruption of INAD-target protein interactions, have at least two effects on phototransduction. First, the overall amplitude of photoresponse is reduced (299). Second, a reduction in the levels of PLC result in slower response termination (301, 302). This defect may be due to loss of the proper stoichiometry between the PLC and the G protein (302). The relative concentrations of these two proteins are critical because the PLC functions as a GTPase-activating protein for the trimeric Gα q subunit (302, 303), in addition to its more recognized phospholipase activity (304). Thus, a reduction in the levels of PLC results in delayed termination, due to slower inactivation of the G protein. Therefore, the signalplex maintains both the proper stoichiometry and absolute concentrations of signaling proteins in the rhabdomeres. A key question is whether the association of any target protein with INAD functions directly in the photoresponse, Fig. 4. Classical TRP proteins associate with signaling complexes. (A) Model of the Drosophila signalplex. independent of any require- INAD is a scaffold protein with five ~90–amino acid PDZ domains that bind directly to TRP, TRPL, ment for retention or protein PLC-β, PKC, rhodopsin (Rh), the NINAC myosin III, and calmodulin (CaM). The signalplex could be stability. Disruption of the linked to actin filaments through NINAC. INAD is also capable of forming homomultimers. (B) Speculative INAD-binding site in PKC model of the TRPC4 signalplex. TRPC4 binds directly to a protein, NHERF (also known as EBP50), decreases the rate of termina- containing two PDZ domains and a C-terminal domain that is an ERM binding domain (EBD). tion of the photoresponse Although NHERF can bind to at least one G protein–coupled receptor (GPCR) and members of the (305), although this effect ERM family, the TRPC4 signalplex has not been shown to include a GPCR, ezrin, or any other may be due to mislocalization actin-binding protein. Thus, these latter interactions are speculative. The complexity of the NHERF of PKC. However, interaction signalplex may be increased by homomultimerization of NHERF and TRPC4.

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REVIEW is most likely indirect, although the molecular link between these two proteins has not been identified. TRPC1 is localized to a subset of lipid rafts referred to as caveolae, where it may also form part of a multicomponent complex. Lipid rafts are glycosphingolipid- and cholesterol-enriched membrane microdomains that appear to concentrate certain transmembrane proteins and proteins with glycosylphosphatidylinositol anchors or hydrophobic modifications (306–309). Caveolae are invaginations in the plasma membrane that for m through coalescence of lipid rafts. Caveolae may have particular importance in Ca2+ signaling because they are enriched with various proteins that participate in Ca2+ regulation and may be sites for Ca2+ entry and sequestration (310). Caveolin, a transmembrane cholesterol-binding protein that is concentrated in caveolae (311–313), may be a scaffolding protein that nucleates signaling complexes (314). TRPC1 appears to be localized to caveolin-containing lipid rafts and coimmunoprecipitates with caveolin, the IP3R, and Gαq from a salivary gland cell line (315). Furthermore, thapsigargin-induced Ca2+ influx is disrupted in this cell line upon depletion of cholesterol from the plasma membrane. Because cholesterol depletion disrupts lipid raft domains, this suggests that TRPC1 function is dependent on association with caveolae. However, there is no direct evidence that the current was mediated by TRPC1, and it remains to be determined whether TRPC1 binds directly to caveolin. In vitro studies indicate that TRPC4 and TRPC5 associate with macromolecular complexes that bear similarities to the Drosophila signalplex (Fig. 4B) (316). The central protein in these complexes is the Na+/H+ exchanger regulatory factor (NHERF, also referred to as EBP50), a protein containing two PDZ domains (317, 318). Mutation of the C terminus of TRPC4 disrupts its interaction with NHERF and translocation of the channel to the plasma membrane (319). In addition to TRPC4 and TRPC5, in vitro NHERF also binds to PLC-β (316). Moreover, TRPC4, PLC, and NHERF coimmunoprecipitate from mouse brain cells. PLC and TRPC4 are unlikely to bind to the same NHERF monomer, because they both interact with NHERF through PDZ1. As with INAD, NHERF appears to self-associate, and such homomultimerization could provide NHERF with the capacity to cluster an array of proteins (316). The complexity of the NHERF signalplex could be further increased by multimerization of TRPC4 or TRPC5 (Fig. 4B). Other known targets for NHERF include a G protein– coupled receptor (GPCR) (320) and members of the ezrin-radixinmoesin (ERM) family (321), which could provide a link to the actin cytoskeleton. It remains to be determined whether these latter classes of proteins are complexed with the same NHERF molecules that associate with TRPC4 and PLC-β. If so, then mammalian TRPC proteins may be organized into signaling complexes that resemble the Drosophila signalplex. The next challenge will be to determine whether the TRPC4/NHERF signalplex contributes to signaling, as well as to the localization and stability of the component proteins, as is the case in Drosophila photoreceptor cells. Several TRPC proteins have been shown to form a multimeric complex with either of several Homer isoforms. Homer is an EVH1 domain scaffold protein that binds to proline-containing sequences similar to PPXXF (322, 323). Homer binds to TRPC1 in the brain, and the interaction appears to occur through two sites in the channel, N- and C-terminal to the transmembrane domains (26). The C-terminal Homer interaction sequence

