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The transient receptor potential (TRP) protein super-family consists of a diverse group of cation channelsthat bear structural similarities to Drosophila TRP.TRP channels play important roles in nonexcitablecells; however, an emerging theme is that many TRP-related proteins are expressed predominantly in thenervous system and function in sensory physiology.The TRP superfamily is divided into seven subfami-lies, the first of which is composed of the “classical”TRPs” (TRPC subfamily). Some TRPCs may be store-operated channels, whereas others appear to beactivated by production of diacylglycerol or regulatedthrough an exocytotic mechanism. Many membersof a second subfamily (TRPV) function in sensoryphysiology and respond to heat, changes in osmolarity,odorants, and mechanical stimuli. Two members ofthe TRPM family function in sensory perception andthree TRPM proteins are chanzymes, which containC-terminal enzyme domains. The fourth and fifthsubfamilies, TRPN and TRPA, include proteins withmany ankyrin repeats. TRPN proteins function inmechanotransduction, whereas TRPA1 is activatedby noxious cold and is also required for the auditoryresponse. In addition to these five closely relatedTRP subfamilies, which comprise the Group 1 TRPs,members of the two Group 2 TRP subfamilies, TRPPand TRPML, are distantly related to the group 1TRPs. Mutations in the founding members of theselatter subfamilies are responsible for human diseases.Each of the TRP subfamilies are represented bymembers in worms and flies, providing the potentialfor using genetic approaches to characterize thenormal functions and activation mechanisms ofthese channels.

IntroductionThe transient receptor potential (TRP) superfamily is distinct fromother groups of ion channels in displaying a daunting diversity inion selectivities, modes of activation, and physiological functions.Nevertheless, they all share the common feature of six transmem-brane domains, varying degrees of sequence similarity, andpermeability to cations. A recurring theme in the TRP field is thatthese channels are of particular importance in sensory physiology.These include roles for TRP channels in each of the sensorymodalities, ranging from vision to taste, smell, hearing,mechanosensation, and thermosensation. TRP channels also serveto 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 tocharacterize the channel that might account for a highly Ca2+-

selective Ca2+ entry mechanism in nonexcitable cells, referredto as store-operated Ca2+ entry (SOCE) (1, 2). Stimulation ofmany nonexcitable cells with growth factors or hormones leadsto activation of phospholipase C (PLC), production of inositol1,4,5-trisphosphate (IP3) plus diacylglycerol (DAG), and releaseof Ca2+ from internal IP3-sensitive stores. This transient releaseof Ca2+ often results in a sustaining rise in Ca2+ through Ca2+

influx across the plasma membrane (3, 4). Interest in findingthe channels responsible for these SOCE pathways garneredconsiderable 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) werecandidate store-operated channels (SOCs), because the channelin flies functions in phototransduction, which is a PLC-mediatedsignaling pathway (7, 8). Moreover, Drosophila TRP wasspeculated to be a SOC. In the 10 years that have elapsedsince the first reports of mammalian TRP channels (1, 2),there have been many surprises in the field. Unanticipatedwere the large size of the superfamily (Table 1), the unusualdomain organization of certain members, the range of cationselectivities, and the panoply of activation mechanisms. Anironic twist is that some of the mammalian TRP channelsappear to be SOCs, yet current evidence indicates that activationof Drosophila TRP is not dependent on release of Ca2+ frominternal stores and is not a SOC.

The critical importance of TRP channels is underscored notonly by their roles in sensory physiology, but also by thedemonstrations that mutations in TRP channels are associatedwith diseases ranging from hypomagnesemia to polycystic kidneydisease and a neurodegenerative disease, mucolipidosis.

Composition of TRP SuperfamilyThe TRP superfamily is composed of seven subfamilies, all ofwhich include six putative transmembrane domains (Fig. 1).The fourth transmembrane domain lacks the complete set ofpositively charged residues necessary for the voltage sensor inmany voltage-gated ion channels (9). The structure of a TRPchannel has not been solved; however, the channels appear toform tetrameric assemblies (10–13), consistent with the structuresof voltage-dependent K+ channels.

The initially identified members of the TRP superfamily arereferred to as the “classical” or “canonical” TRPs and fall intothe TRPC subfamily. The names of the remaining subfamiliesare based on the original designation of the first recognizedmembers of each subfamily (14). The Group 1 TRPs—TRPC,TRPV, TRPM, TRPN, and TRPA—share substantial sequenceidentity in the transmembrane domains. The TRPC, TRPM, andTRPN (except for NOMPC) proteins include a 23– to 25–aminoacid “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 lowsequence similarity and a large extracellular loop between thefirst and second transmembrane domains (Fig. 1). The TRPP

The TRP Superfamily of Cation ChannelsCraig Montell

(Published 22 February 2005)

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Department of Biological Chemistry, Johns Hopkins UniversitySchool of Medicine, 725 North Wolfe Street, Baltimore, MD 21205,USA. E-mail: [email protected]


and TRPML subfamilies are both included in Group 2 becauseof similarities in their primary amino acid sequences andpredicted topologies.

Six of the subfamilies include members that are conservedin organisms as divergent as worms, flies, and humans. Theremaining subfamily, TRPN, contains members in invertebrates(15) and in zebrafish (16), although no mammalian TRPN proteinhas been described. The TRP superfamily has ancient originsbecause distantly related proteins are expressed in fungi(17–19). These latter proteins belong to the TRPY subfamily, sonamed after the first reported member, the yeast vacuolar protein,Yvc1 (18). Owing to the weak relationship of the TRPY proteinsto other TRPs, they are not classified along with either Group 1or Group 2 TRPs.

Group 1 Subfamilies: TRPC SubfamilyInvertebrate TRPC proteins. TRPC proteins contain three tofour ankryin repeats and extensive amino acid similarity toDrosophila TRP, and are PLC-dependent nonselective cationchannels. The founding member of the TRP family was discoveredas a key component required for the light response in Drosophilaphotoreceptor cells. Mutations in trp cause the response to lightto be transient (20) and result in a ~10-fold decrease in the level

of light-induced Ca2+ influx (21). This phenotype, combinedwith the observation that fly vision requires PLC (7), raised thepossibility that trp might encode the archetypal PLC-operatedCa2+ channel. This hypothesis was confirmed with the cloningand functional characterization of TRP. The gene trp encodes an

R E V I E W

TRPV1TRPMTRPV

P

N

C

P

NC

Intracellular

Kinasedomain

C

P

N

TRPNTRPC

P

N

A

cc TRPdomain

AAA

C

TRPC1 TRPN1

N

C

P

N

C

P P

N C

TRPP TRPML

TRPATRPA1TRPM7

PKD2 MLN1

Intracellular

Extracellular

Extracellular

Group 1 TRPs

Group 2 TRPs

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).

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

TRPP2 1 13 3 3

TRPML 1 1 3 3

Total 17 13 28 27

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


eye-enriched protein with four N-terminal ankyrin repeats andan overall predicted topology similar to that of many membersof the superfamily of voltage-gated and second messenger–gatedion channels (5, 6). Consistent with the structural similaritiesbetween TRP and known ion channels, in vitro studies havedemonstrated that TRP is a cation channel with moderateselectivity for Ca2+ relative to Na+ (PCa:PNa ~ 10:1) (6, 22, 23).

In addition to TRP, two other TRPC proteins are expressed inDrosophila, TRPL (24) and TRPγ (25), each of which shares~45 to 50% amino acid identity with TRP over the N-terminal800 to 900 amino acids. The sequence similarity encompassesall six transmembrane segments and decreases after the TRPdomain (Figs. 1 and 2). The most highly conserved regions ofthe 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 twobinding sites for a scaffold protein (Homer) in mammalianTRPC1 (26). The functional implications of the interactionbetween TRPC and Homer are intriguing and are discussed inthe last section of this Review. As is the case with TRP (6), bothTRPL (27) and TRPγ (25) are highly enriched in photoreceptorcells. Thus, all the TRPC members in Drosophila are expressedpredominantly in the visual system.

Analyses of loss-of-function and dominant-negative formsof the Drosophila TRPCs indicate that all three contribute to thelight-dependent cation influx. Flies devoid of TRPL were originallyreported to be indistinguishable from the wild type (27). However,the light response in trpl mutant flies displays several differencesfrom that of the wild type, including changes in the permeabilityratios for several cations (28) and a reduced response to alight stimulus of long duration (29). Double mutants lackingboth TRP and TRPL are completely unresponsive to light (27),indicating that TRPγ cannot function independently of TRP andTRPL. TRPγ may function in combination with TRPL becausethe light response is nearly eliminated in trp mutant fliesexpressing a dominant-negative form of TRPγ (25).

Caenorhabditis elegans encodes three TRPC proteins, one ofwhich, TRP-3, has been subjected to genetic analysis (30). Thisprotein is required in sperm for fertility in hermaphrodites andin males. The sperm are motile, but appear to be defective in astep following gamete contact, possibly gamete fusion.

Activation of Drosophila TRP. Drosophila TRP is capable offunctioning as a SOC, because it can be activated in tissueculture systems using drugs, such as thapsigargin, that causerelease of Ca2+ from the internal Ca2+ stores (22, 23, 31).Thapsigargin treatment results in Ca2+ release because itinhibits the smooth endoplasmic reticulum (ER) Ca2+-ATPase(adenosine triphosphatase) that normally counterbalances theconstant leak of Ca2+ from the ER stores (32, 33).

Despite the observation that TRP appears to function as aSOC in cultured cells (in vitro) (22, 23, 31), TRP is not activatedthrough a store-operated mechanism in vivo. Introduction ofeither thapsigargin (34, 35) or IP3 (36) to photoreceptor cellsdoes not activate cation influx. In addition, the Drosophilagenome encodes a single relative of the mammalian IP3 receptor(IP3R) (37, 38), and mutations that eliminate this gene have nodiscernible effect on the photoresponse (39, 40). In addition,there exists a second Ca2+-release channel, the ryanodinereceptor, which is distantly related to the IP3R (37, 41). As isthe case for the IP3R, there is only one ryanodine receptorhomolog in Drosophila, and mutations in this locus have noeffect on phototransduction (42). Thus, TRP function is not

dependent on either of the known Ca2+-release channels.Moreover, the lack of concordance between the in vitro and invivo studies raises questions concerning the physiologicalrelevance of using thapsigargin to address whether or not a givenchannel is a SOC.

