I. INTRODUCTION

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The role of plasma membrane channel proteins is to regulate the flow of different ions between the cell and its environment. Each channel has a typical permeability to different ions, conductance, mechanism of opening and closing (gating), typical agonists and antagonists, voltage dependence, and additional regulatory characteristics. The gating of channels may be direct by voltage or ligand binding, or indirect via cascade of molecular events and production of second messengers that lead to channel opening. A large volume of data and detailed understanding has been accumulated on voltage- and ligand-gated channels. However, channel proteins of the TRP family do not strictly fit to any of the above categories. The activation and regulation mechanisms of TRP channels are largely unknown and highly diverse. Channel activity is affected by several physical parameters such as osmolarity, pH, mechanical force, as well as biochemical interactions with external ligands or cellular proteins.

The structure of TRP channels relates them to the superfamily of voltage-gated channel proteins characterized by six transmembrane segments (S1-S6), a pore region (between S5 and S6), and a voltage sensor, although the voltage sensor in S4 is missing in TRPs. Functionally, they may be categorized as nonselective cation channels, although they demonstrate large diversity in the characteristics of their permeability to ions and some of them are highly selective for Ca²⁺. TRP channels have been found in many organs, mainly in the brain but also in heart, kidney, testis, lung, liver, spleen, ovaries, intestine, prostate, placenta, uterus, and vascular tissues. They have been found in many cell types, including both neuronal cells, such as sensory, primary afferent neurons and nonneuronal tissues such as vascular endothelial cells, epithelial cells, and smooth muscle cells. Functionally, the most prominent cellular signaling pathways in which TRPs play a role are also mediated via PLC. (for reviews, see Refs. 70, 107, 111, 118, 119, 152, 165).

The archetypal TRP protein was found and extensively studied with relation to Drosophila phototransduction (107, 111, 118, 165). The phototransduction cascade provides a unique and powerful model system to study functions of specific signaling proteins with relevance to many transduction cascades. Studying phototransduction in Drosophila offers many advantages. The small size of the genome, ease of growth, and short generation make Drosophila ideally suited for screening large numbers of mutagenized individuals. Furthermore, the creation of powerful genetic tools such as balancer chromosomes and P-element-mediated germline transformation combined with tissue-specific promoters allow the introduction of cloned genes back into the organism, providing a way to study in vitro mutagenized genes in the living fly (107, 175). In Drosophila it is thus easy to screen for mutants that have physiological or morphological defects (133). Studies of single cells using electrophysiology and microfluorimetry allow characterizing different functional aspects of responses in great detail. The high sensitivity to light allows detection and characterization of responses to activation of single receptor molecules. Moreover, responses to higher light intensities enable the study of fast response kinetics and adaptation. Several important and novel signaling proteins have been identified in Drosophila, and their role was established using the genetic dissection approach (107, 111, 118, 165). TRP is an illuminating example for a novel signaling protein of prime importance whose function has been elucidated due to the powerful genetic tools and functional tests that have been developed in Drosophila. These studies have led to identification and characterization of TRP as a light-sensitive and Ca²⁺-permeable channel (60, 126, 140), although the mechanism that leads to channel opening is not yet clear. The studies of Drosophila phototransduction have thus indicated that TRP is permeable to Ca²⁺, and it is the target of a phosphoinositide cascade, leading to the suggestion that phototransduction in Drosophila might be analogous to the general and widespread process of phosphoinositide-mediated Ca²⁺ influx (61).

Inositol lipid signaling is one of the most widespread signal transduction cascades, which exists in virtually every eukaryotic cell. This transduction cascade that is mediated via G protein-activated phospholipase C (PLC) produces two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP₃) (14). This cascade begins by activation of a surface membrane receptor, followed by activation of InsP₃ receptors in the endoplasmic reticulum (ER), leading to Ca²⁺ release, and ends by the opening of surface membrane channels and Ca²⁺ influx, which activates a large variety of specific and critical cellular functions (Fig. 1). A great deal is known about the initial stages of this ubiquitous cascade; however, the molecular mechanism underlying activation and gating of the channels is elusive (152).

View larger version (57K): [in this window] [in a new window]   Fig. 1. The phosphoinositide cascade of vision. Cloned genes (for all of which mutants are available) are shown in italics, alongside their corresponding proteins. Upon absorption of light, rhodopsin (ninaE gene) is converted to the active metarhodopsin state, which activates a heterotrimeric G protein (dGq). This leads to activation of phospholipase C (PLC, norpA gene) and subsequent opening of two classes of light-sensitive channels encoded at least in part by trp and trpl genes, by an as yet unknown mechanism. TRP and TRPL opening leads to the light-induced current (LIC). Deactivation of channel activity is regulated by protein kinas C (PKC, inaC gene). PLC, PKC, and the TRP ion channel form a supramolecular complex with the scaffolding protein INAD (inaD gene, not shown). PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into the soluble second messenger inositol 1,4,5-trisphosphate (IP3) and the membrane-bound diacylglycerol (DAG). DAG is recycled to PIP2 by the phosphatidylinositol (PI) cycle shown in an extension of the smooth endoplasmic reticulum called submicrovillar cisternae (SMC, shown on the bottom). DAG is converted to phosphatidic acid (PA) via DAG kinase (DGK, rdgA gene) and to CDP-DAG via CD synthetase (not shown). After conversion to PI, PI is presumed to be transported back to the microvillar membrane by the PI transfer protein (PITP encoded by the rdgB gene). Both RDGA and RDGB have been immunolocalized to the SMC. At the base of the rhabdomere, a system of submicrovillar cisternae has been proposed by analogy to other insects, to represent Ca2+ stores endowed with IP3 receptors (IP3R), which is an internal Ca2+ channel that opens and releases Ca2+ upon binding of IP3.

Currently, the surface membrane nonexcitable Ca²⁺ influx channels are classified into two categories: store-operated channels (SOC) (100, 152) and store-independent channels (70, 152). Both channels require PLC for their activation. SOC channels are activated by the reduction in stored Ca²⁺. Store-independent channels include channels that are activated by Ca²⁺ elevation in the cytosol due to Ca²⁺ release and channels that are activated by the DAG branch of the inositol-lipid signaling pathway. In both cases the mechanism of channel activation is unknown (107). The Ca²⁺ release activated current (I_CRAC) represents activation of the classical SOC channels (134), and recently a TRP-related channel was suggested as a molecular candidate for I_CRAC channels (215). The paradigm that has been used to demonstrate SOC channel activation rather than store-independent channels is based on a bypass of InsP₃ production. Thus depletion of the internal stores by a specific Ca²⁺-ATPase inhibitor, thapsigargin, which is known to deplete InsP₃-sensitive stores (83) or by the Ca²⁺ ionophore ionomycin in Ca²⁺-free medium in the presence of strong Ca²⁺ buffers in the cell, activates SOC channels. Accordingly, after store depletion, application of Ca²⁺ to the external medium results in Ca²⁺ influx. The store-independent channels are activated by signaling mechanisms not directly related to the Ca²⁺ stores and, therefore, are not sensitive to thapsigargin or other Ca²⁺-mobilizing agents.