corresponds to TRP box 2 (Fig. 2). Both Homer and several TRPCs bind to the IP3R (79, 81, 323), and the Homer/TRPC1 interaction promotes the direct interaction between the TRPC1 and the IP3R (26). Interestingly, depletion of Ca 2+ stores disrupts the association of TRPC1 with Homer and the IP3R, resulting in increased Ca2+ influx. The TRPC/Homer/IP3R complex may also contain group 1 metabotropic glutamate receptors (mGluR1) because these GPCRs bind to Homer (322). Moreover, mGluR1 associates in a complex with TRPC1 (264), although it is not known whether the mGluR1/TRPC1 interaction is Homer-dependent. TRPV5 and TRPV6 also appear to be complexed with a macromolecular assembly because these channels associate with a complex that includes annexin 2 and S100A10 (324). Annexins are phospholipid-binding proteins, some of which shuttle between intracellular membranes and the cell surface in a Ca2+-dependent manner. The association between S100A10 can augment phospholipid binding of annexin 2. The interaction between TRPV5 or TRPV6 and the annexin 2/S100A10 complex promotes surface localization of the channels. However, it remains to be determined whether this reflects a role for annexin 2/S100A10 in trafficking or retention of TRPV5/6. In addition to TRPC and TRPV complexes mentioned above, it seems likely that most TRP channels may be linked to massive protein complexes. In addition to altering TRP channel localization, stability, and activity, it is possible that macromolecular assemblies may also contribute to the speed and specificity in signaling. Now that the members of the TRP superfamily have been identified, one of the many challenges in the field will be to define the nature and functions of each channel and the associated supramolecular complexes. References 1. P. D. Wes, J. Chevesich, A. Jeromin, C. Rosenberg, G. Stetten, C. Montell, TRPC1, a human homolog of a Drosophila store-operated channel. Proc. Natl. Acad. Sci. U.S.A. 92, 9652–9656 (1995). 2. X. Zhu, P. B. Chu, M. Peyton, L. Birnbaumer, Molecular cloning of a widely expressed human homologue for the Drosophila trp gene. FEBS Lett. 373, 193–198 (1995). 3. A. B. Parekh, R. Penner, Store depletion and calcium influx. Physiol. Rev. 77, 901–930 (1997). 4. J. W. Putney Jr., R. R. McKay, Capacitative calcium entry channels. Bioessays 21, 38–46 (1999). 5. C. Montell, K. Jones, E. Hafen, G. Rubin, Rescue of the Drosophila phototransduction mutation trp by germline transformation. Science 230, 1040–1043 (1985). 6. C. Montell, G. M. Rubin, Molecular characterization of the Drosophila trp locus: A putative integral membrane protein required for phototransduction. Neuron 2, 1313–1323 (1989). 7. C. Montell, Drosophila visual transduction. Annu. Rev. Cell Dev. Biol. 15, 231–268 (1999). 8. R. C. Hardie, P. Raghu, Visual transduction in Drosophila. Nature 413, 186–193 (2001). 9. W. A. Catterall, From ionic currents to molecular mechanisms: The structure and function of voltage-gated sodium channels. Neuron 26, 13–25 (2000). 10. H. Amiri, G. Schultz, M. Schaefer, FRET-based analysis of TRPC subunit stoichiometry. Cell Calcium 33, 463–470 (2003). 11. J. G. Hoenderop, T. Voets, S. Hoefs, F. Weidema, J. Prenen, B. Nilius, R. J. Bindels, Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6. EMBO J. 22, 776–785 (2003). 12. N. Kedei, T. Szabo, J. D. Lile, J. J. Treanor, Z. Olah, M. J. Iadarola, P. M. Blumberg, Analysis of the native quaternary structure of vanilloid receptor 1. J. Biol. Chem. 276, 28613–28619 (2001). 13. E. V. Kuzhikandathil, H. Wang, T. Szabo, N. Morozova, P. M. Blumberg, G. S. Oxford, Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation. J. Neurosci. 21, 8697–8706 (2001). 14. C. Montell, L. Birnbaumer, V. Flockerzi, R. J. Bindels, E. A. Bruford, M. J. Caterina, D. E. Clapham, C. Harteneck, S. Heller, D. Julius, Y. Mori, R.

www.stke.org/cgi/content/full/sigtrans;2005/272/re3

Page 17

REVIEW

15. 16. 17.

18.

19. 20. 21. 22.

23.

24.

25.

26.

27.

28.

29.

30. 31.

32.

33.

34.

35.

36.

37.

38.

39.