Because IP3 is not sufficient to activate TRP, an alternativeproposal is that activation of TRP is coupled to PLC activitythrough hydrolysis of the PLC substrate, phosphatidylinositol-4,5-bisphosphate (PIP2) (43). Another proposal is that productionof DAG rather than IP3 is essential for opening of the TRPchannels. Consistent with this latter proposal, polyunsaturatedfatty acids (PUFAs), which can be derived from DAG, lead toactivation of TRP either in vitro or after application to isolatedDrosophila photoreceptor cells (44). In addition, TRP is consti-tutively active in a mutant, rdgA, that disrupts an eye-enrichedDAG kinase (45). Elimination of the DAG kinase also increasesthe small light response in strong norpA alleles, which disruptthe activity of PLC (46, 47). These results were interpreted as

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Fig. 2. The TRP domain. This region is a highly conserved 23– to25–amino acid region in TRPC, TRPN, and TRPM proteins, whichis C-terminal to the transmembrane segments (see Fig. 1). TRPbox 1 is invariant in the TRPC proteins. A variation of TRP box 1is present in zTRPN1 and cNOMPC. A less conserved version ofTRP box 1 is in each TRPM protein. The TRP box 2 is the proline-rich region. Sequences are from mammals, except for zTRPN1(zebrafish), cNOMPC (C. elegans), and TRP, TRPL, TRPγ, anddTRPM (Drosophila).


additional evidence that PUFAs gate TRP, because eliminationof the DAG kinase should, in principle, result in higher levelsof PUFAs. However, it has not been demonstrated that the levelsof PUFAs are increased in rdgA, and it cannot be excluded thatthe effects of PUFAs on TRP may be indirect (48). Thus, themechanism through which activation of PLC is coupled toactivation of TRP remains controversial.

Given that Ca2+ release from internal stores does not functionin Drosophila visual transduction, the transient response tolight in trp mutant flies could be due to rapid Ca2+-dependentinactivation of the remaining influx channels in trp mutantphotoreceptor cells (49). Consistent with this proposal, mutationof one of the calmodulin-binding sites in TRPL results in asustained rather than a transient light response in trp mutantflies (49). Furthermore, it was reported that the trp photoresponsewas similar to that of the wild type in the absence of extracellularCa2+. However, this latter result has been challenged (50).

An intriguing proposal that may account for the transientlight response in trp flies is that it results from depletion of thesubstrate for PLC, PIP2, in trp photoreceptors (51). Using theinwardly rectifying K+ channelKir2.1 as a biosensor, it ap-pears that PIP2 concentrationsare lower in trp cells than inwild-type photoreceptor cells.The decrease in PIP2 is pro-posed to be a consequence of arequirement for Ca2+ influx todown-regulate PLC activityand up-regulate PIP2 recycling(51). However, direct evidencethat the PIP2 concentration isreduced in trp photoreceptorcells and that this decreaseresults in the trp phenotyperemains to be demonstrated.

Mammalian TRPC proteins.A total of seven TRPC proteinshave been described in mammals(1, 2, 52–60). In contrast to miceand rats, humans express onlysix TRPCs, because TRPC2 is apseudogene (1). The seven TRPCproteins are subdivided into foursubsets on the basis of their primary amino acid sequences (Table 2and Fig. 3). As is the case for the three Drosophila TRPCs, themammalian TRPC proteins include three to four ankyrin repeats,six putative transmembrane domains, and amino acid sequenceidentity (≥30%) over the N-terminal ~750 to 900 amino acids,which extends to and includes the TRP domain. The sequencesof the mammalian TRPC proteins are quite variable in the regionC-terminal to the TRP domain. However, the lengths of theC-terminal tails and the overall size of the mammalian TRPCproteins (Table 1) are typically smaller than those of the DrosophilaTRPCs (1124 to 1275 residues). Some of the TRPC proteins, suchas TRPC1, are widely expressed, although each of the TRPCs isexpressed in the nervous system and some are highly enriched inneurons. As is the case with the Drosophila TRPC proteins (23,25), most of the mammalian TRPCs appear to form hetero-multimers. The functional consequences of these heteromultimericassemblies are discussed later in this Review.

Activation mechanisms of mammalian TRPC channels. Acommon feature of the mammalian TRPC channels is that theyare activated in cultured cells through pathways that engagePLC [for example, see (52, 58–64)]. All of the TRPC-dependentconductances are nonselective cation channels, although thereare differences in the permeabilities of Ca2+ relative to Na+

and other cations (Table 3). As with the Drosophila TRPs, acontroversial issue concerns the mechanism through whichstimulation of PLC and production of IP3 and DAG activates orpotentiates TRPC-dependent conductance. Three models havebeen investigated, one of which is that TRPC channels areactivated by production of IP3 and release of Ca2+ from internalstores. Another proposal is that a rise in DAG leads to openingof TRPC channels. Alternatively, TRPC channels may be activatedthrough an exocytotic mechanism. According to this latter model,PLC activation leads to a regulated translocation of the channelsfrom intracellular vesicles to the plasma membrane. Evidencehas been presented supporting each of these proposals, althoughin only a few cases has the activation mechanisms underlying amammalian TRPC channel been addressed in vivo.

At least four TRPC proteins (TRPC1, -2, -4, and -5) may beactivated through a store-operated mechanism, because applicationof IP3 or thapsigargin results in increases in cation influx intissue culture cells expressing any one of these proteins (52, 56,57, 61, 65). The mechanism underlying SOCE is unresolved.One proposed mechanism is that a diffusible messenger (Ca2+

influx factor, CIF), which is generated upon release of Ca2+

from the internal stores, leads to Ca2+ influx (66–68). Anotherpossibility is that SOCE depends on a conformational couplingbetween the IP3R and the influx channels (69). According tothis second model, there is a direct interaction between theIP3R, situated in the intracellular Ca2+ stores, and the Ca2+

influx channels in the plasma membrane. Upon release of Ca2+

from the internal stores, there is a change in conformation inthe IP3R that induces a conformational shift in the SOCs, resultingin activation of Ca2+ influx. In support of the conformationcoupling model is the demonstration that the addition of

R E V I E W

Subset Members Length(aa)

Percent IDwithin same set

Percent ID tomTRPCs outside set

Percent ID tofly TRPs4

1 TRPC1 759–810 N/A 31–44 33–36

2 TRPC2 psuedo1

4322

876–1172

N/A 33–38 33–34

3 TRPC3 828–848 71–793 37–38 33–38

TRPC6 815–931 70–72 35–37 32–37

TRPC7 862 69–793 35–37 30–36

4 TRPC4 836–1077 65 35–45 42–47

TRPC5 966–975 65 34–43 40–43

Table 2. Four sets of mammalian TRPC proteins. In many cases, the lengths of the TRPC proteinsdiffer owing to alternative RNA splicing. The percent identities apply to the N-terminal ~750 to 900amino acids. This portion of the proteins includes all six transmembrane segments and the TRPdomain. 1Human TRPC2 is a pseudogene (1). 2A bovine form of TRPC2 is predicted to encodeonly four transmembrane segments (53). 3TRPC3 and TRPC7 share slightly greater amino acididentities to each other than to TRPC6. 4TRPγ is the most related to each mammalian TRPCprotein and then to a lesser degree to TRPL and TRP. TRPγ shares 50% amino acid identity toeither TRP or TRPL over the NH2-terminal ~800 residues. TRP and TRPL are 45% identical overthe N-terminal ~900 residues. mTRPC, mouse TRPC.


calyculin A, which causes condensation of cortical actinbundles at the plasma membrane, precludes SOCE and TRPC3activation in vitro (70, 71). This manipulation of the actincytoskeleton was interpreted as interfering with SOCE bypreventing access of the ER to the plasma membrane.Alternatively, the reorganization of the cytoskeleton maydisrupt Ca2+ entry by causing internalization of either TRPC1,TRPC3, or TRPC4 (72, 73).

In contrast to some TRPC channelsthat may be SOCs, other TRPC proteins,notably TRPC6 and -7, are activated invitro by DAG (60, 74). These results arereminiscent of the report that PUFAsactivate Drosophila TRP channels (60).However, it remains unclear whetherDAG and PUFAs function directly orindirectly in gating TRP channels. Indirectactivation of TRPC proteins by DAGcould occur through production of long-chain fatty acid metabolites, which canlead to mitochondrial uncoupling.Metabolic stress induced by mitochondrialuncoupling can activate TRPC proteins(75) by depletion of adenosine 5´-triphosphate (ATP), as is the case withDrosophila TRP (48).

The findings that some TRPC channelsmay be store-operated whereas othersmay be activated through production ofDAG would suggest that different TRPCproteins are gated through distinctmechanisms. However, such a conclusionbecomes murky with regard to TRPC2and TRPC3. Evidence has been providedthat mouse TRPC2 is activated invomeronasal neurons through a rise inDAG (76). However, TRPC2 appears tobe activated by Ca2+ release in mousesperm (77). Potentially, the differences inthese modes of activation could resulteither from heteromultimerization ofTRPC2 with distinct subunits in spermand vomeronasal neurons or could bedue to differences in TRPC2 isoforms,which are generated by alternative mRNAsplicing (61, 78).

Different modes of activation have alsobeen ascribed to TRPC3. According to onereport, activation of TRPC3 depends onproduction of DAG (74), whereas otherstudies indicate that TRPC3 is store-operated (52). Conformational couplingmay activate TRPC3 because this channelinteracts directly with the type I IP3R invitro (79). The association between TRPC3and IP3R occurs through two regions in theIP3R, which are situated between the N-terminal IP3 binding siteand the transmembrane domains, and a small portion of TRPC3C-terminal to the transmembrane domains (80, 81). Additionalevidence consistent with the conformational coupling model is thatintroduction of IP3 and the IP3R appears to restore regulation of

TRPC3 by IP3 in excised patches after the native IP3R was removedby extensive washing (79). Direct interactions between the IP3Rand TRPC channels may be a common phenomenon, becauseTRPC1 can coimmunoprecipitate with the type II IP3R from humanplatelets (82). Ca2+ release through another Ca2+-release channel,the ryanodine receptor, can also lead to activation of TRPC3 (83).Distinct TRPC3 channels appeared to be functionally coupled toeither the ryanodine receptor or the IP3R, but not both (83).

The disparate observations that TRPC3 may be store-operatedin some studies and gated by DAG in others may reflect differencesin the cell types used for the expression studies. Different celllines may express distinct sets of endogenous proteins that interactwith TRPC3 and affect its mode of regulation. Thus, it is critical

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Fig. 3. Dendrogram showing the relatedness of the TRP proteins. The phylogenetic treewas generated using MacVector 6.5.3 and includes all of the human TRPs, mouse TRPC2,and zebrafish TRPN1 (open box). In addition, one Drosophila and one C. elegans memberof each subfamily are included (indicated by filled boxes).


to characterize the mechanisms regulating TRPC proteins in vivo.Unfortunately, there is a paucity of such studies because of thedifficulties inherent in ascribing native conductances to specificTRPC channels.

