InteractiveFly: GeneBrief

transient receptor potential: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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).

Photomechanical responses in Drosophila photoreceptors

Phototransduction in Drosophila microvillar photoreceptor cells is mediated by a G protein-activated phospholipase C (PLC). PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), leading by an unknown mechanism to activation of the prototypical transient receptor potential (TRP) and TRP-like (TRPL) channels. This study found that light exposure evokes rapid PLC-mediated contractions of the photoreceptor cells and modulates the activity of mechanosensitive channels introduced into photoreceptor cells. Furthermore, photoreceptor light responses are facilitated by membrane stretch and are inhibited by amphipaths, which alter lipid bilayer properties. These results indicate that, by cleaving PIP₂, PLC generates rapid physical changes in the lipid bilayer that lead to contractions of the microvilli, and suggest that the resultant mechanical forces contribute to gating the light-sensitive channels (Hardie, 2012b).

In most invertebrate photoreceptor cells, the visual pigment (rhodopsin) and other components of the phototransduction cascade are localized within tightly packed microvilli (tubular membranous protrusions), together forming a light-guiding rod-like stack (rhabdomere). After photoisomerization, rhodopsin activates a heterotrimeric guanine nucleotide-binding protein (Gq protein), releasing its guanosine triphosphate-bound α subunit, which in turn activates phospholipase C (PLC). How PLC activity leads to gating of the light-sensitive transient receptor potential channels (TRP and TRPL) in the microvilli is unresolved. PLC hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), yielding soluble inositol 1,4,5-trisphosphate (InsP₃), diacylglycerol (DAG, which remains in the inner leaflet of the microvillar lipid bilayer), and a proton. The light-sensitive channels in Drosophila photoreceptors can be activated by a combination of PIP₂ depletion and protons, but it remains unclear how PIP₂ depletion might contribute to channel gating. It has been speculated that changes in membrane properties play a role, and members of the TRP ion channel family have been repeatedly, although controversially, implicated as mechanosensitive channels. This led the current study to ask whether cleavage of the bulky, charged inositol head group of PIP₂ from the inner leaflet might alter the physical properties of the lipid bilayer in the microvilli, resulting in mechanical forces that contribute to channel gating (Hardie, 2012b).

Remarkably, Drosophila photoreceptors responded to light flashes with small (<1 μm) but rapid contractions that are directly visible in dissociated cells via bright-field microscopy. To obtain improved temporal and spatial resolution, these photomechanical responses were recorded with an atomic force microscope (AFM), positioning the AFM cantilever on the distal tips of photoreceptors in a whole excised retina glued to a coverslip. Contact force (~100 pN) was maintained constant, so that changes in sample height resulted in immediate, matching changes in the cantilever's z-position. Contractions were elicited indefinitely by repeated brief flashes of modest intensity, with kinetics similar to those of electrical responses recorded from dissociated photoreceptors. The latencies of contractions induced by the brightest stimuli (4.9 ± 0.9 ms) were significantly shorter than the latencies of voltage responses to the same stimuli (6.6 ± 0.6 ms). Only a few hundred effectively absorbed photons per photoreceptor are required to elicit detectable contractions, which saturate with flashes containing ~10⁶ photons, corresponding to ~30 effectively absorbed photons per microvillus. This intensity dependence overlapped with that of the electrical response. Like the electrical responses, the contractions were eliminated in mutants lacking PLC, which shows that they too required PLC activity (Hardie, 2012b).

Because PLC activity is normally terminated by Ca²⁺ influx through the light-sensitive channels, net PIP₂ hydrolysis is enhanced when the light-sensitive current is blocked. Therefore contractions were measured in trpl mutants expressing only TRP light-sensitive channels before and after blocking TRP channel activity with La³⁺ and ruthenium red (RR). Indeed, after blocking the light-sensitive channels the contractions are enhanced, more sensitive to light, and saturate at lower intensities, corresponding to only ~1 to 5 effectively absorbed photons per microvillus. In the absence of Ca²⁺ influx, such intensities deplete virtually all microvillar PIP₂, resulting in temporary loss of sensitivity to light. After such saturating flashes, the photomechanical response is also temporarily refractory, recovering sensitivity with a time course (t_1/2 ~ 40 s) similar to that of PIP₂ resynthesis. By contrast, without channel blockers, sensitivity recovers within ~10 s (Hardie, 2012b).

