Drosophila transient receptor potential (Trp) serves dual roles as an essential cation channel during response to light and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering Trp channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study the isolation of a set of new trp alleles is described, including trp¹⁴ with an amino acid substitution juxtaposed to the Trp domain. The trp¹⁴ flies stably express Trp and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp^Delta1272, has minor effects on retinal morphology whereas disruption of channel function causes profound light-induced cell death. This retinal degeneration is greatly suppressed by elimination of the Na⁺/Ca²⁺ exchanger, CalX, indicating that the cell death was due primarily to deficient Ca²⁺ entry rather than disruption of the Trp-anchoring function. The mechanism through which decreased Ca²⁺ influx causes cell death in trp appears to be due at least in part from increased rhodopsin-arrestin complexes that ensues from decreased Ca²⁺ (T. Wang, 2005b).

The Ca²⁺ channel Trpl requires interaction with other TRP channels for regulated activity. A third TRP-related channel is expressed in fly photoreceptor cells, TRPγ; TRPL form obligatory heteromultimers with either TRP or TRPγ (Xu, 1997; Xu, 2000). TRP is 10-fold more abundant that TRPL or TRPγ and the full composition of TRPC channels in photoreceptor cells are TRP homomultimers and heteromultimers composed of TRP/TRPL and TRPγ/TRPL. Neither of these latter heteromultimers is constitutively open, but is activated through stimulation of PLC-dependent signalling. This is particularly notable with respect to the TRPγ/TRPL heteromultimer since in vitro expression of each of the individual channels leads to constitutively activity. Currently, a trpγ loss-of-function mutation is not available, although a dominant negative form of TRPγ suppresses most of the remaining light-dependent conductance in trp photoreceptor cells (Xu, 2000; Montell, 2005a and references therein).

Phototransduction in Drosophila is mediated by a phospholipase C (PLC) cascade culminating in activation of transient receptor potential (TRP) channels. Ca(2+) influx via these channels is required for light adaptation, but although several molecular targets of Ca(2+)-dependent feedback have been identified, their contribution to adaptation is unclear. By manipulating cytosolic Ca(2+) via the Na(+)/Ca(2+) exchange equilibrium, it was found that Ca(2+) inhibits the light-induced current (LIC) over a range corresponding to steady-state light-adapted Ca(2+) levels [0.1-10 microM Ca(2+)] and accurately mimics light adaptation. The results indicate that light adaptation is primarily mediated downstream of PLC by Ca(2+)-dependent inhibition of TRP channels. This is interpreted as a strategy to prevent inhibition of PLC by global steady-state light-adapted Ca(2+) levels, whereas rapid inhibition of PLC by local Ca(2+) transients is required to terminate the response and ensures that PIP(2) reserves are not depleted during stimulation (Gu, 2005).

The transient receptor potential (Trp) superfamily is comprised of a large group of related cation channels that function in sensory processes ranging from phototransduction to touch, hearing, taste, olfaction, osmosensation, and thermosensation (Montell, 2005b). As such, dissecting the specific contributions of Trp channels is central to understanding each of these sensory modalities. It is generally accepted that the roles of mammalian Trp proteins is to mediate influx of cations, such as Ca²⁺ and Na⁺. However, in the case of Drosophila Trp, which is the founding member of the superfamily that is required for phototransduction (Montell, 1989), the protein functions both as a Ca²⁺-permeable channel and as a molecular anchor (Li, 2000; Tsunoda, 2001; T. Wang, 2005b and references therein).

Many of the proteins that are essential for fly visual transduction are organized into a large macromolecular complex, referred to as the signalplex (for review see Montell, 2005a). The molecular scaffold that nucleates the signalplex, inactivation no afterpotential D (INAD) consists of multiple PDZ protein interaction modules and appears to be constantly associated with Trp as well as two other important signaling proteins, PLC (encoded by norpA [no receptor potential A, encodes PLC]) and PKC (encoded by inaC [inactivation no afterpotential C, encodes PKC]). These three core binding proteins depend on INAD for localization in the phototransducing compartment of the fly photoreceptor cells, the rhabdomeres. In addition, at least five other signaling proteins appear to associate with INAD, and these latter proteins may interact dynamically with INAD. However, none of these noncore binding proteins requires interaction with INAD for normal localization (T. Wang, 2005b).