A native TRPC3-dependent conductance current has beencharacterized from the brains of neonatal rats, which is activatedby 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 inhibitorsof the IP3R. Thus, at least one endogenous TRPC3 conductancerequires activity of the IP3R and is not gated by DAG.

TRPC3, as well as several other TRPC channels, appear to besituated primarily in intracellular compartments in native cells(84–86). These results raise the possibility that some TRPC channelsare held in vesicular membranes and may be activated by regulatedshuttling to the plasma membrane. If correct, such a proposal might

account for the observation that many TRPC channels are constitu-tively active in vitro. The possibility that activation of some SOCsmight involve exocytosis is supported by the finding that SOCE isblocked by inhibitors of vesicular trafficking (87) and is preventedby inhibition of a protein, SNAP-25, that is required for the fusionof vesicles with their target membranes (88).

Several in vitro studies provide evidence that exocytosis andendocytosis can alter the concentration of TRPC proteins in theplasma membrane and thereby modulate TRPC-dependent cationinflux (72, 73, 89, 90). The reorganization of the cytoskeletonappears to participate in controlling the insertion and removal ofseveral TRPC proteins from the plasma membrane (72, 73).Stimulation of receptors coupled to the activation of PLC can alsoincrease the concentration of TRPC channels in the plasmamembrane (89). Furthermore, proteins involved in the exocytoticmachinery contribute to the movement of TRPC proteins to theplasma membrane (90–92).

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TRPC subfamily

Genename

Chromosomallocation

SelectivityPCa:PNa

Mode(s) of activation Adult tissues with highestexpression

Functions and diseases

TRPC1 3q22–q24 Nonselectivecation

Store-operated? Heart, brain, testis,ovaries

Required for EPSC inPurkinje cells

TRPC2 11p15.3–p15.4 2.7 Store-operated?, DAG VNO, testis Acrosome reaction, maleaggression, pheromoneresponse (human TRPC2 isa pseudogene)

TRPC3 4q27 1.6 Store-operated, DAG,exocytosis

Brain –

TRPC4 13q13.1–q13.2 7 (>1003) Store-operated? Brain, endothelia, adrenalgland, retina, testis

Vasorelaxation,neurotransmitter release

TRPC5 Xq23 9.5 Store-operated?,exocytosis

Brain Modulating neuriteextension

TRPC6 11q21–q22 5 DAG Lung, brain –TRPC7 5q31.2 1.91; 52 Store-operated, DAG Eye, heart, lung –

TRPV subfamily

Name Chromosomallocation

SelectivityPCa:PNa


Functions and genetics

TRPV1 17p13.3 9.6(vanilloids)3.8 (heat)

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

Trigeminal (TG) anddorsal root ganglia (DRG),urinary bladder

Hot pain sensor 43°C

TRPV2 17p11.2 3 Heat (52°C), exocytosis,membrane stretch

DRG, spinal cord (SC),brain, spleen, small andlarge intestine, vascularmyocytes

Very hot pain sensor 52°C

TRPV3 17p13.3 2.6 Warm (30°–39°C) DRG, kerotinocytes,tongue, TG, SC, brain

Warm temperature sensor(30°-39°C)

TRPV4 12q24.1 6 Osmotic cell swelling,phorbol esters, warm(27°C), 5´6´-EET,

Kidney, lung, spleen,testis, endothelia, liver,heart, DRG, keratinocytes

Osmosensorwarm temperature sensor(27°C)

TRPV5 7q35 >100 Low intracellullar Ca2+;hyperpolarization,exocytosis

Kidney, duodenum,jejunum, placenta,pancreas

Ca2+ reabsorption in kidneys

TRPV6 7q33–q34 >100 Similar to TRPV5, store-operated (CRAC?),exocytosis

Small intestine, pancreas,placenta, prostate4

–

Table 3. Properties of vertebrate TRPC and TRPV proteins. All of the chromosomal locations apply to the human genes. Thebiophysical 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.


Regulated translocation of TRPC proteins also occurs in vivoin both invertebrates and vertebrates. In Drosophila photoreceptorcells, TRPL shuttles from the microvillar membranes to the cellbodies in response to light, and this movement functions inlong-term adaptation (93). The TRPC protein in worms, TRP-3,is expressed in spermatids in intracellular vesicles; upon spermactivation, TRP-3 is detected in the plasma membrane (30).This translocation is commensurate with an increase in SOCEin the sperm, suggesting that exocytosis is a mechanism regulatingCa2+ influx. In mammals, both TRPC3 and TRPC5 appear toundergo regulated insertion into the plasma membrane inhippocampal neurons (91, 92). In the case of TRPC5, exocytosisof the channel inhibits neurite outgrowth (92, 94).

Group 1: TRPV SubfamilyInvertebrate TRPVs. The first reported members of the TRPVsubfamily include C. elegans OSM-9 (95) and the mammalianprotein, TRPV1 (96). Mammals express six distinct TRPVproteins, whereas worms and flies encode five and two members,respectively (Table 1). The proteins that comprise the TRPVsubfamily contain three to five ankyrin repeats and share ~25%amino acid identity to TRPC proteins over a span that includestransmembrane segments V and VI and the TRP box (Fig. 1).

The osm-9 locus was originally shown to function in osmoticavoidance, olfaction, and mechanosensation (95, 97). In addition,osm-9 is required for “social feeding” (98), a phenomenon inwhich certain strains of worms feed in groups rather thanindividually, depending on a variation in a single amino acid inthe putative G-protein–coupled neuropeptide receptor, NPR-1(99). Mutation of osm-9 eliminates “social feeding” in theaggregation-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 cationchannels, which are activated in vitro by hypoosmolarity (100,101). Nan and Iav are expressed in a sensory structure, thechordotonal organ, which participates in the auditory response,and mutations in either loci disrupt hearing.

Mammalian TRPV1. The archetypal mammalian TRPVmember, TRPV1, was identified through an expression cloningstrategy for a channel activated by vanilloid compounds, suchas capsaicin, which are present in spicy foods (for example, hotchili peppers) (96). Moderate heat (≥43°C) also activates TRPV1,demonstrating that thermal heat and the pungent ingredientin “hot” foods elicit their effects through the same channel.Anandamide, which is an endogenous compound structurallyrelated to capsaicin, also is capable of activating TRPV1 (102).

TRPV1 is a molecular integrator for multiple types of sensoryinput, particularly those that function in the sensation of pain.Protons (pH ≤5.9) reduce the heat threshold for activation of theTRPV1-dependent cation conductance (103, 104). A decreasein PIP2 concentration, which occurs through production of thepro-algesic agents bradykinin and nerve growth factor, potentiatesthe capsaicin-evoked response and decreases the temperaturethreshold for channel activation (105, 106).

Analyses of TRPV1-deficient mice confirmed that the channelis activated in vivo through multiple mechanisms (107, 108).In wild-type animals, TRPV1 is enriched in small- to medium-diameter dorsal root ganglia (DRG) neurons, which respond tomoderate 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). Further-more, the mutant mice showed a reduced behavioral response tovanilloid compounds and a reduced withdrawal response aftertail immersion in a hot waterbath (107). However, using ahotplate to perform the assay, there was no reported differencebetween wild-type and TRPV1−/− mice (108). Nevertheless,the knockout mice exhibited a clear and pronounced deficitin thermal hyperalgesia resulting from prior inflammation(107, 108).

It is possible that regulated insertion of TRPV1 into the plasmamembrane may contribute to hyperalgesia. In support of this,TRPV1 binds to the vesicular SNARE proteins snapsin andsynaptotagmin IX, which participate in exocytosis (110), andTRPV1 colocalizes in vesicles in the cell bodies and processesof DRG neurons along with synaptotagmin IX and VAMP2.These may correspond to cytoplasmic transport packets similarto those reported for TRPC5. Phosphorylation of TRPV1 byprotein kinase C (PKC) promotes the insertion of the channelinto the plasma membrane, and this translocation appears to beregulated by SNARE proteins.

Mammalian TRPV2, -3, and -4. At least three additional TRPVchannels (TRPV2, TRPV3, and TRPV4) are activated by thermalheat, but with thresholds distinct from that of TRPV1(111–116). The four thermally activated channels form the firstof two TRPV subsets [TRPV1-4 (Fig. 3)] on the basis of theirprimary amino acid similarity, modes of activation, and ionselectivities. 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°Cand 27°C, respectively. TRPV1, -2, -3, and -4 channels areexpressed 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 thermosensorsis activated by capsaicin; however, at least two of these channelsrespond to additional stimuli. Mouse TRPV2 has been reportedto participate in cation influx after translocating from intracellularpools to the plasma membrane in response to stimulation witheither insulin growth factor I or neuropeptide head activator(119, 120). However, these studies were performed using an invitro expression system, and it remains to be determinedwhether TRPV2 displays similar growth factor–inducedtranslocation in vivo. Decreases in osmolarity and cell swellingcan also lead to TRPV2 activation (121).

TRPV4 is another polymodally activated channel that wasinitially shown to be activated by hypertonic cell swelling(122–124). TRPV4 can also be activated by phorbol esters andby chemicals, such as anandamide and arachidonic acid, whichlead to the formation of the cytochrome P450 epoxygenase,5´6´-epoxyeicosatrienoic acid (5´6´-EET) (125, 126). The variousmodes of TRPV4 activation do not appear to occur through asingle mechanism because a point mutation in the thirdtransmembrane domain inhibits activation by heat and phorbolesters, 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 phorbolesters 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 restoresbehavioral responses to hypertonicity and light mechanicalstimuli to the nose, but not avoidance to noxious odorants (128).

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These results suggest that there is substantial evolutionary con-servation in the mechanisms leading to activation of these channels.

Mammalian TRPV5 and -6. The remaining two TRPVchannels, TRPV5 and TRPV6, form a second TRPV subset[TRPV5-6 (Fig. 3)]. These two channels are distinct from allother mammalian TRPs in displaying very high selectivity forCa2+ (PCa:PNa>100) (129–131). As with other Ca2+-selectivechannels, the TRPV5-6 channels become permeable to monovalentcations in the absence of divalent cations. It has been proposedthat TRPV6 may be the highly Ca2+-selective, store-operatedchannel, referred as CRAC (132), which was originally describedin mast cells and T lymphocytes. However, several biophysicalproperties of the TRPV6-dependent current appear to be distinctfrom those of ICRAC (133). Nevertheless, it remains possible thatTRPV5 or TRPV6 may be one subunit of a CRAC channel. Asappears to be the case for several TRPC proteins, exocytosis ofTRPV5 and TRPV6 may also contribute to activation of thesechannels (11).