Blockade of all light-sensitive current in these experiments also shows that the contractions cannot result from any downstream effects of Ca²⁺ influx or osmotic changes caused by ion fluxes associated with the light response. Therefore, these results indicate that the contractions result from hydrolysis of PIP₂. Although other downstream effects of PLC cannot be excluded, the speed of the contractions supports a simple and direct mechanism. Cleavage of the bulky head groups from PIP₂ molecules, which represent 1% to 2% of lipids in the plasma membrane, leaves DAG in the membrane, which occupies a substantially smaller area than PIP₂. This should increase membrane tension, leading to shrinkage of the microvillar diameter, as reported for the action of PLC on the diameter of artificial liposomes. Integrated over the stack of ~30,000 microvilli, such a mechanism seems capable of accounting for the observed macroscopic contractions, which represent at most ~0.5% of the rhabdomere length. Within each microvillus, it is suggested that the alteration to the mechanical properties of the lipid bilayer may contribute to channel gating (Hardie, 2012b).

To test whether phototransduction generates sufficient mechanical forces to gate mechanosensitive channels (MSCs), whole-cell patch-clamp recordings were made from dissociated photoreceptors lacking all light-sensitive channels (trpl;trp double mutants, or trpl mutants exposed to La³⁺ and RR). The photoreceptors were then perfused with gramicidin, a monovalent (Ca²⁺-impermeable) cation channel and one of the best-characterized MSCs, which is known to be regulated by changes in bilayer physical properties. Incorporation of gramicidin channels into the membrane generated a constitutive inward current that stabilized after a few minutes. Despite having replaced the native light-sensitive channels with MSCs, the photoreceptors still responded to light, with a rapid increase in the gramicidin-mediated current. Like the photomechanical responses recorded after blocking Ca²⁺ influx through the light-sensitive channels, these gramicidin-mediated responses inactivate slowly, are temporarily refractory to further stimulation and have a similar intensity dependence (Hardie, 2012b).

To test whether the light-sensitive channels were mechanically sensitive, membrane tension was manipulated osmotically. Channels were not directly activated by perfusing cells with hyper- or hypo-osmotic solutions; however, it was reasoned that it would be impossible to mimic the exact physical effects of PIP₂ hydrolysis, which would include a specific combination of changes in membrane tension, curvature, thickness, lateral pressure profile, charge, and pH. Therefore whether osmotic manipulation could enhance or suppress light-sensitive channel activity was tested. In wild-type photoreceptors, light-induced currents are rapidly and reversibly facilitated by ~50% after perfusion with hypo-osmotic solutions (200 mOsm), which, like PIP₂ depletion, would be expected to alleviate crowding between phospholipids, increase tension, and reduce membrane thickness. Conversely, responses in hyperosmotic (400 mOsm) solutions were about half those in control solutions. Analysis of single-photon responses (quantum bumps) indicated that modulation resulted from changes in both quantum efficiency (fraction of rhodopsin photoisomerizations generating a quantum bump) and bump amplitude. Recordings from trp and trpl mutants showed that both TRP and TRPL channels were modulated, although facilitation of currents mediated by TRPL channels (in trp mutants) was more pronounced. Modulation of the light response by osmotic manipulation was at least as pronounced in Ca²⁺-free bath, indicating that facilitation by membrane stretch did not result from leakage of Ca²⁺ into the cell from the extracellular space (Hardie, 2012b).

To test whether modulation might have been mediated by effects on upstream components of the cascade such as PLC, the activity of spontaneously active TRP channels was measured in recordings made with pipettes lacking adenosine triphosphate. Under these conditions, PIP₂ becomes depleted (thereby removing PLC’s substrate), sensitivity to light is lost, and the TRP channels enter a constitutively active ('rundown') state uncoupled from the phototransduction cascade. Nonetheless, the channels were still similarly modulated by osmotic manipulation, whereas single-channel conductance, estimated by noise analysis, was unaffected. These results indicate that osmotic pressure directly modulates the open probability of both TRP and TRPL channels (Hardie, 2012b).

MSCs such as gramicidin are sensitive to amphiphilic compounds, which insert into the lipid bilayer. Because they are attracted to anionic phospholipids, cationic amphipaths insert preferentially into the inner leaflet, where they increase crowding, promote negative (concave) curvature, and decrease membrane stiffness. This study found that four structurally unrelated cationic amphipaths were all effective, reversible inhibitors of the light-induced current. Neither light-induced PLC activity (measured using a genetically targeted PIP₂-sensitive biosensor to monitor PIP₂ hydrolysis) nor single-channel conductance were substantially affected. The 50% inhibitory concentrations (IC₅₀ values) were much higher than those of their traditional drug targets and ranged over approximately three orders of magnitude. However, after correcting for pK_a and partitioning, the effective concentration of the compounds in the membrane was similar (~5 mM) in each case. Thus, their mode of action is likely related to their physicochemical properties rather than conventional drug-receptor interactions. Because cationic amphipaths are also lipophilic weak bases, and because it is proposed that protons are also critical for activating the light-sensitive channels, an alternative but not mutually exclusive possible mechanism of action is as lipophilic pH buffers of the membrane environment. It was also noted that polyunsaturated fatty acids (PUFAs) such as arachidonic and linolenic acid (effective activators of both TRP and TRPL) are not only anionic amphipaths (predicted to have opposite effects to cationic amphipaths) but also, as weak acids, natural protonophores; such a dual action could account for their agonist effect (Hardie, 2012b).