A surprising finding is that there is a reciprocal requirement for association of Trp and INAD for concentration of these two proteins in the rhabdomeres (Li, 2000; Tsunoda, 2001). Deletion of the COOH-terminal four residues in Trp destroys the PDZ binding site and results in mislocalization of INAD (Li, 2000). In turn, the rhabdomeral distributions of PKC and PLC are also disrupted. The interaction between Trp and INAD is not necessary for targeting of these proteins, but rather for subsequent retention in the rhabdomeres. Also unexpected was the finding that interference with the direct interactions between Trp and INAD had no major impact on activation of the Trp channels (Li, 2000), which in Drosophila photoreceptor cells is very rapid and occurs within milliseconds. These data demonstrate that the Trp channel functions as a molecular anchor, in addition to its more appreciated role as a cation channel (T. Wang, 2005b).

The dual roles of Trp raise the question as to the impact of altering the channel activity independent of effects on the anchoring function. Null mutations in Trp result in light-dependent retinal degeneration, in addition to causing a transient response to bright light (Cosens, 1972; Chevesich, 1997). Retinal degeneration in fly photoreceptor cells is a common phenomenon that occurs as a result of mutations in nearly any protein important for phototransduction. However, in most cases the mechanism underlying the retinal degeneration has not been clarified. In some mutants retinal degeneration occurs as a result of formation of stable rhodopsin-arrestin complexes, which in turn lead to endocytosis of rhodopsin. Ca²⁺ overload due to expression of a constitutively active Trp channel can also lead to rapid cell death in fly photoreceptor cells (Yoon, 2000; T. Wang, 2005a). However, the mechanism underlying the retinal degeneration in trp-null mutant flies is not known. In particular, it is not clear whether the light-dependent retinal degeneration due to loss of trp function results from disruption of the anchoring role, or from lower Ca²⁺ influx during light stimulation. This question has not been possible to address because the existing loss-of-function mutations in trp have major impacts on protein levels and consequently disrupt both Trp functions (Montell, 1989; T. Wang, 2005b and references therein).

In addition to Trp, there are two related cation channels expressed in photoreceptors: Transient receptor potential-like (Trpl), and Transient receptor potential gamma (Trpgamma; Phillips, 1992: Xu, 2000). Currently, there are no loss-of-function mutations in Trpgamma and elimination of Trpl has only subtle effects on the photoresponse (Niemeyer, 1996; Reuss, 1997; Leung, 2000). Nevertheless, Trpl contributes to phototransduction since flies that are missing both Trp and Trpl are blind (Niemeyer, 1996; Reuss, 1997; T. Wang, 2005b and references therein).

Multiple new trp alleles have been isolated, including one (trp¹⁴) that specifically affected the channel function, but not the molecular anchoring role. In contrast to the wild-type light response, it was found that in trp¹⁴ photoreceptor cells, the light response is transient. This phenotype results from a missense mutation in Trp juxtaposed to the highly conserved Trp domain (for review see Montell, 2005b). In addition, it was found that the light-induced retinal degeneration is as severe in trp¹⁴ flies as in trp-null flies, trp^P343. Conversely, elimination of the Trp-INAD interaction has relatively minor effects on the morphology of the photoreceptor cells. Finally, the retinal degeneration associated with either trp¹⁴ or trp^P343 is suppressed by a loss-of-function mutation in the Na⁺/Ca²⁺ exchanger, CalX. These results demonstrate that the cell death in trp mutant photoreceptor cells is due primarily to disruption of Trp channel activity and decreased light-dependent Ca²⁺ influx, rather than elimination of the Trp anchoring role (T. Wang, 2005b).

Drosophila Trp is a multifunctional protein because it serves both as a cation channel and a molecular anchor required for the retention of the scaffold protein, INAD, in the rhabdomeres. The Trp scaffold function is critical because the consequent mislocalization of INAD in turn causes instability and mislocalization of Trp, PLC, and PKC. Thus, in the absence of the scaffold function, over time the core proteins in the signalplex are lost from the rhabdomeres and the visual response is reduced. In addition to Trp, other related proteins may also have dual roles because several vertebrate Trps, such as TrpM2, TrpM6, and TrpM7 consist of channel domains fused to enzyme domains. In the case of Drosophila Trp, the specific role of the anchoring function on the photoresponse has been charactized, using transgenic flies expressing a derivative of Trp that is missing the INAD binding site (trp^Delta1272 (Li, 2000). Surprisingly, young trp^Delta1272 flies display a normal photoresponse, although as the flies age, INAD and the core binding proteins are not retained in the rhabdomeres (T. Wang, 2005b and references therein).