Penner, D. Prawitt, A. M. Scharenberg, G. Schultz, N. Shimizu, M. X. Zhu, A unified nomenclature for the superfamily of TRP cation channels. Mol. Cell 9, 229–231 (2002). R. G. Walker, A. T. Willingham, C. S. Zuker, A Drosophila mechanosensory transduction channel. Science 287, 2229–2234 (2000). S. Sidi, R. W. Friedrich, T. Nicolson, NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Science 301, 96–99 (2003). V. Denis, M. S. Cyert, Internal Ca2+ release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue. J. Cell Biol. 156, 29–34 (2002). C. P. Palmer, X. L. Zhou, J. Lin, S. H. Loukin, C. Kung, Y. Saimi, A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+permeable channel in the yeast vacuolar membrane. Proc. Natl. Acad. Sci. U.S.A. 98, 7801–7805 (2001). M. Bonilla, K. W. Cunningham, Calcium release and influx in yeast: TRPC and VGCC rule another kingdom. Sci. STKE 2002, pe17 (2002). D. J. Cosens, A. Manning, Abnormal electroretinogram from a Drosophila mutant. Nature 224, 285–287 (1969). R. C. Hardie, B. Minke, The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron 8, 643–651 (1992). L. Vaca, W. G. Sinkins, Y. Hu, D. L. Kunze, W. P. Schilling, Activation of recombinant trp by thapsigargin in Sf9 insect cells. Am. J. Physiol. 266, C1501–C1505 (1994). X.-Z. S. Xu, H.-S. Li, W. B. Guggino, C. Montell, Coassembly of TRP and TRPL produces a distinct store-operated conductance. Cell 89, 1155–1164 (1997) Cell 89, 1155–1164 (1997. A. M. Phillips, A. Bull, L. E. Kelly, Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene. Neuron 8, 631–642 (1992). X. Z. Xu, F. Chien, A. Butler, L. Salkoff, C. Montell, TRPγ, a Drosophila TRP-related subunit, forms a regulated cation channel with TRPL. Neuron 26, 647–657 (2000). J. P. Yuan, K. Kiselyov, D. M. Shin, J. Chen, N. Shcheynikov, S. H. Kang, M. H. Dehoff, M. K. Schwarz, P. H. Seeburg, S. Muallem, P. F. Worley, Homer binds TRPC family channels and is required for gating of TRPC1 by IP3 receptors. Cell 114, 777–789 (2003). B. A. Niemeyer, E. Suzuki, K. Scott, K. Jalink, C. S. Zuker, The Drosophila light-activated conductance is composed of the two channels TRP and TRPL. Cell 85, 651–659 (1996). H. Reuss, M. H. Mojet, S. Chyb, R. C. Hardie, In vivo analysis of the Drosophila light-sensitive channels, TRP and TRPL. Neuron 19, 1249–1259 (1997). H. T. Leung, C. Geng, W. L. Pak, Phenotypes of trpl mutants and interactions between the transient receptor potential (TRP) and TRP-like channels in Drosophila. J. Neurosci. 20, 6797–6803 (2000). X. Z. Xu, P. W. Sternberg, A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization. Cell 114, 285–297 (2003). C. C. H. Petersen, M. J. Berridge, M. F. Borgese, D. L. Bennett, Putative capacitative calcium entry channels: Expression of Drosophila trp and evidence for the existence of vertebrate homologues. Biochem. J. 311, 41–44 (1995). O. Thastrup, A. P. Dawson, O. Scharff, B. Foder, P. J. Cullen, B. K. Drobak, P. J. Bjerrum, S. B. Christensen, M. R. Hanley, Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Actions 27, 17–23 (1989). O. Thastrup, P. J. Cullen, B. K. Drobak, M. R. Hanley, A. P. Dawson, Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. U.S.A. 87, 2466–2470 (1990). R. Ranganathan, B. J. Bacskai, R. Y. Tsein, C. S. Zuker, Cytosolic calcium transients: Spatial localization and role in Drosophila photoreceptor cell function. Neuron 13, 837–848 (1994). R. C. Hardie, Excitation of Drosophila photoreceptors by BAPTA and ionomycin: Evidence for capacitative Ca2+ entry? Cell Calcium 20, 315–327 (1996). R. C. Hardie, P. Raghu, Activation of heterologously expressed Drosophila TRPL channels: Ca 2+ is not required and InsP 3 is not sufficient. Cell Calcium 24, 153–163 (1998). G. Hasan, M. Rosbash, Drosophila homologs of two mammalian intracellular Ca2+-release channels: Identification and expression patterns of the inositol 1,4,5-trisphosphate and the ryanodine receptor genes. Development 116, 967–975 (1992). S. Yoshikawa, T. Tanimura, A. Miyawaki, M. Nakamura, M. Yusaki, T. Furuichi, K. Mikoshiba, Molecular cloning and characterization of the inositol 1,4,5-trisphosphate receptor in Drosophila melanogaster. J. Biol. Chem. 267, 16613–16619 (1992). P. Raghu, N. J. Colley, R. Webel, T. James, G. Hasan, M. Danin, Z. Selinger, R. C. Hardie, Normal phototransduction in Drosophila photoreceptors lacking an InsP 3 receptor gene. Mol. Cell. Neurosci. 15, 429–445 (2000).