At present, the only candidates for the inositide-mediated Ca²⁺ influx channels, which include both SOC channels and store-independent channels, are proteins of the TRP family. In this review we emphasize the importance of studying TRP functions and properties in native cells and, therefore, first outline the research on TRP and TRP homologs that have been performed on Drosophila photoreceptors. Then we describe the fast-growing literature on a wide variety of TRP homologs that have been recently found in other species. The in vivo properties and possible functions of TRP channels and their relevance to signal transduction processes are the focus of this review.

	II. HISTORICAL BACKGROUND

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A spontaneously occurring Drosophila mutant was designated transient receptor potential (trp) by Minke et al. (113) because of its unique phenotype. In this mutant the response to prolonged illumination declines to baseline during light (38, 106, 113) (Fig. 2). Drosophila photoreceptors of wild-type and trp mutants have become a preparation of extensive research because of the ability to study TRP within the well-characterized phototransduction cascade in the native tissue (63, 107, 116, 157).

View larger version (7K): [in this window] [in a new window]   Fig. 2. The trp phenotype. Light-induced currents in response to prolonged intense orange lights were recorded in voltage-clamped photoreceptors of wild type (WT), the trp mutant, and WT treated with La3+. A peak response and a plateau characterize the light response of WT. The rapid peak-plateau transition is a manifestation of Ca2+-dependent light adaptation. The response of the trp photoreceptor decays close to baseline during light due to exhaustion of excitation. A similar decay of the light response close to baseline is obtained by application of 10 µM La3+ to the extracellular medium of WT photoreceptors. The horizontal bar indicates the light monitor. The intensity of the orange light stimulus is 10-fold dimmer in WT than in the trp mutant or WT treated with La3+. [From Minke and Selinger (111).]

The trp phenotype was originally explained by the hypothesis that some critical factor, which is required for excitation and needs to be constantly replenished during illumination, is in short supply in the mutant and cannot be replenished fast enough due to a mutation in the trp gene. Indeed, several lines of evidence indicate that the decline of the light response in trp mutants is due to exhaustion of excitation. Accordingly, the response to continuous intense light becomes equivalent to a response to dim light and then to darkness during illumination (106, 113).

A correlation between the trp phenotype and exhaustion of cellular Ca²⁺ was provided by exposure of isolated Drosophila ommatidia to Ca²⁺-free medium for a long (1 h) period of time in cells buffered with EGTA to ~50 nM intracellular Ca²⁺. The typical trp phenotype was obtained under such conditions (60). Similar apparent cellular Ca²⁺ deprivation could be obtained in the isolated ommatidia of wild-type (WT) Drosophila during a critical period of development. At this developmental stage (P14), no response to light can be observed unless Ca²⁺ (µM) is applied by the whole cell recording pipette. Interestingly, the light response under such conditions has the typical characteristics of the trp phenotype (65). Presumably under such conditions cellular Ca²⁺ is the limiting factor of excitation as in La³⁺-treated or Ca²⁺-deprived WT cells.

A recent study has proposed that the transient photoresponse of the trp mutants is due to Ca²⁺-dependent inactivation of the TRP-like (TRPL) channels rather than exhaustion of Ca²⁺-dependent excitation (164). According to this model, light-induced influx of Ca²⁺ through the TRPL channels (which remain operative in the trp mutant and have a considerable permeability to Ca²⁺) activates calmodulin (CaM), which in turn binds to CaM binding sites of TRPL (147) and deactivates the channels. In contrast, other experiments revealed that the trp phenotype is exacerbated in Ca²⁺-free medium, thus indicating that the negative feedback hypothesis probably does not underlie the trp phenotype (37). This conclusion was strongly supported by Hardie et al. (67), who found that trp decay and the associated inactivation are correlated with a drastic Ca²⁺-dependent reduction of phosphatidylinositol 4,5-bisphosphate (PIP₂) levels in the microvilli, while recovery from inactivation is abolished in mutants of the PIP₂ recycling pathway (rdgB and cds).

A turning point in understanding the trp phenotype and the possible role of TRP in phototransduction was brought about by the discovery that lanthanum (La³⁺) mimics the trp phenotype in WT flies (72, 181).

Application of La³⁺ in micromolar concentrations to the extracellular space of several species of WT flies accurately mimics the trp phenotype (Fig. 2) (60, 72, 181). The rate of occurrence of the single photon responses decreases in La³⁺-treated WT flies with incrementing intensity of long light stimulation in a similar manner to the effect of light found in the mutant. Furthermore, application of La³⁺ to the mutant has virtually no effect (181). The effect of La³⁺ is reversible upon removal of La³⁺ from the external solution. Because La³⁺ is a known nonspecific Ca²⁺ channel blocker, it was suggested that exhaustion of excitation in La³⁺-treated WT flies or in the trp mutant, which underlies the trp phenotype, is Ca²⁺ dependent. Thus, with the assumption that cellular Ca²⁺ is required for sustained excitation, it has been suggested that a TRP-mediated mechanism is responsible for replenishing cellular Ca²⁺ fast enough during strong illumination and, therefore, TRP is a Ca²⁺ channel/transporter (109, 110).

Whole cell voltage-clamp recordings applied to isolated Drosophila ommatidia (55, 156) revolutionized the field of Drosophila phototransduction and made it possible to examine the hypothesis that TRP is a Ca²⁺ channel/transporter (109). The experiments of Hardie and Minke (60) revealed that the fundamental defect in the trp mutant is a change in the ionic selectivity of the light-sensitive conductance. The relative Ca²⁺ permeability of the light-sensitive conductance in trp mutant was reduced by a factor of ~10 (60, 151, 158), along with a significant change in the relative permeability to different monovalent ions. The reduced Ca²⁺ permeability in the trp mutant has also been corroborated by the demonstration of a reduced light-induced Ca²⁺ influx using Ca²⁺ indicator dyes (59, 140) or Ca²⁺-selective microelectrodes (139). The effect of the trp mutation on ionic selectivity could be mimicked in WT photoreceptors by bath application of La³⁺ in micromolar quantities (60, 181).

The effects of the trp mutation on the permeability properties of the light-sensitive channels and the fact that in null trp mutants a significant response to light is preserved led Hardie and Minke (60, 61) to propose that there are at least two light-sensitive channels. Accordingly, one channel has the permeation properties of the channels remaining in the trp mutant, and a second class has high Ca²⁺ permeability, which is absent in trp mutants and blocked by La³⁺.