Group 1: TRPM SubfamilyThe TRPM subfamily is composed of eight mammalian members(Table 4), three of which are highly unusual “chanzymes”consisting of C-terminal enzyme domains. TRPM proteinsshare ~20% amino acid identity to TRPCs over a ~325-residueregion that includes the C-terminal f ive transmembranesegments and the TRP domain (Fig. 2). The N-terminal domainof TRPM proteins is devoid of ankyrin repeats and is considerablylonger (~750 residues) than the corresponding regions in TRPCand TRPV proteins (~325 to 450 residues). The total length ofTRPM proteins (~1000 to 2000 amino acids) varies primarilybecause of diversity in the regions C-terminal to the transmembranesegments. 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 biophysicalproperties, 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 ofthe other subsets. TRPM proteins are encoded in the Drosophilaand C. elegans genomes, and one such protein, GON-2, isrequired for mitotic cell divisions of the gonadal precursor cellsin worms (134).

TRPM1 and –3. The designation of the TRPM subfamily isbased on the former name of the founding member, melastatin(TRPM1), which is a putative tumor suppressor protein. TRPM1was isolated in a screen for genes whose level of expressioncorrelated with the severity of metastatic potential of mousemelanoma cell lines (135, 136)). TRPM1 expression in the celllines and in melanocytic neoplasms is inversely correlated withmelanoma aggressiveness (135, 137). An alternatively splicedisoform of TRPM1 encodes a short protein (~500 residues;TRPM1-S), which consists exclusively of the N-terminal region ofthe full-length form (TRPM1-L) and is devoid of any predictedtransmembrane segments (135, 138). TRPM1-S associates withTRPM1-L and suppresses the activity of TRPM1-L by inhibitingits translocation to the plasma membrane (139). TRPM1-L activitywas assayed by Ca2+ imaging because patch-clamp recordings ofeither TRPM1 or its close relative, TRPM3, have been difficult toperform. However, there is a report indicating that the constitutivecation currents resulting from expression of TRPM3 are augmentedby hypotonic conditions (140). Alternatively, it has been suggestedthat TRPM3 may be a SOC (141).

TRPM4 and -5 are VCAMs. The biophysical features of thecurrents mediated by the TRPM4 and TRPM5 channels aredistinct from those of other TRPs and may account for the Ca2+-activated monovalent currents identified in various excitableand nonexcitable cells. TRPM4 is expressed as at least twoisoforms, TRPM4a and TRPM4b (139, 142), which consist ofshorter and longer N-terminal regions, respectively. WhereasTRPM4a displays little activity (139, 143), TRPM4b andTRPM5 appear to be directly activated by an increase inintracellular Ca2+ concentration (142–145). Continuous exposureto Ca2+ desensitizes TRPM5, and this effect is partially reversedby 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 activatemore slowly at positive potentials and deactivate at negativepotentials (143–146). The voltage dependence, Ca2+ activation,and monovalent cation selectivities of these currents (IVCAM)are all features distinct from those of other TRP-dependentcurrents.

Ca2+-activated monovalent cation currents (ICAM) have beendescribed in a diversity of cell types, such as pancreatic acinarcells, cardiac myocytes, and neurons of the central nervoussystem and peripheral nervous system (147, 148). Moreover,these currents have been implicated in processes ranging fromcontrolling the firing properties of pyramidal cells to mediatingthe slow oscillations in thalamocortical neurons during sleep(149, 150). Whether these endogenous Ca2+-activated monovalentcation conductances display voltage dependencies similar tothat of ICAM remains to be addressed; however, it is intriguing tospeculate that they are due to TRPM4b and TRPM5. Among themany cell types expressing TRPM5 are taste receptor cells, andTRPM5 functions in the chemosensory responses to sugars,glutamate, and quinine (151, 152).

TRPM6 and -7 chanzymes. The domain organization ofthe TRPM6/7 proteins is striking, owing to the presence ofa C-terminal atypical protein kinase domain (153, 154).The protein kinase was identified in one report in a screen forPLC-interacting proteins (153). This domain is also expressedas a separate 347–amino acid enzyme independently from theTRPM domain. The protein kinase contains a FYVE (Fab1,YOTB, Vac1, and EEA1) domain zinc finger motif (155) andis most related to the atypical α-kinase family (156), whichincludes myosin heavy chain kinase A (157) and elongationfactor–2 kinase (158). Despite a lack of apparent sequencesimilarity with conventional protein kinases, the three-dimensional structure of α-kinases bears similarities with thatof conventional protein kinases (159).

The specific function of the TRPM protein kinase has beencontroversial, but it appears that the enzymatic activity is not astrict requirement for channel activity. According to one study,TRPM7 channel function was augmented by the addition ofATP and inhibited by an inactivating mutation in the enzyme(75). Another study concluded that TRPM7 is suppressed byintracellular Mg2+ and that the addition of ATP promotes activationof TRPM7 by lowering the concentration of free Mg2+ (154).Point mutations or deletions in the protein kinase domain didnot substantially impair channel activation, but affected thesensitivity of the channel to Mg2+ suppression (160). However,for unknown reasons, the point mutations resulted in an increasein Mg2+ sensitivity, whereas the deletion had the opposite effect.Nevertheless, the findings that TRPM7 conducts Mg2+ and is

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suppressed by increasing concentrations of Mg2+ provide for anefficient mechanism of negative-feedback regulation.

The C-terminal protein kinase domain mediates the effectsof adenosine 3´,5´-monophosphate (cAMP) on channel activity(161). An increase in cAMP levels elevates TRPM7-dependentcurrents, and this effect is eliminated by mutations that abolishthe TRPM7 kinase activity.

In addition to Mg2+, channel activity of TRPM7 is alsosuppressed by hydrolysis of PIP2 (162). Whether this latterregulatory mechanism is related to the finding that TRPM7

binds to PLC remains to be resolved (153). It is intriguing tospeculate on whether Mg2+ activates the PLC associated withthe chanzyme, leading to diminution of PIP2 concentration. Infact, Mg2+-activated PLCs have been described in certain celltypes, such as B lymphocytes (163).

The highly related chanzyme, TRPM6, was identified asthe mutated gene in patients with hypomagnesemia (164, 165).The biophysical features of the current resulting from in vitroexpression of TRPM6 are quite similar to those of TRPM7. Aswith TRPM7, the TRPM6-dependent current is carried mostly

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TRPM subfamily


SelectivityPCa:PNa



TRPM1 15q13–q14 Nonselectivecation

Translocation? Eye –

TRPM2 21q22.3 ~0.3 ADP-ribose, NAD, H2O2 Brain Redox sensorTRPM3 9q21.11 1.6 Decrease in osmolarity,

store-operated?Human kidney, mousebrain

–

TRPM4 19q13.33 Monovalentselective cation

Ca2+-activated, voltage-modulated (VCAM)

Prostate, colon, heart,kidney,

–

TRPM5 11p15.5 Monovalentselective cation

Ca2+-activated, voltage-modulated (VCAM)

Small Intestine, liver,lung, taste receptor cells

Taste1

TRPM6 9q21.13 Primarilydivalent, e.g.,Mg2+ and Ca2+

Mg2+ inhibited,translocation

Kidney, small intestine Mg2+ absorptionhypomagnesemia,hypocalcemia

TRPM7 15q21 Divalent-specific, e.g.,Mg2+ and Ca2+

Phosphorylation,Mg2+-ATP

Kidney, heart Cell viability

TRPM8 2q37.2 3.3 Menthol, icilin,cool (23°–28°C)

Prostate, TG, DRG Cool temperature sensor(23°–28°C)

TRPA and TRPN


SelectivityPCa:PNa



TRPA1 8q13 0.8 Cold (17°C), icilin,mustard oil, wasabi,cannabinoids (THC),cinnaldehyde,bradykinin, mechanicaldeflection?

DRG, hair cells Cold pain sensor (17°C),hearing1,2

zTRPN1 – ? Mechanical stimuli? Ear, eye Hearing2

TRPP and TRPML


SelectivityPCa:PNa



TRPP2 4q21-q23 Nonspecificcation

Modulated by Ca2+,translocation?,interaction withpolycystin-1, protons

Kidney, widely expressed Polycystic kidney disease

TRPP3 10q24 4.3 Modulated by Ca2+,inhibited by troponin I

Kidney, heart –

TRPP5 5q31 – – Testis, heart –TRPML1 19p13.2–

p13.3Nonspecific

cationInhibited by reduction inpH, modulated by Ca2+?

Brain, heart, skeletalmuscle

Lysosomal trafficking?,mucolipidosis type IV

TRPML2 1p22 – – – –TRPML3 1p22.3 – – Cochlea hair cells1 Hearing1

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


by divalent cations, such as Mg2+ and Ca2+, and is inhibited byMg2+ (166). TRPM6 is capable of efficiently heteromultimerizingwith TRPM7, and this interaction appears to promote translocationof TRPM6 from an intracellular compartment to the plasmamembrane (167).

The TRPM6 and TRPM7 chanzymes represent the f irstchannels shown to be highly permeable to Mg2+ (154, 166).The TRPM7-dependent Mg2+ influx contributes to cell survivalbecause TRPM7 knockdown cells are not viable unless theyare bathed in a medium rich in Mg2+ (160). TRPM7 is alsopermeable to trace and toxic metals, such as Zn2+ and Ni2+

(168). Thus, entry of Zn2+ and Ni2+ through TRPM7 maycontribute to the accumulation and toxicity of these metals.TRPM6 or TRPM7 may correspond to a Mg2+-inhibited divalentcurrent, referred to as MIC or MagNuM, that has been identifiedin T lymphocytes and rat basophilic leukemia (RBL) cells (163,169–171).

TRPM2 and -8. TRPM2 is a second type of chanzymerepresented by members of the TRPM family. TRPM2 iscomposed of a C-terminal ADP-ribose pyrophosphatase(NUDT9) domain (172, 173). TRPM2 is activated by eitherADP-ribose or agents, such as H2O2, which affect the redoxstate (172, 173). The alteration in redox state appears to activateTRPM2 through binding of β-NAD+ (nicotinamide adeninedinucleotide) to the NUDT9 domain (174). Such a mode ofactivation has important implications in terms of the mechanismunderlying hypoxia-induced cell death (see below: TRPs andhypoxia-induced cell death). A short form of TRPM2, which iscomposed of the N terminus and the first two transmembranesegments, binds to and suppresses the activity but not the local-ization of the long form of TRPM2 (175). These data are consistentwith the f indings that the N-terminal fragments of theDrosophila and mammalian TRPCs can bind to and suppressthe activities of full-length TRPC proteins (23, 25, 75, 176).