Null mutations in trp have at least three major consequences in photoreceptor cells. These include the inability to maintain a light response, mislocalization of INAD, PLC, and PKC, and light-induced retinal degeneration. However, it has not been possible to determine the physiological consequences resulting from specifically disrupting the Trp channel function independent of the anchoring role, since all of the previously described loss-of-function mutations (with the exception of trp^Delta1272) virtually eliminate the Trp protein. The trp¹⁴ allele expresses relatively high levels of the Trp protein and exhibits a normal anchoring role since INAD coimmunoprecipitates with the Trp¹⁴ protein as effectively as with wild-type Trp. Furthermore, the spatial distributions of the core members of the signalplex are normal in trp¹⁴ photoreceptor cells (T. Wang, 2005b).

Rather than affecting the anchoring role, the mutation of the basic residue situated between the sixth transmembrane segment and the Trp domain disrupts Trp channel function such that the response to light stimulation is transient. Though the molecular basis for the defect in Trp channel function is unclear, mutation of the corresponding basic residue in Trpl also disrupts the activity of this latter channel. Thus, this region would appear to play a critical role in TrpC channel function in vivo. The transient light response in trp¹⁴ is not a simple consequence of the slightly lower expression of the mutant protein (60% of wild-type levels) since it was found that expression of wild-type Trp at 4% the normal levels does not cause a transient light response, though the amplitude of the ERG is reduced. The Trp¹⁴ protein also displays a wild-type rhabdomeral expression pattern, so that the phenotype is not due to mislocalization of the protein (T. Wang, 2005b).

Of significance here, it was found that the retinal degeneration associated with loss-of-function mutations in trp is due primarily to defects in channel function, rather than disruption of the anchoring role. This finding is surprising because elimination of the Trp scaffold function causes time-dependent instability and mislocalization of all four core proteins in the signalplex. Thus, low levels of Trp, INAD, PLC, and PKC result in less pronounced cell death than an amino acid substitution in Trp that disrupts channel function, but has no impact on the concentrations of the core proteins in the signalplex (T. Wang, 2005b).

The basis for the retinal degeneration was decreased light-dependent Ca²⁺ influx because the cell death in either trp¹⁴ or trp-null mutant flies (trp^P343) is greatly reduced by strong loss-of-function mutations in the gene encoding the Na⁺/Ca²⁺ exchanger, CalX. This effect is not a consequence of suppression of the anchoring defect because the core signalplex proteins are still mislocalized in calx;trp^P343 double mutant flies. Given that the strong light-dependent retinal degeneration in calx is reciprocally suppressed by the trp^P343 or trp¹⁴ mutations, these data also indicate that the cell death in calx resulted from Ca²⁺ overload (T. Wang, 2005b).

The mechanism through which decreased Ca²⁺ influx causes cell death in trp appears to be due at least in part from increased rhodopsin-arrestin complexes. Stable rhodopsin-arrestin complexes and endocytosis of rhodopsin has been associated with degeneration resulting from mutations in the PLC and rhodopsin phosphatase. In the current study, it was found that the trp-dependent retinal degeneration is partially suppressed by mutations in arr2. Because Ca²⁺/calmodulin-dependent phosphorylation of arrestin promotes the release of arrestin from rhodopsin, it is suggested that a consequence of decreased light-dependent Ca²⁺ influx in trp¹⁴ is reduced phosphorylation of arrestin, which in turn results in increased stability of arrestin-rhodopsin complexes. Alternatively, the reduced Ca²⁺ influx could result in increased arrestin-rhodopsin complexes due to effects on the rhodopsin phosphatase, RDGC (retinal degeneration C). The activity of RDGC is dependent on Ca²⁺/calmodulin and loss of function mutations in rdgC result in stable rhodopsin-arrestin complexes and retinal degeneration (T. Wang, 2005b).

The observation that decreased Trp-dependent Ca²⁺ influx underlies retinal degeneration in fly photoreceptor cells has potential implications in terms of the possible effects on cell survival resulting from loss-of-function mutations in vertebrate Trps. It appears that constitutive activity of Drosophila and mammalian Trp leads to cell death due to Ca²⁺ overload (Yoon, 2000; Hara, 2002; Wehage, 2002; Aarts, 2003; T. Wang, 2005a). Moreover, constitutive activity of Trps by anoxic conditions has been proposed to underlie the massive cell death in the mammalian brain that can occur under anoxic conditions, such as occurs as a result of stroke (Agam, 2000; Yoon, 2000; Hara, 2002; Aarts, 2003; T. Wang, 2005a; T. Wang, 2005b and references therein).