A significant step forward in the study of TRP became possible after the trp gene was cloned and sequenced by Montell and Rubin (121) and described as encoding a novel membrane protein. Subsequent reanalysis of the sequence by Kelly and colleagues (147) revealed significant homologies of the transmembrane domain to voltage-gated Ca²⁺ channels, the dihydropyridine receptors, and thus raised the hypothesis that TRP and its homolog, TRPL, may be channel proteins (147). A second putative channel gene, trp-like (trpl), was isolated using a screen for calmodulin binding proteins and was found to show ~40% overall identity with trp (147; for review, see Refs. 111, 116). This homology suggested that trpl might encode a second class of light-sensitive channel, responsible for the light-induced current recorded in the trp mutant (60, 147). Subsequent isolation of a null mutant of the trpl gene strongly supported the hypothesis that TRPL functions as a second light-sensitive channel (126). Under physiological conditions, the trpl mutation has a relatively small but distinct influence on the light response (92). Importantly, eliminating TRP-dependent current in trpl, either by La³⁺ or by examining the double mutant trpl;trp, results in complete abolishment of the response to light. Therefore, TRP and TRPL channels make up all the light-activated channels or are required for their activation (for details, see Refs. 158, 164; for review, see Refs. 107, 111, 116). The isolation of the null trpl mutant allows investigating whether the trp phenotype is a property inherent in the TRPL channels (164). An answer to this question was provided by the creation of the double mutants in which only a small amount of functional TRP channels remain (126, 158). Interestingly, these mutant alleles show the typical trp phenotype (92), indicating that the trp phenotype does not arise only from a specific property unique to the TRPL channel, as previously suggested (164), but also from low level of TRP in the absence of TRPL.

The first report that TRP-related proteins might also be found outside invertebrate photoreceptor came from Petersen et al. (143), who identified partial sequences of TRP homologs from Xenopus oocyte and murine brain cDNA libraries. Shortly thereafter, the full sequence of a human homolog (TRPC1) was reported following homology searches of EST databases (199, 216). Meanwhile, a large number of vertebrate TRP homologs have been cloned and sequenced (see Table 1). The functional role in the native tissues of vertebrate TRP homologs that have high similarity to Drosophila TRP is largely unknown. They show very widespread tissue distribution, but their properties have been mainly inferred from heterologous expression studies. The function of other members of the TRP family with low identity to Drosophila TRP can be hypothesized from their specific tissue distribution and production of knockout mice, which suggests important functions in both neuronal and nonneuronal cells (Table 1).

	III. MOLECULAR ANALYSIS OF THE TRP FAMILY

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The trp gene encodes a 1,275-amino acid membrane protein (121). Expression of trp genomic DNA in a trp mutant (trp^CM) background by germ-line transformation rescued the mutant phenotype (120), indicating that the gene is responsible for the trp mutant phenotype. The molecular structure of the TRP proteins as depicted in Figure 3 shows multiple domains that are likely to play important roles in cellular functions.

View larger version (60K): [in this window] [in a new window]   Fig. 3. Putative domain structure and topology of Drosophila TRP, TRPL, and human TRPC1. The protein sequences contain six putative transmembrane helices S1-S6 (TMD; A) and a putative pore region between S5 and S6 (shaded sphere between S5 and S6 in B and C); S3-S6 shows sequence fragments identical to the equivalent regions of the dihydropyridine receptor (a vertebrate voltage-gated Ca2+ channel). Both TRP and TRPC1 have four consensus ankyrin motifs (ankyrin) toward the NH2 terminus. In TRP there is one putative calmodulin-binding site (CaM) in the sequence. This site is missing in TRPC1. The TRP protein has a curious and unique hydrophilic sequence near the COOH terminus consisting of nine repeats of the sequence DKDKKPG/AD (8 × 9). The other features of TRP are PEST sequence, a proline-rich region with the dipeptide KP repeating 27 time (KP), and an INAD binding domain at the end of the COOH terminus. The special structural features of TRPC1 are the coiled-coil (cc) domain near the NH2 terminus and a dystrophin domain (dystrophin). [Modified from Minke and Selinger (111).]

At the NH₂ terminal of TRP there are three or four ankyrin repeats (147). Ankyrin repeats are 33-amino acid residue motifs that mediate specific protein-protein interactions with a diverse repertoire of macromolecular targets (166). Ankyrin has been shown to inhibit InsP₃ and ryanodine receptor-mediated Ca²⁺ release from the internal stores (21, 22). Ankyrin also provides a mechanism for linking membrane proteins to the cytoskeleton and plays a role in subunit interactions of proteins with two or more subunits (105). If TRP is a subunit of an oligomeric channel, the ankyrin repeat may play a role in subunit interactions or in TRP localization. Importantly, the ankyrin repeat is highly conserved throughout evolution in most members of the TRP family, strongly suggesting that it plays an essential, although still unknown, role in TRP function.

The most prominent structure of TRP is the transmembrane segments S1-S6 and the pore region loop between transmembrane segments S5 and S6, which is typical of voltage-gated channels. The identification of this region was the first molecular confirmation of the hypothesis that TRP may function as a channel (147). Furthermore, parts of this sequence are the most conserved region of TRP in all members of this family (see below).

A highly conserved region of TRP is found COOH terminal to S6 and includes 25 amino acids, six of which (Glu-Trp-Lys-Phe-Ala Arg) are referred to as the TRP box because of their invariant sequence. The function of this unique region is still unknown (119).

TRP contains a putative CaM binding domain somewhere in the region of the COOH terminal (residues 628-977) (147). A more defined fragment of the TRP sequence has been reported to bind CaM in a Ca²⁺-dependent manner (32). Sequence analysis suggests that the most likely CaM binding site in this region would be residues 705-725, which constitute the equivalent location to the CaM binding site in TRPL, even though there is no amino acid identity in this region between TRP and TRPL (see below). Drosophila photoreceptors contain an abundant amount of CaM and have specific proteins (NINAC) that stock-pile large amounts of CaM in the cell (149), yet the function of CaM in Drosophila phototransduction seems to be diverse and not entirely clear (6, 164).

The COOH-terminal region near the putative CaM binding domain contains a PEST sequence, which is a signal for protein degradation by the calcium-dependent protease calpain, typical of CaM binding proteins. Thus it has been expected that TRP will show a fast turnover rate. In contrast to this expectation, recent studies that measured the turnover of TRP in flies between 1.5 and 9.5 days old revealed only 25% turnover of TRP during that time, indicating a slow turnover of TRP (94).

At the COOH-terminal region there is also a proline-rich sequence in which the dipeptide KP is repeated 27 times (KP, in Fig. 3). Near the end of the COOH terminal, TRP contains the following amino acid sequence repeated in tandem nine times: D K D K K P G/A D. This 8 × 9 repeats region is likely to be important for protein-protein interactions. This region is unique to TRP and has not been found in any other member of the TRP family.

At the end of the COOH terminal there is a binding domain for the PDZ scaffold protein INAD (169, 187, 208) (see sect. VI and Fig. 3).

The TRP homolog protein TRP-like (TRPL) was isolated using a screen to detect CaM binding proteins and shared an overall 40% identity with TRP with much greater similarity (70%) in the putative transmembrane regions. Two regions that contain a typical CaM binding site were found in the trpl-encoded protein (Fig. 3A). A region homologous to the first putative CaM binding site in TRPL is not present in TRP. One putative CaM binding regions is capable of forming amphiphilic helices, which is typical for CaM bindings sites (147, 169, 187). Localization of the two CaM binding sites (designated CBS1 and CBS2) was determined by CaM overlays on fusion protein fragments (198). CBS1 is localized within residues 710-728, while CBS2 lies within residues 865-895, where there is no predicted amphiphilic helix. CBS2 is also unconventional in binding CaM in the absence of Ca²⁺ (198). The COOH termini of TRP and TRPL show very little homology (17%); nevertheless, both proteins show a large number of proline residues and thus contain a proline-rich domain at the COOH terminus part.