40. J. K. Acharya, K. Jalink, R. W. Hardy, V. Hartenstein, C. S. Zuker, InsP3 receptor essential for growth and differentiation but not for vision in Drosophila. Neuron 18, 881–887 (1997). 41. H. Takeshima, M. Nishi, N. Iwabe, T. Miyata, T. Hosoya, I. Masai, Y. Hotta, Isolation and character ization of a gene for a r yanodine receptor/calcium release channel in Drosophila melanogaster. FEBS Lett. 337, 81–87 (1994). 42. K. M. Sullivan, K. Scott, C. S. Zuker, G. M. Rubin, The ryanodine receptor is essential for larval development in Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. 97, 5942–5947 (2000). 43. M. Estacion, W. G. Sinkins, W. P. Schilling, Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms. J. Physiol. 530, 1–19 (2001). 44. S. Chyb, P. Raghu, R. C. Hardie, Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Nature 397, 255–259 (1999). 45. P. Raghu, K. Usher, S. Jonas, S. Chyb, A. Polyanovsky, R. C. Hardie, Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA. Neuron 26, 169–179 (2000). 46. B. T. Bloomquist, R. D. Shortridge, S. Schneuwly, M. Perdew, C. Montell, H. Steller, G. Rubin, W. L. Pak, Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction. Cell 54, 723–733 (1988). 47. R. C. Hardie, F. Martin, G. W. Cochrane, M. Juusola, P. Georgiev, P. Raghu, Molecular basis of amplification in Drosophila phototransduction: Roles for G protein, phospholipase C, and diacylglycerol kinase. Neuron 36, 689–701 (2002). 48. K. Agam, M. von Campenhausen, S. Levy, H. C. Ben-Ami, B. Cook, K. Kirschfeld, B. Minke, Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo. J. Neurosci. 20, 5748–5755 (2000). 49. K. Scott, Y. Sun, K. Beckingham, C. S. Zuker, Calmodulin regulation of Drosophila light-activated channels and receptor function mediates termination of the light response in vivo. Cell 91, 375–383 (1997). 50. B. Cook, B. Minke, TRP and calcium stores in Drosophila phototransduction. Cell Calcium 25, 161–171 (1999). 51. R. C. Hardie, P. Raghu, S. Moore, M. Juusola, A. Baines, S. T. Sweeney, Calcium influx via TRP channels is required to maintain PIP2 levels in Drosophila photoreceptors. Neuron 30, 149–159 (2001). 52. X. Zhu, M. Jiang, M. Peyton, G. Boulay, R. Hurst, E. Stefani, L. Birnbaumer, trp, a novel mammalian gene family essential for agonistactivated capacitative Ca2+ entry. Cell 85, 661–671 (1996). 53. U. Wissenbach, G. Schroth, S. Philipp, V. Flockerzi, Structure and mRNA expression of a bovine trp homologue related to mammalian trp2 transcripts. FEBS Lett. 429, 61–66 (1998). 54. B. Vannier, X. Zhu, D. Brown, L. Birnbaumer, The membrane topology of human transient receptor potential 3 as inferred from glycosylationscanning mutagenesis and epitope immunocytochemistry. J. Biol. Chem. 273, 8675–8679 (1998). 55. E. R. Liman, D. P. Corey, C. Dulac, TRP2: A candidate transduction channel for mammalian pheromone sensory signaling. Proc. Natl. Acad. Sci. U.S.A. 96, 5791–5796 (1999). 56. S. Philipp, A. Cacalié, M. Freichel, U. Wissenbach, S. Zimmer, C. Trost, A. Marquart, M. Murakami, V. Flockerzi, A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO J. 15, 6166–6171 (1996). 57. S. Philipp, J. Hambrecht, L. Braslavski, G. Schroth, M. Freichel, M. Murakami, A. Cavalie, V. Flockerzi, A novel capacitative calcium entry channel expressed in excitable cells. EMBO J. 17, 4274–4282 (1998). 58. T. Okada, S. Shimizu, M. Wakamori, A. Maeda, T. Kurosaki, N. Takada, K. Imoto, Y. Mori, Molecular cloning and functional characterization of a novel receptor- activated TRP Ca2+ channel from mouse brain. J. Biol. Chem. 273, 10279–10287 (1998). 59. G. Boulay, X. Zhu, M. Peyton, M. S. Jiang, R. Hurst, E. Stefani, L. Birnbaumer, Cloning and expression of a novel mammalian homolog of Drosophila Transient Receptor Potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J. Biol. Chem. 272, 29672–29680 (1997). 60. T. Okada, R. Inoue, K. Yamazaki, A. Maeda, T. Kurosaki, T. Yamakuni, I. Tanaka, S. Shimizu, K. Ikenaka, K. Imoto, Y. Mori, Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca 2+ -permeable cation channel that is constitutively activated and enhanced by stimulation of G proteincoupled receptor. J. Biol. Chem. 274, 27359–27370 (1999). 61. B. Vannier, M. Peyton, G. Boulay, D. Brown, N. Qin, M. Jiang, X. Zhu, L. Birnbaumer, Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel. Proc. Natl. Acad. Sci. U.S.A. 96, 2060–2064 (1999). 62. R. S. Hurst, X. Zhu, G. Boulay, L. Birnbaumer, E. Stefani, Ionic currents

www.stke.org/cgi/content/full/sigtrans;2005/272/re3

Page 18

REVIEW

63.

64.

65.

66.