The opposite of constitutive activation is elimination of Trp channel function and whether loss of vertebrate Trp-dependent Ca²⁺ influx leads to cell death has not been addressed. However, the results of the current analysis indicate that this is a likely possibility. Elimination TrpM7 from chicken DT40 cells results in cell death (Nadler, 2001), but the basis for the requirement for TrpM7 is not known. Given that TrpM7 consists of a Trp channel domain, fused to a COOH-terminal protein kinase domain, the cell death due to loss of TrpM7 could reflect a requirement for either the channel or kinase functions. Moreover, since TrpM7 is highly permeable to both Mg²⁺ and Ca²⁺, it is unclear whether the Mg²⁺ or Ca²⁺ influx is most important for viability. It will be of interest to determine whether the TrpM7-dependent cell death can be suppressed by inhibition of the Na⁺/Ca²⁺ exchanger, as was observed for Drosophila Trp and CalX (T. Wang, 2005b).

GENE STRUCTURE

cDNA clone length - 4130

Bases in 5' UTR - 226

Exons - 14

Bases in 3' UTR - 76

PROTEIN STRUCTURE

Amino Acids - 1275

Structural Domains

Drosophila Trp and Trpl can be functionally expressed in Sf9 insect cells using baculovirus expression vectors. The trp gene encodes a Ca2+-permeable channel that is activated by thapsigargin, blocked by low micromolar Gd3+, and is relatively selective for Ca2+ versus Na+ and Ba2+. In contrast, trpl encodes a Ca2+-permeable cation channel that is constitutively active, not affected by thapsigargin, blocked by high micromolar Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The region of lowest sequence identity between Trp and Trpl occurs in the COOH-terminal domain. To test the hypothesis that this region is responsible for the differential sensitivity of these channels to thapsigargin, chimeric constructs of Trp and Trpl were created in which the COOH-terminal tail region of each protein was exchanged. The Trp construct with the Trpl COOH-tail was constitutively active, insensitive to thapsigargin, but retained selectivity for Ca2+ over Na+ and Ba2+. In contrast, the Trpl construct with the Trp COOH-tail was not constitutively active, could be activated by thapsigargin, but remained non-selective with respect to Ca2+, Ba2+, and Na+. These results suggest that the COOH-terminal domain of Trpl plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin (Sinkins, 1996).

The TRP superfamily includes a diversity of non-voltage-gated cation channels that vary significantly in their selectivity and mode of activation. Nevertheless, members of the TRP superfamily share significant sequence homology and predicted structural similarities. Currently, most of the genes and proteins that comprise the TRP superfamily have multiple names and, in at least one instance, two distinct genes belonging to separate subfamilies have the same name. Moreover, there are many cases in which highly related proteins that belong to the same subfamily have unrelated names. Therefore, to minimize confusion, a unified nomenclature for the TRP superfamily is proposed (Montell, 2002).

The current effort to unify the TRP nomenclature focuses on three subfamilies (TRPC, TRPV, and TRPM) that bear significant similarities to the founding member of this superfamily, Drosophila TRP, and that include highly related members in worms, flies, mice, and humans. Members of the three subfamilies contain six transmembrane segments, a pore loop separating the final two transmembrane segments, and similarity in the lengths of the cytoplasmic and extracellular loops. In addition, the charged residues in the S4 segment that appear to contribute to the voltage sensor in voltage-gated ion channels are not conserved. The TRP-Canonical (TRPC) subfamily (formerly short-TRPs STRPs) is comprised of those proteins that are most highly related to Drosophila TRP. The TRPV sub family (formerly OTRPC), is so named based on the origi nal designation, Vanilloid Receptor 1 (VR1), for the first member of this subfamily (now TRPV1). The name for the TRPM subfamily (formerly long-TRPs or LTRPs) is derived from the first letter of Melastatin, the former name (now TRPM1) of the founding member of this third subfamily of TRP-related proteins. Based on amino acid homologies, the mammalian members of these three subfamilies can be subdivided into several groups each (Montell, 2002).

The numbering system for the mammalian TRPC, RPV, and TRPM proteins takes into account the order of their discovery and, in as many cases as possible, the number that has already been assigned to the genes and proteins. In the case of the TRPV proteins, the numbering system is also based in part on the groupings of the TRPV proteins. New members of each subfamily will maintain the same root name and, with the exception of TRPV3, will be assigned the next number in the sequence. Currently, TRPV3 is unassigned to maintain the TRPV1/ TRPV2 and TRPV5/TRPV6 groupings and so that the former OTRPC4 could be renamed TRPV4. The next TRPV protein will be designated TRPV3 (Montell, 2002).

date revised: 12 June 2006

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