TRPM8 is most related to TRPM2, although it is not achanzyme. TRPM8 is a “thermoTRP,” which is activated bymoderately cool temperatures and by chemicals, such as mentholand icilin, that evoke the sensation of thermal coolness (177,178). Interestingly, the molecular bases for cold activation ofTRPM8 and heat activation of TRPV1 are conceptually similar.Activation of both of these channels by temperature involveschanges in the voltage-dependent activationcurves by temperature (179, 180). In addition,menthol and capsaicin, ligands that activateTRPM8 and TRPV1, respectively, do so byshifting the activation curves. Whether theother thermoTRPs are activated through asimilar mechanism remains to be addressed.

Group 1: TRPA SubfamilyThe TRPA proteins are characterized by thepresence of large numbers of ankyrin repeatsin the N-terminal domain (8-18) (Fig. 1).There is only one TRPA member in mammalianorganisms, although there are four inDrosophila (Table 1). The founding TRPAprotein, TRPA1, was isolated in a screen forgenes down-regulated in fibroblasts that haveundergone oncogenic transformation (181).

Several TRPA proteins have been reportedto be thermosensors (Tables 4 and 5); however,

there is controversy concerning the activation mechanism ofmammalian TRPA1. According to one group, TRPA1 is activatedby cold temperatures below 17°C (182). Although this thermo-activation has not been reproduced in another study (183),there is agreement that various chemical agonists lead toactivation of TRPA1 (182, 183) (Table 5). These includepungent compounds present in mustard oil and other naturalsources and the psychoactive compound in marijuana ∆9-tetrahydrocannabinol (THC). In addition, TRPA1 is activatedby an agonist (bradykinin) that initiates a PLC-coupled signalingpathway (184).

The mouse and zebrafish TRPA1 proteins are required forhearing and may be mechanically gated channels (185). Amechanically gated channel is thought to be activated by agating spring, and it has been proposed that the large number ofankyrin repeats in near the N terminus may serve this purpose(185, 186).

Two Drosophila TRPAs appear to be thermosensors. TheDrosophila homolog of TRPA1 is also thermally activated;however, it is activated in vitro by warm rather than coldtemperatures (187). A role for dTRPA1 in vivo has not beendescribed. A phenotypic analysis of another Drosophila TRPA,referred to as Painless, demonstrates that it functions in larvaein the avoidance of noxious heat above ~38°C (188). Currently,there is no evidence that the Drosophila TRPA channelsfunction in mechanotransduction.

Group 1: TRPN SubfamilyThe TRPN subfamily includes a single member in worms, flies,and zebrafish (15, 16, 189), although no TRPN protein has beenreported in mammals. Each of the TRPN proteins include 29ankyrin repeats N-terminal to the six transmembrane segments(15, 16, 189) (Fig. 1). TRPN proteins share ~20% amino acididentity to TRPC proteins over a ~400–amino acid segment thatspans the six transmembrane domains. However, TRPN proteinsdiffer from TRPC, TRPV, and TRPM proteins in that they donot include a TRP domain.

Several lines of evidence indicate that TRPN channels maybe mechanically gated. The founding member of the TRPNsubfamily, Drosophila NOMPC, is most likely a subunit for amechanically gated channel because it is expressed in

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Mammalian thermal-sensitive channelsChannel Threshold Chemicals that evoke thermal-like response

TRPV1 ≥43 Capsaicin

TRPV2 ≥52 –TRPV3 ≥30-39 –TRPV4 ≥27 –TRPM8 23-28 Menthol, icilinTRPA1 17 Icilin, mustard oil

Drosophila thermal-sensitive channelsPainless(TRPA subfamily)

≥39 –

TRPA1 ≥27 –

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


mechanosensory organs and the mechanosensory response isgreatly reduced in loss-of-function mutants (15). In addition, aC. elegans TRPN protein appears to be expressed inmechanosensory neurons (15). The zebrafish homolog, TRPN1,is expressed in hair cells of the inner ear and is required inlarvae for hearing (16). However, these TRPN proteins have yetto be characterized in vitro, and it is not yet clear whether eitherof these proteins is capable of functioning independently as achannel. Nevertheless, the large number of ankyrin repeats inTRPN has been proposed to function as a gating spring (186).

Group 2 SubfamiliesOverview. The archetypal TRPP and TRPML proteins, TRPP2(polycystin-2, PKD2, PC2) and TRPML1 (mucolipin,MCOLN1, ML1), respectively, were identified in screens forthe genes disrupted in human diseases. The TRPP and TRPMLproteins are designated as Group 2 TRPs, owing to their rela-tively high similarity to each other but low primary sequencesimilarity to Group 1 TRPs. Nevertheless, as is the case with theGroup 1 TRPs, the Group 2 members contain six predictedtransmembrane domains and appear to be cation-permeablechannels.

Group 2: TRPP subfamily. TRPP2 was discovered as one ofthe gene products mutated in many cases of autosomal dominantpolycystic kidney disease (ADPKD) (190). ADPKD results inkidney failure in ~1 in 1000 individuals, due to the formation ofbenign, fluid-filled cysts (191–193). Human TRPP2 interactswith TRPP1 (polycystin-1, PKD1, PC1) (194, 195), and mutationsin one or the other of these two proteins account for ~95%of autosomal-dominant PKD (191). Both proteins are widelyexpressed (190, 196–198), and cyst formation may arise in othertissues, as well. Mice with targeted mutations in TRPP2 die inutero and display cyst formation in the maturing nephrons andpancreatic ducts (199) and defects in the cardiac septum (200).Thus, the mouse model recapitulates many of the features ofhuman ADPKD. In addition, TRPP2-deficient mice display alaterality phenotype (201, 202), which is described in moredetail below.

TRPP proteins appear to be present throughout the animalkingdom and, in addition to TRPP2, include two othermammalian proteins predicted to contain six transmembranedomains, referred to as TRPP3 (PKD2L1, polycystin-L, PCL)(203, 204) and TRPP5 (PKD2L2) (205, 206), and proteins inC. elegans (LOV-2) (207, 208), flies (AMO) (209, 210), andsea urchins (suPC2) (211). TRPP proteins share ~25% aminoacid identity to the most closely related TRPC proteins, TRPC3and TRPC6, over a region spanning transmembrane segmentsIV, V, and the pore-loop (H5 segment), which is a hydrophobicdomain between segments V and VI that contributes to ion se-lectivity (212) (Fig. 1). Mammalian TRPP2 contains a Ca2+-binding motif (EF-hand) and a coiled-coil domain near the Cterminus, but does not include any ankyrin repeats or a TRPdomain. In addition, TRPP proteins include a large extracellularloop between the first and second presumed transmembranesegments (Fig. 1).

TRPP1 is a very large protein consisting of 11 predictedtransmembrane domains, with amino acid sequence similarityto the transmembrane domains in TRPP2. In addition, TRPP1includes an ~2500–amino acid extracellular domain and anintracellular C-terminal domain (196) that interacts with the Cterminus of TRPP2 (194, 195). The interaction between the two

polycystins appears to be critical for function. According to onestudy, introduction of TRPP2 into Chinese hamster ovary(CHO) cells does not result in any discernible channel activity(213). However, coexpression of TRPP1 along with TRPP2induces translocation of TRPP2 to the plasma membrane andproduction of a Ca2+-permeable nonselective cation conductance(213). According to another study analyzing TRPP2 in lipidbilayers, the cytoplasmic tail of TRPP1 increased the channelactivity of TRPP2. Nevertheless, controversy exists as towhether TRPP2 is a cation influx channel (213–215) or a newtype of Ca2+-release channel (216).

TRPP3 has also been functionally expressed and shown tobe a nonselective cation influx channel that is positively regulatedby intracellular Ca2+ (217). Currently, there is no evidence thatTRPP3 Ca2+ influx activity requires TRPP1. Evidence has beenpresented raising the possibility that TRPP1 may also be acation influx channel (218). However, it was not excludedthat the currents induced by expression of TRPP1 were due toindirect effects on an endogenous channel. Nevertheless, thispossibility is consistent with the substantial similarity betweenTRPP2 and the C-terminal six transmembrane domains ofTRPP1.

ADPKD primarily affects the kidneys; however, it is alsofrequently associated with clinical manifestations in variousother tissues (219). TRPP1 and TRPP2 are concentrated inmono- or primary cilia (201, 220–222). In mammals, monociliaare present on the surface of most cells and consist of ninedoublets of microtubules distributed near the cell periphery (9+0structure). Motile cilia are similar in structure and include twocentral pairs of microtubules, in addition to the nine peripheraldoublets (9+2 structure).

Analyses of the mammalian TRPPs and their invertebratehomologs suggest a conserved role for TRPPs in both motileand nonmotile axonemal structures. The C. elegans homologsof both the TRPP1 and TRPP2 homologs (LOV-1 and PKD2,respectively) are required for male sensory behavior and areenriched in sensory cilia (207, 208). In the sea urchin, TRPP1and TRPP2 homologs are present in the acrosomal region ofsperm (211, 223), and in Drosophila the TRPP2 homolog,AMO, is concentrated in the sperm tail (209). Mutation of amodoes not affect sperm development, mating, or entry of thesperm into the females (209, 210). Rather, there is a defect intransfer of the sperm from the uterus to sperm storage organs.In female flies, sperm is subsequently released from the spermstorage organs so that eggs can be fertilized over an extendedperiod following mating.

The localization of TRPPs to both motile and primary ciliasuggest that TRP channels may play a role not only in the sensorymodalities, but in allowing individual cells to sense their localenvironment. TRPPs may be situated on primary cilium, whichprotrude from the surface of most vertebrate cells, to facilitatethe detection of extracellular signaling molecules or to sensechanges in fluid flow, osmolarity, or other mechanical stress.Consistent with this proposal, cultured kidney cells from wild-type, but not TRPP1 mutant, cells display an increase in Ca2+

influx in response to fluid flow (222). Similarly, the flow-induced Ca2+ influx was disrupted in wild-type cells bathed inantibodies that recognize the extracellular domains of eitherTRPP2 or TRPP1 (222).

Interestingly, a role for TRPPs in detecting fluid flow isconsistent with the findings that TRPP2 knockout mice display

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a defect in left-right asymmetry (201, 202). Wild-type verte-brates, such as mice and humans, display lateral asymmetryin the heart and several other organs. This left-right asymmetryis established during embryogenesis and requires leftwardflow of fluid over the node, which is generated by motile cilia.TRPP2 is normally expressed in node monocilia, and TRPP2-deficient embyros do not display the flow-induced rise in intra-cellular Ca2+ (201, 202). The combination of studies on TRPP2indicates that it may be a mechanosensor that responds tofluid flow.