67.

68.

69. 70. 71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK293 cells. FEBS Lett. 422, 333–338 (1998). X. Zhu, M. Jiang, L. Birnbaumer, Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry. J. Biol. Chem. 273, 133–142 (1998). C. Zitt, A. G. Obukhov, C. Strubing, A. Zobel, F. Kalkbrenner, A. Luckhoff, G. Schultz, Expression of TRPC3 in chinese hamster ovary cells results in calcium-activated cation currents not related to store depletion. J. Cell Biol. 138, 1333–1341 (1997). C. Zitt, A. Zobel, A. G. Obukhov, C. Harteneck, F. Kalkbrenner, A. Lückhoff, G. Schultz, Cloning and functional expression of a human Ca2+permeable channel activated by calcium store depletion. Neuron 16, 1189–1196 (1996). C. Randriamampita, R. Y. Tsien, Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature 364, 809–814 (1993). D. Thomas, M. R. Hanley, Evaluation of calcium influx factors from stimulated Jurkat T-lymphocytes by microinjection into Xenopus oocytes. J. Biol. Chem. 270, 6429–6432 (1995). T. Smani, S. I. Zakharov, P. Csutora, E. Leno, E. S. Trepakova, V. M. Bolotina, A novel mechanism for the store-operated calcium influx pathway. Nat. Cell Biol. 6, 113–120 (2004). R. F. Irvine, ‘Quantal’ Ca2+ release and the control of Ca2+ entry by inositol phosphates—a possible mechanism. FEBS Lett. 263, 5–9 (1990). R. L. Patterson, D. B. van Rossum, D. L. Gill, Store-operated Ca2+ entry: Evidence for a secretion-like coupling model. Cell 98, 487–499 (1999). H. T. Ma, R. L. Patterson, D. B. van Rossum, L. Birnbaumer, K. Mikoshiba, D. L. Gill, Requirement of the inositol trisphosphate receptor for activation of store-operated Ca 2+ channels. Science 287, 1647–1651 (2000). T. Lockwich, B. B. Singh, X. Liu, I. S. Ambudkar, Stabilization of cortical actin induces internalization of transient receptor potential 3 (Trp3)-associated caveolar Ca2+ signaling complex and loss of Ca2+ influx without disruption of Trp3-inositol trisphosphate receptor association. J. Biol. Chem. 276, 42401–42408 (2001). K. Itagaki, K. B. Kannan, B. B. Singh, C. J. Hauser, Cytoskeletal reorganization internalizes multiple transient receptor potential channels and blocks calcium entry into human neutrophils. J. Immunol. 172, 601–607 (2004). T. Hofmann, A. G. Obukhov, M. Schaefer, C. Harteneck, T. Gudermann, G. Schultz, Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259–263 (1999). M. Balzer, B. Lintschinger, K. Groschner, Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells. Cardiovasc. Res. 42, 543–549 (1999). P. Lucas, K. Ukhanov, T. Leinders-Zufall, F. Zufall, A diacylglycerol-gated cation channel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice: Mechanism of pheromone transduction. Neuron 40, 551–561 (2003). M. K. Jungnickel, H. Marrero, L. Birnbaumer, J. R. Lemos, H. M. Florman, Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3. Nat. Cell Biol. 3, 499–502 (2001). T. Hofmann, M. Schaefer, G. Schultz, T. Gudermann, Cloning, expression and subcellular localization of two novel splice variants of mouse transient receptor potential channel 2. Biochem. J. 351, 115–122 (2000). K. Kiselyov, X. Xu, G. Mozhayeva, T. Kuo, I. Pessah, G. Mignery, X. Zhu, L. Birnbaumer, S. Muallem, Functional interaction between InsP3 receptors and store-operated Htrp3 channels. Nature 396, 478–482 (1998). K. Kiselyov, G. A. Mignery, M. X. Zhu, S. Muallem, The N-terminal domain of the IP3 receptor gates store-operated hTrp3 channels. Mol. Cell 4, 423–429 (1999). G. Boulay, D. M. Brown, N. Qin, M. Jiang, A. Dietrich, M. X. Zhu, Z. Chen, M. Birnbaumer, K. Mikoshiba, L. Birnbaumer, Modulation of Ca2+ entry by polypeptides of the inositol 1,4, 5- trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): Evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry. Proc. Natl. Acad. Sci. U.S.A. 96, 14955–14960 (1999). J. A. Rosado, S. O. Sage, Coupling between inositol 1,4,5-trisphosphate receptors and human transient receptor potential channel 1 when intracellular Ca2+ stores are depleted. Biochem. J. 350, 631–635 (2000). K. I. Kiselyov, D. M. Shin, Y. Wang, I. N. Pessah, P. D. Allen, S. Muallem, Gating of store-operated channels by conformational coupling to ryanodine receptors. Mol. Cell 6, 421–431 (2000). H.-S. Li, X.-Z. S. Xu, C. Montell, Activation of a TRPC3-dependent cation current channel through the neurotrophin BDNF. Neuron 24, 261–273 (1999). C. Strübing, G. Krapivinsky, L. Krapivinsky, D. E. Clapham, TRPC1 and TRPC5 form a novel cation channel in mammalian brain. Neuron 29, 645–655 (2001).