Both TRP and TRPL have weak but significant homologies to a known channel subunit of the vertebrate voltage-gated Ca²⁺ channel, the dihydropyridine receptor. However, TRP and TRPL contain only one of the four channel motifs with the six putative transmembrane domains S1-S6. By analogy with voltage-gated K⁺ channels and the cyclic nucleotide-gated channels, both TRP and TRPL represent subunits of putative tetrameric channels. Because null trp and trpl mutants both respond to light, each can function without the other. However, heterologous coexpression studies and coimmunoprecipitation experiments led to the suggestion that in WT flies the light-dependent conductance was mediated by at least three types of channels, two of which were TRP homomultimers and TRP-TRPL heteromultimer (209). Biophysical measurements of WT, trp, and trpl mutants questioned this conclusion, but these experiments did not exclude the possibility that heteromultimeric TRP-TRPL channels are important for specific, still uninvestigated features of the response to light (158).

A recent study has revealed an additional channel subunit called TRP (207), which is highly enriched in the photoreceptor cells. The NH₂-terminal domain of TRP dominantly suppressed the TRPL-dependent transient receptor potential on a trp mutant background, suggesting that TRPL-TRP heteromultimers contribute to the photoresponse. Furthermore, TRPL and TRP coimmunoprecipitate, suggesting that a physical interaction between these proteins forms a heteromeric channel (207). Further studies will be required to unequivocally determine the subunit composition of the light-sensitive channels.

The trp gene of the closely related blowfly Calliphora (79) and a more distantly related squid, Loligo (115), have been cloned and sequenced. The Calliphora sequence shows 77% sequence identity with Drosophila TRP, indicating that it is the ortholog of TRP rather than TRPL. The greatest difference is in the COOH terminal in which the KP proline-rich sequence in Calliphora is somewhat shorter (79). The squid sequence is more distantly related, showing only 46% identity to TRP and TRPL, and thus is equally similar to both proteins. As in TRP and TRPL, squid TRP and Calliphora TRP have CaM binding domains on the COOH terminus, multiple ankyrin repeats are found in the NH₂ termini, which are common feature of all TRP homologs, including those in vertebrates (see below).

Immunogold labeling indicates that the putative light-sensitive channels TRP and TRPL are localized to the microvillar membrane (187). One immunofluorescence study (148) indicates that TRP is particularly localized to the base of the microvilli in contrast to other studies (32, 187, 214). The reason for these differences in immunofluorescence localization of TRP is not clear. In addition, TRP is found in photoreceptor axons up to the lamina and medulla regions in the brain (148). Functional TRP is found in the antenna of the developing but not in the mature olfactory system of Drosophila (176). The possible localization of TRP to the base of the microvilli in juxtaposition to the putative InsP₃-sensitive Ca²⁺ stores bears important implications on the possible gating mechanism of TRP (see below). It should be noted that TRP is an abundant protein with amounts equal to that of PLC (i.e., ~10⁷ copies per cell, Ref. 79). This is unusual for channel proteins that are usually expressed in low amounts. Because the maximal light-induced current (LIC) in response to very intense light is ~20 nA (at 60 mV holding potential) and single-channel conductance of TRP is ~4 pS, it follows that ~7 × 10⁴ channels are sufficient to account for the maximal LIC. Accordingly, a small fraction (i.e., <1%) of TRP channels is sufficient to mediate the maximal LIC measured during very intense light. A possible reason for the unusually high levels of TRP is the second function of this protein as an anchor that localizes signaling complexes to the microvillar membrane (see sect. VI). A support for the notion that the large amount of TRP is not required for its function as a channel stems from experiments in which TRP was dissociated genetically from the INAD scaffold protein in the inaD^P215 mutant. In inaD^P215 mutants there is a large reduction of TRP in the microvilli, with minor effects on the response to light that manifests in somewhat slow response termination (32). Similarly, transgenic Drosophila trp¹²⁷², in which the binding site of TRP with INAD was deleted, shows a gross mislocalization of TRP in young flies. However, there was no effect on the response to light (94, 169). These data implicate that a large portion of TRP proteins are not necessary for production of the response to light and thus may have additional long-term roles (see below).

The localization of TRPL has not been extensively studied. Nevertheless, TRPL was reported to localize to the rhabdomere of Drosophila (126). Interestingly, TRP is 10-fold more abundant than TRPL (52, 209), and according to Hardie and colleagues (158), TRP and TRPL contribute about equally to the LIC under certain conditions.

By searching Caenorhabditis elegans genome database using the sequence that encodes the sixth putative transmembrane segment (S6) and part of the adjacent COOH-terminal cytoplasmic domain of Drosophila TRP, Harteneck et al. (70) identified 13 C. elegans TRP members. These were divided into three groups: short, long, and osm. The "short" group has a considerable similarity to Drosophila TRP and TRPL and therefore will be referred to in this review as the "TRP-homolog" group or TRPC. This group was recently designated classical TRPs by Montell to indicate the very significant similarity of the mammalian members to the original Drosophila member of this group (119). Full-length murine homologs, which constitute most of the known mammalian members of the TRP-homolog group have been cloned and characterized by Birnbaumer and colleagues (218) and Schultz and colleagues (70). Recently, Montell (119) has proposed to classify the nonclassical TRP channels into five subfamilies, which include the members of the "long" and "osm" groups. These five subfamilies are distantly related to the Drosophila TRP and TRPL and are referred to as the "TRP-related" group in this review.

Based on the released Drosophila genomic sequence, which accounts for ~95% of the euchromatic sequences, a total of 14 members of the TRP family have been reported (2). However, it appears that only TRP, TRPL, and TRP belong to the TRP-homolog group while the rest are TRP-related genes (207).

On the basis of similarity to Drosophila TRP and TRPL sequences, new TRP isoforms have been cloned using database searches of expressed sequence tags (EST), RT-PCR, or expression-cloning strategies. Seven major groups termed TRPC1-7 have been cloned and sequenced (Table 1; for review, see Ref. 152). The TRPC homologs have been cloned from human, mouse, rat, rabbit, bovine, and Xenopus. Five characteristic features of the Drosophila TRP and TRPL proteins have been found to be common to most members of the TRP-homolog group. 1) The predicted topology of six (but see Ref. 203) transmembrane segments (S1-S6) includes the typical pore region loop between transmembrane regions S5 and S6. 2) The charged residues in the putative S4 helix, which usually underlies voltage gating, are replaced by noncharged residues. 3) Three or four ankyrin repeats are found at the NH₂ terminal. 4) A proline-rich sequence is found in the COOH-terminal domain (70). 5) The TRP domain exists in all members of this group (119).

Birnbaumer and colleagues and Schultz and colleagues (16, 70, 218) have classified the vertebrate members of the TRP-homologs group into four subgroups according to their primary amino acid sequence (see Fig. 4). Type 1 includes all isoforms of TRPC1. Type 2 includes the TRPC2 homologs, which have the lowest similarity to the other groups. Type 3 includes TRPC3, TRPC6, and TRPC7 channel proteins. Type 4 includes TRPC4 and TRPC5 channels and has a higher similarity to the TRPC1 group relative to the other groups.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Pairwise similarity phylogenetic tree of the TRP family. Two distinct subgroups can be identified by their phylogenetic relationship. The top group (except for Drosophila TRP and TRPL) shows murine members of the "TRP homolog" (TRPC) group. The bottom group includes several members of the "TRP-related" group. The scale represents evolutionary distance calculated by Clustal analysis. [Modified from Strotmann et al. (177).]