Group 2: TRPML subfamily. The TRPML subfamily is definedby a human protein, TRPML1 (mucolipidin1; ML1), encodedby the MCOLN1 gene (224–226). Mutations in MCOLN1 areresponsible for a lysosomal storage disorder, mucolipidosis typeIV (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 to580 residues), relative to other TRP-related proteins, and thelevel of primary amino acid sequence identity to TRPC proteinsis quite limited. Analysis of TRPML1 using Prof ileScan(http://myhits.isb-sib.ch/cgi-bin/motif_scan), an algorithm thatcompares proteins to known motifs and patterns (227), reveals astronger relation to TRPCs than to other proteins in the databases.The similarity of TRPML1 (amino acids 331 to 521) to TRPspans the region that includes transmembrane segments three tosix and the putative pore-loop region. TRPML1 has strongersequence identity to members of the TRPP subfamily (≥20%amino acid identity over a region that includes transmembranesegments four to six) and includes a large extracellular loopbetween transmembrane domains one and two similar to that ofTRPP proteins (Fig. 1). Notable sequence motifs in TRPML1include a lipase serine active-site domain, a bipartite nuclearlocalization signal, and a putative late endosomal-lysosomaltargeting signal.

The subcellular distribution of the TRPML proteins isunresolved, but one hypothesis is that TRPML1 is an endosomal-and lysosomal-associated cation channel, which is required forthe re-formation of lysosomes (228). According to one expressionstudy in Xenopus oocytes, TRPML1 is a cation channel,which translocates to the plasma membrane upon treatmentwith the Ca2+ ionophore, ionomycin (229). TRPML1 has alsobeen reported to be inhibited by a decrease in pH (230). Twomutations associated with weak or moderate forms of MLIV donot eliminate channel activity, but prevent the pH-mediatedinhibition of the current (230).

The roles of TRPML2 and TRPML3 in humans are notknown; however, mutations in mouse Mcoln3 are responsiblefor the hearing loss and pigmentation defects associated withvaritint-waddler mice (231). Consistent with a role in hearing,TRPML3 is expressed in hair cells, both in a vesicular compartmentand in the stereocilia. Currently, the specific role of TRPML3 inhearing and pigmentation is not known.

Single representatives of the TRPML subfamily are encodedin worms and flies and are between 40 and 44% identitical tomammalian TRPMLs over most of the proteins (224–226).Within a domain encompassing transmembrane segments threeto six, the percent identity between TRPML1 and these invertebratehomologs rises to nearly 60%.

The C. elegans TRPML gene, cup-5, has been subjected togenetic analyses (232, 233). As appears to be the case in MLIV

(232), cup-5 mutant worms accumulate large vacuoles and displaydefects in endocytosis and lysosome biogenesis (234). In addition,introduction of either the human MCOLN1 or MCOLN3 genesinto worms rescues the cup-5 phenotype to a great extent(233, 235). The ability of MCOLN3 to rescue cup-5 suggeststhat the ML1 and ML3 proteins may be serving similar functionsat the molecular level, even though they are not redundant inmice or humans.

Functions of TRP ProteinsTRP proteins in vascular endothelial cells and myocytes. SustainedCa2+ entry in vascular endothelial cells leads to changes in cellshape (236) and affects vessel tone and permeability (237, 238),angiogenesis (239), and leukocyte trafficking (240). TRPCchannels may mediate these Ca2+ entry pathways becausedifferent TRPCs are expressed in various endothelial cells (158,176, 236, 241, 242). Furthermore, a dominant-negative form ofTRPC3 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, butthey display impaired vasorelaxation of the aortic rings (242).This defect may be a consequence of a perturbation in SOCEbecause agonist-induced Ca2+ influx is almost eliminated inaorta endothelial cells isolated from the TRPC4−/− mice.

TRPC proteins are also expressed in other nonexcitable cellsthat are proposed to be regulated by SOCE. These includepancreatic β cells (243), human platelets (82), rabbit portal veinsmooth muscle (244), and salivary gland cells (245). TRPC1 is acandidate for modulating the secretion of fluids and electrolytesin salivary glands, because SOCE is reduced in salivary glandcells transfected with antisense TRPC1 RNA (245).

TRP proteins may also function in vascular smooth musclecells. TRPC6 is expressed in rabbit portal vein myocytes, andintroduction of TRPC6 antisense oligonucleotides to suchprimary cells inhibits the nonselective cation channel activatedby α1-adrenoreceptor agonists (244). The α1-adrenoreceptorfunctions in the control of systemic blood pressure (246), and itis possible that it may do so through activation of TRPC6.TRPV2 is also expressed in vascular myocytes, where it appearsto be activated by membrane stretch (121).

TRPC proteins in fertilization. Fertilization of a mammalianegg is a multistep process that begins with association of thesperm with a glycoprotein, ZP3, in the egg’s extracellularmatrix (247). The sperm-ZP3 interaction triggers the release ofhydrolytic enzymes from the sperm acrosome and remodelingof the sperm surface. These events, referred to as the acrosomalreaction, are critical for penetration of the egg by the sperm,ultimately leading to zygotic development. Association of thesperm with ZP3 initiates the acrosomal reaction through asignaling cascade that involves G proteins (heterotrimeric GTP-binding proteins) (248), PLC-δ4 (249), transient Ca2+ influxthrough voltage-gated channels, and a more sustained influxthrough a SOC (250). The identity of the SOC has been elusive,although mouse TRPC2 has been suggested to be a subunit ofthe ZP3-triggered channel. A TRPC2 isoform is highly enrichedin the sperm, and antibodies to an extracellular domain ofTRPC2 inhibit the ZP3-induced Ca2+ influx and acrosomalreaction (77). However, a knockout of mouse TRPC2 does notshow defects in fertilization (251). TRPC2 cannot participate in

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fertilization in humans, because the human TRPC2 is a pseudo-gene (1). At least four TRPC proteins have been localized inhuman sperm (252), raising the possibility that one or moreTRPCs functions in fertilization. Consistent with a possible rolefor mammalian TRPCs in fertilization, a TRPC-related proteinin C. elegans, TRP-3, is expressed in sperm and is required forfertility (30).

TRP proteins in the kidneys, urinary bladder, and smallintestine. In humans, TRPM6 is present at the highest levels inthe kidneys and small intestine, and mutations in this chanzymeresult in hypomagnesemia with secondary hypocalcemia(164–166). The defect in Mg2+ absorption results in a variety ofsymptoms, including seizure and muscle spasms during infancy.TRPM7 is also permeable to Mg2+, and interactions betweenTRPM6 and TRPM7 may also contribute to the disease (seebelow).

Several members of the TRPV subfamily are present in thekidneys, one of which, TRPV4, is predominantly in the distalnephron of the kidneys (122–124, 253). TRPV4 is activated invitro by decreases in osmolarity and is present in a region of thekidneys that may be exposed to hypotonic fluid (122–124).TRPV4-mutant mice display an impairment in osmoticsensation; however, this phenotype is attributed to a role ofTRPV4 in the brain, which leads to an increased level of serumarginine vasopressin in response to hyperosmolarity (254).Whether TRPV4 has a role in the regulation of electrolyte orfluid transport in distal nephron has not been demonstrated.

TRPV5 and TRPV6 are present primarily in the kidneys andsmall intestine, respectively, and may play important roles inCa2+ absorption (130, 255). A definitive demonstration thatTRPV5 functions in Ca2+ reabsorption in the kidney has beenprovided by an analysis of TRPV5-deficient mice (256). Thedefect was observed along the kidney’s distal convolution,where TRPV5 normally is present. In addition, the mutant miceexcreted about six times more Ca2+ in the urine than did wild-type animals. Interestingly, the expression of genes encodingCa2+ transport proteins, such as the Na+/Ca2+ exchanger(NCX1), was reduced in the kidneys. The dysfunction in Ca2+

homeostasis in the TRPV5−/− animals also resulted in defects inbone structure.

In addition to a requirement for TRPV protein in the kidneys,at least one member of this subfamily, TRPV1, is required fornormal urinary bladder function (257). TRPV1-knockout micedisplay normal bladder morphology and excrete similarvolumes of urine as wild-type animals do. However, the mutantanimals show a higher frequency of small volume eliminationsand an increase in bladder capacity and nonvoiding bladdercontractions (257). The bases of these phenotypes are unresolved,but it is noteworthy that ATP and purinergic signaling has beenlinked to bladder function (258–261) and stretch-evoked ATPrelease is greatly reduced in urothelial cells from TRPV1−/−

mice (257). Currently, there is no evidence that TRPV1 is activatedby membrane stretch or hypotonicity, although it is plausiblethat heteromultimers composed of TRPV1 are stretch-activatedchannels.

TRPC proteins in neuronal development and plasticity.Mammalian TRPC RNAs and proteins are each expressed in thebrain (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 additionto the central nervous system (1, 2). TRPC1 is required for the

excitatory postsynaptic conductance (EPSC) in cerebellar Purkinjecells, through a pathway initiated by glutamate stimulation andinvolving activation of a group I metabotropic glutamate receptors(mGluR1), Gαq, and PLC-β (264). Although the function of thispostsynaptic current is unresolved, these data raise the possibilitythat TRPC1 functions in neuronal plasticity.

TRPC3 may participate in activity-dependent changesthat occur in the mammalian brain around the time of birth. TheTRPC3 protein is detected primarily in the brain immediatelybefore and after birth and is activated in vivo by a pathwaythat is initiated by BDNF (84). BDNF functions in neuronaldifferentiation and survival (265, 266), which occurs throughchanges in transcription many hours after exposure to theneurotrophin. However, there is evidence that BDNF is involvedin synaptic plasticity and can cause rapid effects, such asmorphological changes at the growth cone and modulation ofneurotransmitter release (267–269). Because these effects aretoo rapid to occur through transcriptional induction, they maybe mediated by BDNF-stimulated Ca2+ influx through TRPC3.

Direct evidence that TRPC channels function in neurotrans-mitter release and growth cone extension has been obtainedfor TRPC4 and TRPC5, respectively. Many dendrites contributeto synaptic transmission through release of the neurotransmitterGABA (γ-aminobutyric acid). The release of GABA is reducedin TRPC4-mutant mice, indicating that this channel promotesthis process (270). Another exciting finding is the observationthat TRPC5 channels appear to function in neurite extensionand affect growth cone morphology (94). Growth conespossess TRPC5 and display a current reminiscent of TRPC5.A dominant-negative form of TRPC5 induces longer growthcones, suggesting that TRPC5 activity inhibits neuriteextension. The pathway leading to TRPC5 activation involvesstimulation of a receptor tyrosine kinase, which in turn engagesphosphoinositide 3-kinase (PI3K), the guanosine triphosphataseRac1, and PI5K (92).