86. M. C. Buniel, W. P. Schilling, D. L. Kunze, Distribution of transient receptor potential channels in the rat carotid chemosensory pathway. J. Comp. Neurol. 464, 404–413 (2003). 87. B. Somasundaram, J. C. Norman, M. P. Mahaut-Smith, Primaquine, an inhibitor of vesicular transport, blocks the calcium- release-activated current in rat megakaryocytes. Biochem. J. 309, 725–729 (1995). 88. Y. Yao, A. V. Ferrer-Montiel, M. Montal, R. Y. Tsien, Activation of storeoperated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell 98, 475–485 (1999). 89. S. Cayouette, M. P. Lussier, E. L. Mathieu, S. M. Bousquet, G. Boulay, Exocytotic insertion of TRPC6 channel into the plasma membrane upon Gq-protein-coupled receptor activation. J. Biol. Chem. 279, 7241–7246 (2004). 90. D. Mehta, G. U. Ahmmed, B. C. Paria, M. Holinstat, T. Voyno-Yasenetskaya, C. Tiruppathi, R. D. Minshall, A. B. Malik, RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+ entry. Role in signaling increased endothelial permeability. J. Biol. Chem. 278, 33492–33500 (2003). 91. B. B. Singh, T. P. Lockwich, B. C. Bandyopadhyay, X. Liu, S. Bollimuntha, S. Brazer, C. Combs, S. Das, A. G. Leenders, Z. Sheng, M. A. Knepper, S. V. Ambudkar, I. S. Ambudkar, VAMP-2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx. Mol. Cell 15, 635–646 (2004). 92. V. Bezzerides, S. Ramsey, A. Greka, D. E. Clapham, Rapid vesicular translocation and insertion of TRP channels. Nat. Cell Biol. 6, 709–720 (2004). 93. M. Bähner, S. Frechter, N. Da Silva, B. Minke, R. Paulsen, A. Huber, Light-regulated subcellular translocation of Drosophila TRPL channels induces long-term adaptation and modifies the light-induced current. Neuron 34, 83–93 (2002). 94. A. Greka, B. Navarro, E. Oancea, A. Duggan, D. E. Clapham, TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nat. Neurosci. 6, 837–845 (2003). 95. H. A. Colbert, T. L. Smith, C. I. Bargmann, OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J. Neurosci. 17, 8259–8269 (1997). 96. M. J. Caterina, M. A. Schumacher, M. Tominaga, T. A. Rosen, J. D. Levine, D. Julius, The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 389, 816–824 (1997). 97. H. A. Colbert, C. I. Bargmann, Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans. Neuron 14, 803–812 (1995). 98. M. de Bono, D. M. Tobin, M. W. Davis, L. Avery, C. I. Bargmann, Social feeding in Caenorhabditis elegans is induced by neurons that detect aversive stimuli. Nature 419, 899–903 (2002). 99. M. de Bono, C. I. Bargmann, Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans. Cell 94, 679–689 (1998). 100. J. Kim, Y. D. Chung, D. Y. Park, S. Choi, D. W. Shin, H. Soh, H. W. Lee, W. Son, J. Yim, C. S. Park, M. J. Kernan, C. Kim, A TRPV family ion channel required for hearing in Drosophila. Nature 424, 81–84 (2003). 101. Z. Gong, W. Son, Y. D. Chung, J. Kim, D. W. Shin, C. A. McClung, Y. Lee, H. W. Lee, D. J. Chang, B. K. Kaang, H. Cho, U. Oh, J. Hirsh, M. J. Kernan, C. Kim, Two interdependent TRPV channel subunits, inactive and Nanchung, mediate hearing in Drosophila. J. Neurosci. 24, 9059–9066 (2004). 102. P. M. Zygmunt, J. Petersson, D. A. Andersson, H. Chuang, M. Sorgard, V. Di Marzo, D. Julius, E. D. Hogestatt, Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400, 452–457 (1999). 103. M. Tominaga, M. J. Caterina, A. B. Malmberg, T. A. Rosen, H. Gilbert, K. Skinner, B. E. Raumann, A. I. Basbaum, D. Julius, The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21, 531–543 (1998). 104. S. E. Jordt, M. Tominaga, D. Julius, Acid potentiation of the capsaicin receptor determined by a key extracellular site. Proc. Natl. Acad. Sci. U.S.A. 97, 8134–8139 (2000). 105. H. H. Chuang, E. D. Prescott, H. Kong, S. Shields, S. E. Jordt, A. I. Basbaum, M. V. Chao, D. Julius, Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature 411, 957–962 (2001). 106. E. D. Prescott, D. Julius, A modular PIP2 binding site as a determinant of capsaicin receptor sensitivity. Science 300, 1284–1288 (2003). 107. M. J. Caterina, A. Leffler, A. B. Malmberg, W. J. Martin, J. Trafton, K. R. Petersen-Zeitz, M. Koltzenburg, A. I. Basbaum, D. Julius, Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288, 306–313 (2000). 108. J. B. Davis, J. Gray, M. J. Gunthorpe, J. P. Hatcher, P. T. Davey, P. Overend, M. H. Harries, J. Latcham, C. Clapham, K. Atkinson, S. A. Hughes, K. Rance, E. Grau, A. J. Harper, P. L. Pugh, D. C. Rogers, S.

www.stke.org/cgi/content/full/sigtrans;2005/272/re3

Page 19

REVIEW

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124. 125.