TRPC1 was the first mammalian homolog of TRP that was cloned independently by two laboratories using EST database of human fetal brain cDNA library. It was initially called TRPC1 (199) or hTRP-1 (216). This TRP isoform is the shortest of all members of the TRP-homolog group (but see TRPC2 pseudogene, below). It has 38-40% identity and 58-62% sequence similarity to Drosophila TRP, TRPL, and the C. elegans isoform of TRP, which was identified during sequencing of chromosome III of C. elegans. This homology does not allow determination if TRPC1 is more similar to Drosophila TRP or to TRPL. In addition to three putative ankyrin repeats, the NH₂ terminal was also predicted to have a coiled coil structure at amino acids 256-300. Ninety amino acids at the COOH terminal of the human TRPC1 showed significant homology to dystrophin (199) (Fig. 3).

A spliced variant of TRPC1 (called TRPC1A) was cloned independently (221). This variant and TRPC1 were expressed in Chinese hamster ovary (CHO; Ref. 221) cells, in Sf9 (173, 221) cells, where they were studied functionally (see below), and in Xenopus oocytes (185). TRPC1 channels have also been found in other mammalian species: mouse, bovine, and rat (for review, see Ref. 152). Recently, a Xenopus isoform of TRPC1 was cloned (called Xtrp). The amino acids sequence of Xtrp was 82% identical to the mouse TRPC1, 81% identical to bovine TRPC1, and 84% identical to rat TRPC1 (18). The cloning of a Xenopus isoform of TRPC1 and its localization to the oocyte surface membrane is important, since the native inositol lipid signaling of Xenopus oocyte has been an important model system to investigate SOCs (142).

The expression pattern of TRPC1 made by several groups (30, 50, 173, 199, 216) shows that TRPC1 is most abundantly expressed in the brain, heart, testes, ovary, bovine aortic endothelial cells, and salivary glands and is barely detectable in the liver or adrenal gland. Interestingly, the rat ortholog (941 amino acid long with 52% identity to TRP) changes its expression pattern during brain development (50), suggesting that it has a role in developmental signaling. Injection of antisense oligodeoxyribonucleotide for mammalian TRPC1 inhibited the native SOC response of the Xenopus oocytes (185); however, the expression pattern of TRPC1 and its physiological properties suggest that it is not I_CRAC that was described in blood cells. Since a variety of SOC channels have been described and some of them are TRPs (46, 142), it is possible that specific TRP homologs mediate I_CRAC or other SOCs with still unidentified molecular identity. Support for this possibility was found in the report of Birnbaumer and colleagues (218) on carbachol-stimulated store-operated Ca²⁺ influx seen in murine Ltk cells expressing the muscarinic M5 receptor. These cells show loss of Ca²⁺ influx upon coexpression of a mixture of murine TRPC1-6 fragments in the antisense orientation. Several additional studies have reported that a reduction of TRPC1 expression using antisense approach led to inhibition of endogenous SOCs (98). Similar results were obtained in studies of TRPC3 (205) and TRPC4 (146) (for review, see Ref. 46).

The human TRPC2 (also designated Trp2), which was the first cloned TRPC2 gene (199), is probably a pseudogene since several independent ESTs show mutations introducing early stop codons. A bovine homolog of this pseudogene, which is mainly expressed in the testes, spleen, and liver, was cloned and sequenced (203). Later, a full-length 1,172-amino acid TRPC2 isoform from mouse (192) and a spliced TRPC2 isoform from bovine (GenBank) were cloned. Thus TRPC2 is the longest of all vertebrate TRP-homolog group, having a longer cytoplasmic NH₂-terminal domain. In contrast to the TRPC1, TRPC2 is tissue specific and in the mouse it was found only in the testes and vomeronasal organ (VNO). In contrast to other TRPC proteins of this group, each having several consensus glycosylation sites within the putative transmembrane domain of the protein, TRPC2 has only one glycosylation site located in the putative pore region. All other sites are localized to the cytosolic NH₂ terminal. TRPC2 shows only 25-30% identity to Drosophila TRP, and it is closer to the TRP-related group than any other TRP member of the TRP-homolog group (see Table 1 and Fig. 4). This is also reflected in the existence of a segment in the NH₂-terminal domain (amino acid 155 to amino acid 238) of the type 2 polycystic kidney disease gene (PKD2). PKD2 gene product is categorized as TRP-related channel (see below). This sequence is unique to TRPC2 and is absent in the other members of the TRP-homolog group. The function of the PKD sequence of TRPC2 is unknown (192).

A rat ortholog of the mouse and bovine TRPC2 genes was cloned by Liman et al. (96). Importantly, the rat ortholog is highly localized and heavily expressed in the VNO. This organ plays a key role in the detection of pheromones, which are chemicals released by other rats, and elicits stereotyped sexual behavior (96). Turning TRPC2 gene into a pseudogene in humans fits well with the hypothesis that the VNO is no longer functional in apes.

Recent studies revealed that InsP₃ is a second messenger of VNO receptor neurons of snake (184). This fact fits with TRPC2 as a channel, which is activated by the inositol-lipid signaling cascade.

A TRPC2 isoform is highly enriched in the sperm and seems to have an important function in the fertilization of mice (see below and Ref. 84).

TRPC3 homologs are predominantly expressed in the brain and at much lower levels also in ovary, colon, small intestine, lung, prostate, placenta, and testes (51, 114, 218). TRPC6 expression is highest in the brain, but it is also expressed in the lungs and ovaries. Interestingly, the development of tumors is associated with downregulation of TRPC6 isoform in a mouse model for an autocrine tumor (24).

TRPC7 from mouse turned out to be very similar (81% identity and 89% similarity) to TRPC3 and also to TRPC6 (75% identity and 84% similarity) while only 33% identical (53% similar) to Drosophila TRP and TRPL. TRPC7 is mainly expressed in the heart, lung, and eye and at a lower level in the brain, spleen, and testes (131).

TRPC4 and TRPC5 channels are similar in structure to TRPC1. Therefore, several investigators consider these three channels types as one group (15, 152), which also share similarity in function (see below). Petersen et al. (143) first cloned a partial sequence of the mouse TRPC4 isoform. Then, full-length mouse bovine and rat orthologs were described (122, 144). Another TRP homolog with high similarity to TRPC4 was cloned and designated TRPC5 (132, 145). The rabbit and mouse orthologs of TRPC5 show 69% sequence identity to bovine TRPC4 and 41% identity to Drosophila TRP. The expression patterns of TRPC4 and TRPC5 are very different. TRPC5 mRNA is expressed predominantly in the brain (145), while TRPC4 is expressed in the brain but also in the adrenal gland and at a much lower level in the heart, lung, liver, spleen, kidney, testes, thymus, aorta, and uterus (132). Of special interest are TRPC4 isoforms, which are expressed in vascular endothelial cells of various species (mouse, human, and bovine) (49; see sect. IVD).