Vertebrate TRP proteins in sensory physiology. An emergingtheme is that many vertebrate members of the TRP superfamilyfunction in sensory perception, as is the case for invertebrateTRPs. These include chemosensation, hearing, osmosensation,and thermosensation. At least two TRP channels functionin chemosensation, one of which (TRPM5) is present in tastereceptor cells and is required in the mouse for the bitter, sweet,and umami responses (151, 152). TRPM5 is also present in othertissues (271) and presumably has additional roles independentof taste transduction. Features that distinguish TRPM5, and thehighly related channel, TRPM4b, from other TRP channels isthat they are voltage-modulated, Ca2+-activated monovalentselective cation channels (142–146). These are properties ofcurrents that have been implicated in diverse processes rangingfrom slow oscillations in the thalamocortical neurons duringsleep (149) to the firing properties of pyramidal cells in the cortex(150) and signaling in cardiomyocytes (272, 273). WhetherTRPM4b or TRPM5 function in these latter processes remainsto be determined.

One TRPC protein, TRPC2, plays a role in the pheromoneresponse. TRPC2 is expressed as at least two alternativelyspliced isoforms, one of which (TRPC2α) is enriched in thevomeronasal organ (VNO) of rodents (55, 61, 78). In the VNO,pheromone presentation activates TRPC2 through a DAG-dependent pathway (76). Surgical ablation of the VNO indicatesthat this organ functions in the initiation of male sexual behavior

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in rodents (274). However, mating in TRPC2−/− mice is normal(251, 275). Rather, TRPC2-deficient mice fail to display thepheromone-induced male-male intraspecies aggression (251,275). TRPC2-knockout males exhibit sexual behavior, withoutpreference, toward castrated intruder males or females. Thus,TRPC2 function is required for sex discrimination. It is note-worthy that the VNO may not be functional in humans (276),and that human TRPC2 is a pseudogene (1). Therefore, TRPC2cannot contribute to sexual discrimination in humans as it doesin mice.

Three vertebrate TRPs function in hearing, one of which,TRPN1, is the zebrafish homolog of the Drosophila mechan-otransduction channel, NOMPC (16). RNA encoding TRPN1is present in embryonic and larval hair cells and in multipletissues in the adult, including the ear. Reduction in TRPN1expression, using morpholino antisense oligonucleotides, doesnot affect larval hair cell number or morphology, but inhibitsauditory and vestibular function. The subcellular distribution ofthe TRPN1 protein is not known, and it is unclear whether itis the mechanotransduction channel. Nevertheless, a TRPNprotein cannot serve as a mechanosensory channel in humansbecause members of the TRPN subfamily are not encoded inthe genomes of mammalian organisms.

A third TRP protein that participates in hearing is a memberof the TRPML subfamily. Mutations that disrupt mouseTRPML3 result in hearing loss, vestibular defects, and otherabnormalities (231). TRPML3 is present in hair cells, althoughit is not a strong candidate mechanotransduction channel becauseit does not appear to be localized to the tips of stereocilia wherethe transduction machinery is situated. In addition to hair cells,TRPML3 is present in other cell types and tissues and has beenproposed to function generally in intracellular ion homeostasis.

TRPA1 has emerged as the elusive mechanically gatedtransduction channel necessary for the auditory response inmammals (185). Disruption of TRPA1 expression causes defectsin hearing, and the protein is present in the tips of stereociliawhere the transduction machinery is situated. Furthermore,there is an increase in TRPA1 expression coincident with theinitiation of transduction in hair cells.

In addition to facilitating an animal’s ability to sense stimuliin the external milieu, some TRP channels enable individualcells to detect changes in the local cell environment. Theseinclude alterations in fluid flow and osmolarity. Both TRPV4(122–124) and TRPP2 (277, 278) appear to participate in detectionof such localized stimuli.

Among the most exciting findings in the TRP field are thedemonstrations that six mammalian members of the TRP super-family respond to thermal temperature [Table 5 (also seeabove)] (96, 111–115, 126, 177, 178, 182). These six TRPchannels belong to three different subfamilies (TRPV, TRPM,and TRPA) and together account for the responses to a widerange of temperatures extending from noxious heat (≥43°C) touncomfortable cold (≤17°C). Furthermore, at least three of thethermoTRPs also respond to chemicals that elicit the sensationof thermal heat or thermal cold (96, 177, 178, 182–184),providing at least part of the molecular mechanisms forthese phenomena. Many questions remain concerning thethermoTRPs. Are there one or more additional TRPs, whichrespond to temperatures in the ultracold range? What are theidentities of other proteins that interact with and modulate theactivities of thermoTRPs?

TRPs and hypoxia-induced cell death. Anoxic conditions,such as occur during metabolic stress, can lead to massive deathof neurons and other cell types. Consequently, understandingthe molecular mechanisms underlying this phenomenon hasimportant implications for treating ischemic cell death. A seriesof studies indicate that uncontrolled activity of TRP channelsmay be a major factor leading to anoxic cell death.

The first evidence linking metabolic stress to cell death wasobtained in studies of TRPC channels. Drosophila TRP andTRPL are constitutively active in vivo under anoxic conditionsor as a result of application of mitochondrial uncouplers ordepletion of ATP (48). Furthermore, mutations that causeconstitutive activation of TRP result in neurodegeneration inDrosophila photoreceptor cells (279). Oxidative stress may alsoresult in activation of mammalian TRPC proteins. Endothelialcells express an oxidant-activated nonselective cation channelthat functions as a redox sensor in the vascular endothelium,and a dominant-negative form of TRPC3 abolishes the oxidant-induced current (75). These experiments suggest that eitherTRPC3 or a channel capable of heteromultimerizing withTRPC3 contributes to this conductance. On the basis of thesestudies, oxidative stress in the mammalian brain could potentiallyresult in constitutive activation of TRP proteins, which in turncould result in cell death due to uncontrolled influx of Ca2+.

The potential contribution of TRP channel to hypoxia-inducedcell death has received considerable support from analyses oftwo TRPM channels: TRPM2 and TRPM7. TRPM2 can beactivated by H2O2, which leads to elevated levels of β-NAD+

(174, 280). Disruption of native TRPM2 expression in a ratinsulinoma cell line suppressed the H2O2-induced cell death(174). Thus, TRPM2 may lead to Ca2+ overload and neuronalcell death as a result of a change in redox status. Prolongedoxygen and glucose deprivation appears to lead to activation ofTRPM7 in neurons, and blocking TRPM7 expression in primaryneurons inhibited the cell death resulting from anoxia (281).Thus, drugs that inhibit TRP proteins would offer a new therapyfor minimizing the neurodegeneration associated with strokesand other traumas that induce oxidative stress.

Heteromultimeric Interactions Among TRP FamilyMembersA recurring theme is that many TRPs in native systemsform heteromultimeric channels composed of two or more TRPsubunits. In Drosophila photoreceptor cells, the three TRPCproteins that mediate the light-activated conductance assembleto form TRP homomultimers and heteromultimers of TRPLand TRP or TRPL and TRPγ (23, 25). TRP is ~10-fold moreabundant than TRPL and TRPγ and forms a regulated channelin the absence of other subunits. Thus, TRP may be expressedin vivo primarily as a homomultimer. By contrast, in vitroexpression of just TRPL or TRPγ results in constitutive activityof these channels. However, TRP/TRPL and TRPL/TRPγheteromultimers are regulated channels with biophysicalproperties distinct from those of the homomultimers. Thehomo- and heteromultimeric interactions between the TRPsubunits occur through the coiled-coiled region in the N-terminalsegments (25). In addition, the transmembrane segments alsocontribute to subunit interactions.

Heteromultimeric interactions also occur among mammalianTRPC channels. Based on analyses in human embryonic kidney293 (HEK293) cells (282) and in the brain (283), there is a

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strong preference for interactions between those channels thatbelong to the same subset: TRPC4/5 and TRPC3/6/7. TRPC1displays a propensity to interact with TRPC4 and TRPC5,whereas TRPC2 does not appear to heteromultimerize withother channels.

An important question concerns the functions of the hetero-multimeric interactions. Possibilities include effects on thechannel localization or stability and alterations in the biophysicalproperties of the currents. Each of the TRPC proteins hasbeen expressed individually in tissue culture cells, and in manycases expression of these proteins results in the appearance ofconstitutive cation influx [for example, see (60, 62–65, 284-286)].This property of many TRPC-dependent currents is similarto the constitutive influx resulting from in vitro expression ofeither Drosophila TRPL or TRPγ. TRPL/TRPγ coassemble toproduce a PLC-regulated channel (25). Thus, it is plausiblethat some TRPC proteins are channel subunits that depend oninteractions with other TRPC proteins for regulated activity.Consistent with this proposal, a TRPC3-dependent conductanceendogenous to pontine neurons is not constitutive; rather, it isactivated through a signaling pathway involving TrkB andPLC-γ (84). Whether TRPC3 interacts with another TRPCprotein in vivo has not been addressed, although TRPC3 doesinteract in vitro with TRPC1 (23). However, in contrast toTRPL/TRPγ heteromultimers, coexpression of TRPC1 andTRPC3 in tissue culture cells generates a larger constitutivelyactive 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 TRPC4or TRPC5 in the mammalian brain. TRPC4 and TRPC5coimmunoprecipitate with TRPC1 from rat brains (85). More-over, coexpression of either TRPC4 or TRPC5 with TRPC1 intissue culture cells results in the production of nonselectivecation conductances distinct from those generated by expressionof the individual proteins (85). The conductances arisingfrom TRPC1/TRPC4 and TRPC1/TRPC5 heteromultimers areaugmented 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 internalstores. However, constitutive activity occurs in the absence ofreceptor activation. Thus, as is the case with TRPC1/TRPC3heteromultimers, it is likely that additional subunits interactwith and participate in the regulation of TRPC1/TRPC4and TRPC1/TRPC5 heteromultimers in vivo. Indeed, there isevidence that TRPC heteromultimers consist of three TRPsubunits (288). These include TRPC1/3/5, as well as othercombinations of subunits: TRPC1 with TRPC4 or -5 and TRPC3or -6. Thus, differential TRPC subunit interactions may give riseto a daunting diversity of channels and conductances.

Heteromultimerization is not limited to TRPC, but appearsto typify members of other TRP subfamilies, such as TRPV andTRPM. The C. elegans TRPV proteins, OSM-9 and OCR-2,may form heteromultimers in vivo, because the two proteinsmutually influence their subcellular localizaton in those sensorycilia that normally coexpress both proteins (289). In a similarfashion, it has been proposed that the Drosophila TRPV channels,Iav and Nan, interact directly, because elimination of one proteinresults in instability of the other in cilia required for the auditoryresponse (101). Several lines of evidence indicate that themammalian TRPV5 and TRPV6 form functional heteromultimers

(11). Both channels are coexpressed in kidneys, and taggedversions of the proteins coimmunoprecipitate from Xenopusoocytes. Moreover, coassembly of TRPV5/6 heteromultimersalters several biophysical properties, including Ba2+ selectivityand Ca2+-dependent inactivation, from those observed forhomomultimeric 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 TRPM6/7heteromultimer display a larger conductance than either homo-multimer. In HEK293 cells, the heteromultimeric interactionsare required for insertion of TRPM6 in the plasma membrane.Interestingly, a mutation in TRPM6 that causes hypomagnesemiaand hypocalcemia disrupts association with TRPM7 and insertionin the plasma membrane.