126.

127.

128.

129.

130.

Bingham, A. Randall, S. A. Sheardown, Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 405, 183–187 (2000). R. J. Helliwell, L. M. McLatchie, M. Clarke, J. Winter, S. Bevan, P. McIntyre, Capsaicin sensitivity is associated with the expression of the vanilloid (capsaicin) receptor (VR1) mRNA in adult rat sensory ganglia. Neurosci. Lett. 250, 177–180 (1998). C. Morenilla-Palao, R. Planells-Cases, N. García-Sanz, A. Ferrer-Montiel, Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity. J. Biol. Chem. 279, 25665–25672 (2004). M. J. Caterina, T. A. Rosen, M. Tominaga, A. J. Brake, D. Julius, A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 398, 436–441 (1999). H. Xu, I. S. Ramsey, S. A. Kotecha, M. M. Moran, J. A. Chong, D. Lawson, P. Ge, J. Lilly, I. Silos-Santiago, Y. Xie, P. S. DiStefano, R. Curtis, D. E. Clapham, TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 418, 181–186 (2002). G. D. Smith, M. J. Gunthorpe, R. E. Kelsell, P. D. Hayes, P. Reilly, P. Facer, J. E. Wright, J. C. Jerman, J. P. Walhin, L. Ooi, J. Egerton, K. J. Charles, D. Smart, A. D. Randall, P. Anand, J. B. Davis, TRPV3 is a temperature-sensitive vanilloid receptor-like protein. Nature 418, 186–190 (2002). A. M. Peier, A. J. Reeve, D. A. Andersson, A. Moqrich, T. J. Earley, A. C. Hergarden, G. M. Story, S. Colley, J. B. Hogenesch, P. McIntyre, S. Bevan, A. Patapoutian, A heat-sensitive TRP channel expressed in keratinocytes. Science 296, 2046–2049 (2002). A. D. Güler, H. Lee, T. Iida, I. Shimizu, M. Tominaga, M. Caterina, Heatevoked activation of the ion channel, TRPV4. J. Neurosci. 22, 6408–6414 (2002). H. Watanabe, J. Vriens, S. H. Suh, C. D. Benham, G. Droogmans, B. Nilius, Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells. J. Biol. Chem. 277, 47044–47051 (2002). M. K. Chung, H. Lee, A. Mizuno, M. Suzuki, M. J. Caterina, TRPV3 and TRPV4 mediate warmth-evoked currents in primary mouse keratinocytes. J. Biol. Chem. 279, 21569–21575 (2004). M. K. Chung, H. Lee, M. J. Caterina, Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J. Biol. Chem. 278, 32037–32046 (2003). M. Kanzaki, Y. Q. Zhang, H. Mashima, L. Li, H. Shibata, I. Kojima, Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nat. Cell Biol. 1, 165–170 (1999). K. Boels, G. Glassmeier, D. Herrmann, I. B. Riedel, W. Hampe, I. Kojima, J. R. Schwarz, H. C. Schaller, The neuropeptide head activator induces activation and translocation of the growth-factor-regulated Ca 2+ permeable channel GRC. J. Cell Sci. 114, 3599–3606 (2001). K. Muraki, Y. Iwata, Y. Katanosaka, T. Ito, S. Ohya, M. Shigekawa, Y. Imaizumi, TRPV2 is a component of osmotically sensitive cation channels in murine aortic myocytes. Circ. Res. 93, 829–838 (2003). W. Liedtke, Y. Choe, M. A. Marti-Renom, A. M. Bell, C. S. Denis, A. Sali, A. J. Hudspeth, J. M. Friedman, S. Heller, Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 103, 525–535 (2000). R. Strotmann, C. Harteneck, K. Nunnenmacher, G. Schultz, T. D. Plant, OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat. Cell Biol. 2, 695–702 (2000). U. Wissenbach, M. Bödding, M. Freichel, V. Flockerzi, Trp12, a novel Trp related protein from kidney. FEBS Lett. 485, 127–134 (2000). H. Watanabe, J. Vriens, J. Prenen, G. Droogmans, T. Voets, B. Nilius, Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature 424, 434–438 (2003). H. Watanabe, J. B. Davis, D. Smart, J. C. Jerman, G. D. Smith, P. Hayes, J. Vriens, W. Cairns, U. Wissenbach, J. Prenen, V. Flockerzi, G. Droogmans, C. D. Benham, B. Nilius, Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives. J. Biol. Chem. 277, 13569–13577 (2002). J. Vriens, H. Watanabe, A. Janssens, G. Droogmans, T. Voets, B. Nilius, Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4. Proc. Natl. Acad. Sci. U.S.A. 101, 396–401 (2004). W. Liedtke, D. M. Tobin, C. I. Bargmann, J. M. Friedman, Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 100 (Suppl. 2), 14531–14536 (2003). R. Vennekens, J. G. Hoenderop, J. Prenen, M. Stuiver, P. H. Willems, G. Droogmans, B. Nilius, R. J. Bindels, Permeation and gating properties of the novel epithelial Ca2+ channel. J. Biol. Chem. 275, 3963–3969 (2000). J. B. Peng, X. Z. Chen, U. V. Berger, P. M. Vassilev, H. Tsukaguchi, E. M. Brown, M. A. Hediger, Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J. Biol. Chem. 274, 22739–22746 (1999).