In contrast to members of the TRP-homolog group that were discovered due to sequence homology to Drosophila TRP, the various members of the "TRP-related" group were found following studies aiming to explore specific sensory transduction pathways or specific genetic diseases. These pathways include olfaction and osmolarity in C. elegans (36), defects in mechanosensory transduction in C. elegans and Drosophila (196), defects in pain mechanism in primary afferents of dorsal root ganglia (27), Ca²⁺ transport in Ca²⁺ transporting epithelial cells (73), polycystic kidney disease in cells expressing the polycystin genes PKD (31), tumor suppression in the skin (reviewed in Ref. 119), and mucolipidosis type IV in cells expressing defective mucolipin (10, 179). The different approaches resulted in a wealth of functional data about TRP-related members, in contrast to most mammalian members of the TRP-homolog group, whose functions in most of the native tissues are largely unknown (but see below). In contrast to mammals, none of the five C. elegans members of the TRP-related group or other four C. elegans members of the TRP-homolog subfamily has been characterized at the cellular level, neither in the native cells (but see CUP5, below) nor in heterologous systems.

The TRP-related group (also referred to as nonclassical TRP by Montell) has been subdivided into five subfamilies by Montell (119). All members of this group share significant homology to Drosophila TRP in the transmembrane segments. However, the degree of structural similarity to TRP varies considerably among the various subfamilies. The TRPV subfamily is the closest to Drosophila TRP (see below). Some of these subfamilies lack the ankyrin repeats or the TRP domain. Nevertheless, they share some similar structural features and some of them also share similar unusual functional features such as permeability to Mg²⁺ and sensitivity to metabolic stress (see below).

This subfamily has all the structural features of the TRP-homolog group and is more similar to this group than any other subfamily of the TRP-related group (see Fig. 4). The sequence identities to Drosophila TRP are primarily limited to homology within ~50 amino acid residues at the COOH-terminal ends of S1-S6, the adjacent COOH-terminal region, and conservation of the TRP domain (119). A gene that functions in a subtype of olfactory neurons of C. elegans and designated osm-9 (36) was the first member of this subfamily.

A) THE C. ELEGANS OSM-9. Six recessive alleles of osm-9 have been identified on the basis of defective response to odorants, high osmotic strength, or light touch to the nose (36). A fusion portion between osm-9 and green fluorescent protein (GFP) revealed that OSM-9 is expressed in a subset of chemosensory, mechanosensory, and osmosensory neurons. There is no overlap in expression pattern between osm-9 and the C. elegans TRP (Z C21.2), which belongs to the TRP-homolog group (199) and is expressed in motor neurons, interneurons, vulval muscles, and intestinal smooth muscles (36). Similarly, it appears that there is no overlap in expression pattern between the C. elegans NOMPC and the above TRPs of C. elegans (196).

B) THE MAMMALIAN VANILLOID RECEPTOR 1, VR1, AND ITS HOMOLOG VRL-1. The VR1 member of the TRP-related group was cloned following a search for pain (noxious)-initiated receptor in the peripheral terminals of subgroup of sensory neurons of the dorsal root ganglia. Julius and colleagues (27) used a functional screening strategy based on capsaicin-induced Ca²⁺ influx to transfected HEK 293 cells. Capsaicin belongs to chemical group called vanilloids that are known to induce noxious stimuli. The receptor was discovered using the above screening library and was therefore named vanilloid receptor 1 (VR1; also designated TRPV1). The cloned VR1 cDNA encodes a protein of 828 amino acid with the six putative transmembrane segments S1-S6. It also has a typical short hydrophobic region between S5 and S6 assumed to form the pore region. Three putative ankyrin repeats are found in the NH₂ terminus (27) and a TRP domain near the COOH terminus (119). VR1 is expressed in a subset of sensory ganglion cells of the dorsal root ganglia (DRG) and trigeminal ganglia. When tested in rat DRG, ~40% of all neurons express VR1, predominantly in a population with small cell bodies. VR1 is only 23% identical to C. elegans osm-9 (Table 1).

C) VARIATIONS OF VR1 HAVE BEEN REPORTED THAT DIFFER DUE TO ALTERNATIVE MRNA SPLICING. Other members of the TRPV subfamily have been reported to have highly similar structure but distinct mode of activation. An interesting mouse member of this subfamily, which has ~80% sequence identity to rat VRL-1, was designated "growth factor-regulated channel" (GRC). GRC translocates between the ER and the plasma membrane upon stimulation of cells by insulin-like growth factor I (IGF-I). The translocation of endogenous GRC was demonstrated in MIN6 insulinoma cells and in heterologous system in CHO cells upon coexpression with GFP (86). Electrophysiological and fluorimetrical measurements showed that IGF-I augmented calcium entry through translocation of GRC to the plasma membrane in CHO cells. However, since the CHO cells were marked by GFP, which is toxic to cells, it was not excluded that the observed currents and Ca²⁺ influx did not arise from activation of endogenous channels, which are sensitive to stress.

D) EPITHELIAL CALCIUM CHANNELS. Epithelial calcium channel (ECaC; also designated ECaC1, CaT2, or TRPV5) (73) and calcium transporter 1 (CaT1, also ECaC2 or TRPV6) (119, 138) were cloned from rabbit kidney and rat duodenum, respectively. These are members of the TRPV subfamily found in 1,25-dihydroxyvitamin D₃-responsive epithelial kidney cells (ECaC1) (73) and in Ca²⁺-transporting cells of the small intestine (CaT1) (138). ECaC1 is a putative apical Ca²⁺ channel in epithelia. In kidney it is abundantly present in the apical membrane of Ca²⁺-transporting cells and it colocalized with 1,25-dihydroxyvitamin D₃-dependent calbindin-D_28K. The ECaC1 cDNA encodes a 730-amino acid protein and shows ~30% homology to the VR1 channel. It has all the molecular features of the TRP-related group including the S1-S6 transmembrane segments with the putative pore region between S5 and S6, three ankyrin binding repeats, and two N-linked glycosylation sites in S1-S6 region and a TRP domain. The protein is predominantly expressed in the kidney, small intestine, and placenta (Table 1).

E) THE PUTATIVE OSMORECEPTOR OTRPC4. OTRPC4 (now designated TRPV3) has structural similarity to VR1, osm-9, and ECaC, and it is expressed in kidney, heart, and liver (177). The protein is composed of 871 amino acid residues. The hydropathy profile predicts the typical six transmembrane segments S1-S6, pore-forming loop between S5 and S6, and three ankyrin repeats at the NH₂ terminal. Like other members of this group, OTRPC4 lacks the proline-rich motif at the COOH terminal. Sequence alignment with VR1, VRL-1, and ECaC channels reveal 41, 39, and 23% overall similarity, respectively. In situ hybridization analysis revealed high levels of OTRPC4 transcripts in the inner cortex of the kidney, while higher resolution microscopy indicated a high density in the distal convoluted tubule. This specific localization and functional tests in heterologous system strongly suggest that OTRPC4 is involved in cellular osmoreception (177).