It remains to be determined whether members of differentTRP subfamilies associate in vivo. However, TRPC1, but notTRPC3, interacts directly with the TRPP2 in vitro (290).The functional implications of this interaction have not beendescribed.

Association of TRP Channels into MacromolecularAssembliesThe Drosophila signalplex. It now appears that many membersof the TRP superfamily exist in macromolecular assembliescomposed of multiple signaling components. The existence of aTRP-containing supramolecular signaling complex (signalplex)was first demonstrated in Drosophila photoreceptor cells (7).The molecular scaffold for the signalplex is INAD (inactivation-no-afterpotential D), a protein that consists of five ~90–aminoacid protein domains referred to as PDZ (PSD-95, DLG, zonaoccludens–1) domains. INAD binds directly to a minimum ofseven 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-inactivation-nor-afterpotential C) myosin III (297). INAD also binds in vitroto the Drosophila homolog of the FK506-binding protein,dFKBP59 (294). This latter protein also interacts with TRPLand down-regulates the channel, although a role for dFKBP59in the photoresponse has not been described. The number ofproteins that bind to INAD exceeds the number of PDZ domains.INAD is capable of forming homomeric interactions (293), thusproviding the binding capacity to simultaneously nucleate alarge array of target proteins.

Of primary importance is the identification of the functionsof the signalplex. Because light-dependent cation influx occurswithin milliseconds of activation, it would seem that couplingof the signaling components into a macromolecular assemblywould serve to facilitate rapid activation. However, deletion ofthe INAD binding site in TRP has no effect on the kinetics ofactivation (298). Thus, a direct association of TRP with INADis not required for the light response. Nevertheless, it has notbeen excluded that the mutated TRP still associates indirectlywith the signalplex and that such an interaction could contributeto activation.

One role of the signalplex is to retain signaling proteinsin the microvillar portion of the photoreceptor cells, therhabdomeres. In wild-type photoreceptor cells, the proteinsthat participate in phototransduction are highly enriched in therhabdomeres (7, 299), which are the functional equivalent of the

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outer segments in mammalian photoreceptor cells. In inaDmutant flies, the localizations of at least three INAD targets,TRP, PKC, and PLC, are severely disrupted (295, 299). INADfunctions in retention rather than targeting of these proteinsto the rhabdomeres (298, 300). In addition, elimination ofINAD or the INAD-binding sites in TRP, PKC, or PLC resultsin instability of these INAD-binding proteins (298, 299, 301).

The requirement for the TRP-INAD interaction for retentionin the rhabdomeres is reciprocal. Mutation of the INAD-bindingsite in TRP results in mislocalization of INAD and, as a conse-quence, mislocalization of PLC, PKC, and TRP (298). Eliminationof any other known INAD-binding protein has no effect on thelocalization of INAD. Thus, it appears that TRP and INAD formthe core complex required for retention of the signalplex in therhabdomeres.

Decreases in the concen-tration of signaling proteinsin the rhabdomeres, due todisruption of INAD-targetprotein interactions, have atleast two effects on photo-transduction. First, the overallamplitude of photoresponseis reduced (299). Second, areduction in the levels ofPLC result in slower responsetermination (301, 302). Thisdefect may be due to loss of theproper stoichiometry betweenthe PLC and the G protein(302). The relative concentra-tions of these two proteins arecritical because the PLCfunctions as a GTPase-acti-vating protein for the trimericGαq subunit (302, 303), inaddition to its more recog-nized phospholipase activity(304). Thus, a reduction inthe levels of PLC results indelayed termination, due toslower inactivation of theG protein. Therefore, thesignalplex maintains boththe proper stoichiometry andabsolute concentrations ofsignaling proteins in therhabdomeres.

A key question is whetherthe association of any targetprotein with INAD functionsdirectly in the photoresponse,independent of any require-ment for retention or proteinstability. Disruption of theINAD-binding site in PKCdecreases the rate of termina-tion of the photoresponse(305), although this effectmay be due to mislocalizationof PKC. However, interaction

of INAD with NINAC has a direct role in signaling. Mutationof the INAD-binding site in NINAC has no effect on proteinstability or rhabdomeral localization, but causes a profounddelay in termination (297). The basis for the requirement forthe NINAC/INAD interaction for response termination is notknown, although the observations that NINAC binds actinand that INAD associates with both NINAC and TRP raises thepossibility that actin or myosin force generation functions inturning off the light-sensitive cation channels.

Mammalian TRP supramolecular complexes. MammalianTRPC proteins also appear to be organized into macromolecu-lar assemblies. For example, TRPC3 is activated through a path-way initiated by the BDNF receptor TrkB, and TRPC3 immuno-precipitates with the TrkB from rat brains (84). This interaction

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Fig. 4. Classical TRP proteins associate with signaling complexes. (A) Model of the Drosophila signalplex.INAD is a scaffold protein with five ~90–amino acid PDZ domains that bind directly to TRP, TRPL,PLC-β, PKC, rhodopsin (Rh), the NINAC myosin III, and calmodulin (CaM). The signalplex could belinked to actin filaments through NINAC. INAD is also capable of forming homomultimers. (B) Speculativemodel of the TRPC4 signalplex. TRPC4 binds directly to a protein, NHERF (also known as EBP50),containing two PDZ domains and a C-terminal domain that is an ERM binding domain (EBD).Although NHERF can bind to at least one G protein–coupled receptor (GPCR) and members of theERM family, the TRPC4 signalplex has not been shown to include a GPCR, ezrin, or any otheractin-binding protein. Thus, these latter interactions are speculative. The complexity of the NHERFsignalplex may be increased by homomultimerization of NHERF and TRPC4.


is most likely indirect, although the molecular link betweenthese two proteins has not been identified. TRPC1 is localizedto a subset of lipid rafts referred to as caveolae, where it may alsoform part of a multicomponent complex. Lipid rafts areglycosphingolipid- and cholesterol-enriched membrane micro-domains that appear to concentrate certain transmembraneproteins and proteins with glycosylphosphatidylinositolanchors or hydrophobic modifications (306–309). Caveolaeare invaginations in the plasma membrane that formthrough coalescence of lipid rafts. Caveolae may have particularimportance in Ca2+ signaling because they are enriched withvarious proteins that participate in Ca2+ regulation and maybe sites for Ca2+ entry and sequestration (310). Caveolin, atransmembrane cholesterol-binding protein that is concentratedin caveolae (311–313), may be a scaffolding protein that nucleatessignaling complexes (314). TRPC1 appears to be localized tocaveolin-containing lipid rafts and coimmunoprecipitates withcaveolin, the IP3R, and Gαq from a salivary gland cell line(315). Furthermore, thapsigargin-induced Ca2+ influx is disruptedin this cell line upon depletion of cholesterol from the plasmamembrane. Because cholesterol depletion disrupts lipid raftdomains, this suggests that TRPC1 function is dependent onassociation with caveolae. However, there is no direct evidencethat the current was mediated by TRPC1, and it remains to bedetermined whether TRPC1 binds directly to caveolin.

In vitro studies indicate that TRPC4 and TRPC5 associatewith macromolecular complexes that bear similarities to theDrosophila signalplex (Fig. 4B) (316). The central protein inthese complexes is the Na+/H+ exchanger regulatory factor(NHERF, also referred to as EBP50), a protein containing twoPDZ domains (317, 318). Mutation of the C terminus of TRPC4disrupts its interaction with NHERF and translocation of thechannel to the plasma membrane (319).

In addition to TRPC4 and TRPC5, in vitro NHERF alsobinds to PLC-β (316). Moreover, TRPC4, PLC, and NHERFcoimmunoprecipitate from mouse brain cells. PLC and TRPC4are unlikely to bind to the same NHERF monomer, becausethey both interact with NHERF through PDZ1. As with INAD,NHERF appears to self-associate, and such homomultimerizationcould provide NHERF with the capacity to cluster an array ofproteins (316). The complexity of the NHERF signalplex couldbe 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-radixin-moesin (ERM) family (321), which could provide a link to theactin cytoskeleton. It remains to be determined whether theselatter classes of proteins are complexed with the same NHERFmolecules that associate with TRPC4 and PLC-β. If so, thenmammalian TRPC proteins may be organized into signalingcomplexes that resemble the Drosophila signalplex. The nextchallenge will be to determine whether the TRPC4/NHERFsignalplex contributes to signaling, as well as to the localizationand stability of the component proteins, as is the case inDrosophila photoreceptor cells.

Several TRPC proteins have been shown to form a multimericcomplex with either of several Homer isoforms. Homer is anEVH1 domain scaffold protein that binds to proline-containingsequences similar to PPXXF (322, 323). Homer binds to TRPC1in the brain, and the interaction appears to occur through twosites in the channel, N- and C-terminal to the transmembranedomains (26). The C-terminal Homer interaction sequence

corresponds to TRP box 2 (Fig. 2). Both Homer and severalTRPCs bind to the IP3R (79, 81, 323), and the Homer/TRPC1interaction promotes the direct interaction between theTRPC1 and the IP3R (26). Interestingly, depletion of Ca2+

stores disrupts the association of TRPC1 with Homer and theIP3R, resulting in increased Ca2+ influx. The TRPC/Homer/IP3Rcomplex may also contain group 1 metabotropic glutamatereceptors (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/TRPC1interaction is Homer-dependent.

TRPV5 and TRPV6 also appear to be complexed with amacromolecular assembly because these channels associatewith a complex that includes annexin 2 and S100A10 (324).Annexins are phospholipid-binding proteins, some of whichshuttle between intracellular membranes and the cell surface ina Ca2+-dependent manner. The association between S100A10can augment phospholipid binding of annexin 2. The interactionbetween TRPV5 or TRPV6 and the annexin 2/S100A10complex promotes surface localization of the channels. However,it remains to be determined whether this reflects a role forannexin 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 tomassive protein complexes. In addition to altering TRP channellocalization, stability, and activity, it is possible that macro-molecular assemblies may also contribute to the speed andspecificity in signaling. Now that the members of the TRPsuperfamily have been identified, one of the many challenges inthe field will be to define the nature and functions of eachchannel and the associated supramolecular complexes.

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Citation: C. Montell, The TRP superfamily of cation channels. Sci. STKE2005, re3 (2005).

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