131. J. G. Hoenderop, R. Vennekens, D. Muller, J. Prenen, G. Droogmans, R. J. Bindels, B. Nilius, Function and expression of the epithelial Ca2+ channel family: Comparison of mammalian ECaC1 and 2. J. Physiol. 537, 747–761 (2001). 132. L. Yue, J. B. Peng, M. A. Hediger, D. E. Clapham, CaT1 manifests the pore properties of the calcium-release-activated calcium channel. Nature 410, 705–709 (2001). 133. T. Voets, J. Prenen, A. Fleig, R. Vennekens, H. Watanabe, J. G. Hoenderop, R. J. Bindels, G. Droogmans, R. Penner, B. Nilius, CaT1 and the calcium release-activated calcium channel manifest distinct pore properties. J. Biol. Chem. 276, 47767–47770 (2001). 134. R. J. West, A. Y. Sun, D. L. Church, E. J. Lambie, C. The, elegans gon-2 gene encodes a putative TRP cation channel protein required for mitotic cell cycle progression. Gene 266, 103–110 (2001). 135. L. M. Duncan, J. Deeds, J. Hunter, J. Shao, L. M. Holmgren, E. A. Woolf, R. I. Tepper, A. W. Shyjan, Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. Cancer Res. 58, 1515–1520 (1998). 136. J. J. Hunter, J. Shao, J. S. Smutko, B. J. Dussault, D. L. Nagle, E. A. Woolf, L. M. Holmgren, K. J. Moore, A. W. Shyjan, Chromosomal localization and genomic characterization of the mouse melastatin gene (Mlsn1). Genomics 54, 116–123 (1998). 137. J. Deeds, F. Cronin, L. M. Duncan, Patterns of melastatin mRNA expression in melanocytic tumors. Hum. Pathol. 31, 1346–1356 (2000). 138. D. Fang, V. Setaluri, Expression and Up-regulation of alternatively spliced transcripts of melastatin, a melanoma metastasis-related gene, in human melanoma cells. Biochem. Biophys. Res. Commun. 279, 53–61 (2000). 139. X. Z. Xu, F. Moebius, D. L. Gill, C. Montell, Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform. Proc. Natl. Acad. Sci. U.S.A. 98, 10692–10697 (2001). 140. C. Grimm, R. Kraft, S. Sauerbruch, G. Schultz, C. Harteneck, Molecular and functional characterization of the melastatin-related cation channel TRPM3. J. Biol. Chem. 278, 21493–21501 (2003). 141. N. Lee, J. Chen, L. Sun, S. Wu, K. R. Gray, A. Rich, M. Huang, J. H. Lin, J. N. Feder, E. B. Janovitz, P. C. Levesque, M. A. Blanar, Expression and characterization of human transient receptor potential melastatin 3 (hTRPM3). J. Biol. Chem. 278, 20890–20897 (2003). 142. P. Launay, A. Fleig, A. L. Perraud, A. M. Scharenberg, R. Penner, J. P. Kinet, TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell 109, 397–407 (2002). 143. T. Hofmann, V. Chubanov, T. Gudermann, C. Montell, TRPM5 is a voltagemodulated and Ca2+-activated monovalent selective cation channel. Curr. Biol. 13, 1153–1158 (2003). 144. D. Liu, E. R. Liman, Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5. Proc. Natl. Acad. Sci. U.S.A. 100, 15160–15165 (2003). 145. D. Prawitt, M. K. Monteilh-Zoller, L. Brixel, C. Spangenberg, B. Zabel, A. Fleig, R. Penner, TRPM5 is a transient Ca2+-activated cation channel responding to rapid changes in [Ca2+]i. Proc. Natl. Acad. Sci. U.S.A. 100, 15166–15171 (2003). 146. B. Nilius, J. Prenen, G. Droogmans, T. Voets, R. Vennekens, M. Freichel, U. Wissenbach, V. Flockerzi, Voltage dependence of the Ca2+-activated cation channel TRPM4. J. Biol. Chem. 278, 30813–30820 (2003). 147. J. Teulon, in Pharmacology of Ionic Channel Function: Activators and Inhibitors, Y. Kurachi, M. Mishina, Eds. (Springer-Verlag, Berlin, 2000), pp. 625–649. 148. O. H. Petersen, Cation channels: Homing in on the elusive CAN channels. Curr. Biol. 12, R520–R522 (2002). 149. S. W. Hughes, D. W. Cope, K. L. Blethyn, V. Crunelli, Cellular mechanisms of the slow (