A) THE NO MECHANORECEPTOR POTENTIAL C CHANNEL, NOMPC. NOMPC was identified following a screen for Drosophila mutants with behavioral defects in coordinated movement. The mechanosensory organs of mutants showing the behavioral defects were examined electrophysiologically, leading to the discovery of the nompC gene. This mutant displays a large loss of mechanoreceptor current relative to control flies. Cloning of the nompC gene was carried out after mapping and rescue of the nompC mutant. NOMPC cDNA encodes a putative channel protein of unusual structure composed of 1,619 amino acid residues. The 1,150 NH₂-terminal amino acid residues consist of 29 ankyrin repeats. The remaining 469 amino acid residues share a significant similarity to the TRP family with S1-S6 transmembrane segments and putative pore region between S5 and S6. Pairwise comparison between NOMPC and the various members of the TRPC family in the channel domain reveals ~20% identity and ~40% similarity. Thus NOMPC is a distant member of the TRP-homolog subfamily. NOMPC is expressed in the mechanosensory organs of Drosophila in agreement with its function as a mechanosensory sensitive channel (196). A C. elegans homolog to NOMPC was identified and revealed high expression in mechanosensitive neurons of the nematode (196). Because no human ortholog of NOMPC has been reported, it would be interesting to see in the future whether the ability to respond to mechanical stimuli is a general feature of TRPs or this activity is specific for invertebrate mechanotransduction.

This subfamily is also distantly related to Drosophila TRP, because it neither has the ankyrin repeats domain nor the TRP domain (in the mammalian members) (119). The founding member of this subfamily was discovered in many cases of polycystic kidney disease (PKD, Ref. 205). TRPP channels share ~25% amino acid identity with two members of the TRP-homolog group TRPC3 and TRPC6 in the S5-S6 transmembrane segments and in the pore region (119).

A) POLYCYSTIN. Polycystin-1 (encoded by PKD1) and polycystin-2 (encoded by PKD2) are membrane proteins that are defective in human autosomal dominant polycystic kidney disease (204). Members of this subfamily are conserved through evolution from C. elegans to human. Studies in C. elegans reveal that homologs of the polycystins act together in signal transduction pathway in sensory neurons (11). Structural analysis of the amino acid sequence predicts that the human polycystin-2 protein, which is composed of 968 amino acids, has putative six transmembrane domains S1-S6 with putative pore-forming loop between S5 and S6 segments, typical of the TRP-related group. However, the ankyrin repeats typical for the TRP family proteins are missing in the polycystin proteins (31). Although PKD-1 and PKD-2 gene products have structural similarities, only polycystin-2 seems to be a cationic channel while polycystin-1 seems to be a larger structural protein (of 4,572 amino acids in human) that interacts directly with polycystin-2 (11; see also Ref. 204). An interaction between polycystin-1 and polycistin-2 was revealed by coexpression of the two channel proteins in CHO cells. This interaction appears to be crucial for polycystin-2 function as a channel because only in the presence of polycystin-1, polycystin-2 translocates to the plasma membrane and function as a channel (54). Thus neither polycystin-1 nor polycystin-2 alone is capable of producing currents. Moreover, disease-associated mutant forms of either polycystin protein that are incapable of heterodimerization do not result in new channel activity (54). In C. elegans, there is a homolog of PKD-1 called LOV-1 that is composed of 3,125 amino acids and a PKD-2 homolog with an overall identity of 27% to the human PKD-2 (11). Vertebrate polycystins have the Ca²⁺ binding motif, EF hand, and a coiled coil domain near the COOH terminal. Heterologous expression of a human polycystin-like (PCL) protein, which shares 50% identity and 71% homology to PKD-2 in Xenopus oocytes, confirms that PCL forms a nonselective cation channel that can be activated by Ca²⁺ (31) (see sect. VD2). PKD2 has modest similarity to the Ca²⁺ transporting channel, CaT1, in the region between amino acid residues 596-687 of CaT1, where 23% identity between these proteins has been found (138).

This subfamily is also distantly related to the TRP-homolog group. The TRPM subfamily is conserved through evolution and exists in C. elegans (CED-11, GON-2) and Drosophila. Members of this subfamily are sharing ~20% amino acid identity to Drosophila TRP, over ~325 residue region that includes S2-S6 transemembrane segments and the TRP domain. This subfamily also lacks the ankyrin repeats (119). Originally this subfamily was designated long TRP (LTRPC) (70) because of the unusually long NH₂-terminal region of ~750 amino acid residues relative to the TRP-homolog group. The total length of this subfamily is 1,000-2,000 amino acids, and it varies considerably because of large diversity of the COOH-terminal domain. Members of the TRPM subfamily have a potential role in cell cycle regulation and in regulation of Ca²⁺ influx in immunocytes (160). Thus C. elegans members of TRPM function in programmed cell death (CED-11) and mitotic cell division (GON-2; reviewed in Ref. 119). Importantly, the melastatin member of this subfamily (TRPM1) has been related to skin cancer as a putative tumor suppressor protein (for review, see Ref. 119). An interesting mode of regulating TRPM gating was recently described for another member of the TRPM subfamily, MLSN (210). It was found that an alternatively spliced short form of MLSN (MLSN-S) interacts directly with and suppressed Ca²⁺ entry of full-length MLSN (MLSN-L) in HEK293 cells. This suppression appears to result from inhibition of translocation of MLSN-L to the plasma membrane (210).

A very interesting member of this subfamily is the bifunctional channel protein designated phospholipase C (PLC)-interacting kinase (TRP-PLIK or TRPM7), which constitutes the first example of a channel with putative kinase activity. This protein has an NH₂-terminal domain >50% identical over 1,250 amino acids residues to that of TRPM1 fused to COOH terminal with protein kinase domain (123, 159). Another member of this subfamily now designated TRPM2 (or LTRPC2) also seems to be bifunctional, and in addition to the channel-forming domain, which is similar to the channel domains of other members of this subfamily, there is a NUDT9 homology domain (NUDT9-H) at the COOH terminal. Sequence analysis of NUDT9 revealed the presence of a signal peptide and Nudix box sequence motif. Nudix boxes are found in a family of diverse enzymes that catalyze the hydrolysis of nucleoside diphosphates derivates (141). This motif is also highly conserved in the C. elegans protein EEED8.8. On the basis of these findings combined with heterologous expression of TRPM2 in HEK-293 cells, Scharenberg and colleagues (141) concluded that TRPM2 functions both as a cation channel activated by ADP ribose (ADPR) and a specific ADP-ribose pyrophosphatase. TRPM2 is mainly expressed in the brain but also in lower amounts in bone marrow, spleen, heart, leukocytes, liver, and lungs (141). A potentially important function of LTRPC2 in the native cells has been recently elucidated when LTRPC2 was identified in immunocytes. Detailed experiments have demonstrated that LTRPC2 mediates Ca²⁺ influx into immunocytes where cellular ADPR and NAD directly activate LTRPC2 and enable Ca²⁺ influx. Importantly, NAD-induced activation was suppressed by ATP, suggesting that NAD activates TRPC2 when ATP is depleted, while the activated LTRPC2 causes Ca²⁺ influx and apoptosis in these cells linking apoptosis with the metabolism of ADPR and NAD (160).

5.  TRPML subfamily

A) MUCOLIPIN-1. Mucolipin-1 (also called mucolipidin, encoded by the MCOLN1 gene) is a novel membrane protein that is defective in mucolipidosis type IV disease, which is a developmental neurodegenerative disorder characterized by lysosomal storage disorder, abnormal endocytosis of lipids, and accumulation of large vesicles (10, 12, 179). The predicted full-length mucolipin-1 is a 580-amino acid protein that has the typical six transmembrane S1-S6 domains with the putative pore-forming loop between S5 and S6. Structure analysis revealed similarity to the TRP family between amino acids 331 and 521, which corresponds to domains S3-S6, while the pore region is located between amino acids 496 and 521. Unlike most members of the TRP family, two proline-rich regions were identified near the NH₂ terminal. A leucine zipper motif is located at S2, a nuclear localization motif at amino acids 43-60, and a serin lipase motive. In the region that shows similarity to TRP there is 38% identity, whereas in the pore region there is 58% identity (179). Mucolipin-1 has no ankyrin repeats or TRP domain. Unlike other subfamilies, no functional analysis of mucolipin is available, and it is still not clear if it functions as a channel protein. Members of the TRPML subfamily are conserved in C. elegans (40% amino acid identity) and Drosophila (44% identity, Ref. 48). A recent study on a C. elegans homolog of mucolipin-1 designated CUP-5 showed that a loss-of-function mutation in CUP-5 resulted in an enhanced rate of fluid-phase markers, decreased degradation of endocytosed protein, and accumulation of large vacuoles (48). Overexpression of the normal CUP-5 caused the opposite effects, thus indicating that CUP-5 is a promising model system for studying conserved aspects of structure and function of mucolipin-1 and mucolipidosis type IV (48).

	IV. FUNCTIONAL ANALYSIS IN THE NATIVE TISSUE

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The discovery of vertebrate TRP channels in a large variety of cells and tissues each having a putative specific function suggests that the channels of the TRP family also have diverse and elaborate gating mechanisms that largely depend on their cellular environment. This fact makes the few cases in which channel proteins have been analyzed in the native cellular environment highly valuable. Genetic manipulations allow conducting studies of structure-function relationship of channel proteins while the other cellular components are intact. Such studies in the native tissue in conjunction with studies in heterologous systems provide powerful tools for studying the function of channel proteins. Nevertheless, due to technical difficulties, heterologous expression is still the main method for studying the function of a large number of TRP isoforms. In the TRP-homolog group, so far only Drosophila TRP, TRPL, and mammals TRPC2, TRPC3, and TRPC4 channels have been studied in detail in the native cells. A few studies in which the effects of knockout or partial knockout using the antisense approach of native TRPs were carried out suggested that some of the TRP homologs contribute to SOC (98, 146, 160, 205, 218). The native Xenopus TRPC1 isoform has been suggested to contribute to SOC activity of the oocytes, since knockout of TRPC1 isoform reduces SOC activity (18).

A.  Drosophila Light-Sensitive Channels TRP and TRPL

1.  TRP has a small single-channel conductance

The subcellular localization of TRP and TRPL to the microvilli imposes a considerable difficulty for access with patch electrodes. Accordingly, only very recently it has become possible to record single-channel activities in patches of Drosophila microvilli (53), as previously reported for Limulus (8) and mollusk photoreceptors (125). The light-sensitive conductance in Drosophila has been studied mainly under whole cell voltage-clamp conditions using the dissociated ommatidial preparation (55, 156).

The newly developed preparation of rhabdomeral membranes from Drosophila photoreceptors has been used only under the restrictive conditions of excised patches in the absence of Ca²⁺. Under these conditions the channels are constitutively open with a wide range of conductance amplitudes. Conductance events as large as 144 pS seem to arise from coordinated gating of many small individual events of 4 pS. This large conductance seems to arise from openings of TRP and not TRPL channels, since they were blocked by 10 µM La³⁺ and were completely absent in a null trp mutant (53). It is not clear if the coordinate openings represent a physiological phenomenon, since quantum bumps, which represent coordinate openings of many channels, are not observed under conditions of constitutive activity of the light-sensitive channels during whole cell recordings (62). It is also not clear why the activity of TRPL channels, which is observed under whole cell recordings in the trp mutant (62), is absent in the dissociated rhabdomeral preparation.

The uncertainty as to the physiological significance of the single-channel recordings emphasizes the importance of noise analysis, which has been used to estimate single-channel properties from the so-called "rundown current" (RDC). The RDC represents a spontaneous opening of light-sensitive channels, which develops after several minutes of whole cell recording accompanied by illuminations (62) or under metabolic stress in the dark (4). In the presence of 4 mM external Mg²⁺, TRPL channels have an apparent conductance of ~35 pS and TRP channels ~4 pS. Both channels, however, are blocked by divalent ions including Mg²⁺, and under divalent free conditions, conductance values increase to ~70 pS (TRPL) and ~35 pS (TRP), respectively (64, 68).

Power spectra have also been calculated from current noise, providing information on channel kinetics. Power spectra of TRPL channels are consistent with a channel with two open states with mean open times of 2 and 0.15 ms. Noise analysis data of heterologously expressed TRPL channels in Drosophila S2 cells were indistinguishable from that measured in Drosophila photoreceptors, and the single-channel conductance of the heterologously expressed TRPL channel was directly confirmed by recordings of single channels (68). Power spectra of TRP channels (in the trpl mutant) are characterized by higher frequencies with a calculated time constant of 0.4 ms.

The light-sensitive channels in invertebrate photoreceptors are essentially nonselective cation channels, and the channels in Drosophila readily permeate a variety of monovalent ions including Na, K, Cs, Li, and even large organic cations such as Tris and TEA (55, 156). Moreover, the reversal potential of the LIC in WT and the trpl mutant shows a marked dependence on extracellular Ca²⁺, indicating that TRP channels are permeable to Ca²⁺.

Our knowledge of the biophysical properties of the TRP and TRPL channels has been facilitated by measurements of the in situ conductance in the trp and trpl mutants. Permeability ratios to monovalent and divalent ions in WT and trp flies have been calculated (60) using the constant current equation and reversal potential data obtained under various ionic conditions. With the assumption of Goldman-Hodgkin-Katz (GHK) constant field theory, the quantitative dependence implies a relative Ca²⁺ permeability (P_Ca:P_Na) of ~40:1 (55, 60). More recently, the permeability ratios for a variety of divalent and monovalent ions ratios have been determined under bi-ionic conditions, where in these experiments Cs⁺ was the only intracellular cation while the bath contained one of a variety of monovalent or divalent cations. It was found that TRPL represents a nonselective cation channel (P_Ca:P_Cs = 4.3) and TRP, a channel highly selective for divalent cations (P_Ca:P_Cs >100:1). The WT values are explained by approximately equal summation of TRP and TRPL conductance. On the assumption of independent mobility of ions, these permeability data imply that in WT, 45% of the LIC at resting potential is mediated by Ca²⁺, 25% by Mg²⁺, and 30% by Na⁺ (using a physiological Ringer solution, Ref. 158).