The mechanism of activation of the light-sensitive channels in invertebrate microvillar photoreceptors has long remained an enigma. Neither InsP₃ nor DAG -- the two obvious products of PIP₂ hydrolysis -- are reliable agonists for the light-sensitive channels. Although PUFAs are effective agonists and might be generated from DAG, a DAG lipase with the appropriate specificity has not been found in the photoreceptors. By contrast, two neglected consequences of PLC activity -- the depletion of its substrate (PIP₂) together with protons released by PIP₂ hydrolysis -- were recently shown to potently activate the light-sensitive channels in a combinatorial manner (Huang, 2010). The current results support the hypothesis that the effect of PIP₂ depletion is mediated mechanically by changes to the physical properties of the lipid bilayer, thereby introducing the concept of mechanical force as an intermediate or 'second messenger' in metabotropic signal transduction (Hardie, 2012b).

REGULATION

Activation and inactivation of TRP

In Drosophila photoreceptors, light induces phosphoinositide hydrolysis and activation of Ca(2+)-permeable plasma membrane channels, one class of which is believed to be encoded by the trp gene. The properties of the light-sensitive channels have been investigated under conditions where they are activated independently of the transduction cascade. Whole-cell voltage clamp recordings were made from photoreceptors in a preparation of dissociated Drosophila ommatidia. Within a few minutes of establishing the whole-cell configuration, there is a massive spontaneous activation of cation-permeable channels. When clamped near resting potential, this 'rundown current' (RDC) accelerates over several seconds, peaks, and then relaxes to a steady-state which lasts indefinitely (many minutes). The RDC is invariably associated with a reduction in sensitivity to light by at least 100-fold. The RDC has a similar absolute magnitude, reversal potential, and voltage dependence to the light-induced current, suggesting that it is mediated by the same channels. The RDC is almost completely blocked by La3+ and is absent, or reduced and altered in the trp mutant (which lacks a La(3+)-sensitive light-dependent Ca2+ channel), suggesting that it is largely mediated by the trp-dependent channels. Power spectra of the steady-state noise in the RDC can be fitted by simple Lorentzian functions consistent with random channel openings. The variance/mean ratio of the RDC noise suggests the underlying events (channels) have conductances of approximately 1.5-4.5 pS in wild-type (WT), but 12-30 pS in trp photoreceptors. Nevertheless, the power spectra of RDC noise in WT and trp are indistinguishable, in both cases being fitted by the sum of two Lorentzians with a major time constant (effective 'mean channel open time') of 1-2 ms and a minor component at higher frequencies (approximately 0.2 ms). This implies that the noise in the WT RDC may actually be dominated by non-trp-dependent channels and that the trp-dependent channels may be of even lower unit conductance (Hardie, 1994a).

Whole-cell voltage clamp recordings were made from photoreceptors of dissociated Drosophila ommatidia under conditions when the light-sensitive channels activate spontaneously, generating a 'rundown current' (RDC). The Ca2+ and voltage dependence of the RDC was investigated by applying voltage steps (+80 to -100 mV) at a variety of extracellular Ca2+ concentrations (0-10 mM). In Ca(2+)-free Ringer large currents are maintained tonically throughout 50-ms-long voltage steps. In the presence of external Ca2+, hyperpolarizing steps elicit transient currents which inactivate increasingly rapidly as Ca2+ is raised. On depolarization, inactivation is removed with a time constant of approximately 10 ms at +80 mV. The Ca(2+)-dependent inactivation is suppressed by 10 mM internal BAPTA, suggesting it requires Ca2+ influx. The inactivation is absent in the trp mutant, which lacks one class of Ca(2+)-selective, light-sensitive channel, but appears unaffected by the inaC mutant which lacks an eye-specific protein kinase C. Hyperpolarizing voltage steps applied during light responses in wild-type (WT) flies before rundown induce a rapid transient facilitation followed by slower inhibition. Both processes accelerate as Ca2+ is raised, but the time constant of inhibition is approximately 10 times slower than that of the RDC inactivation. The Ca(2+)-mediated inhibition of the light response recovers in approximately 50-100 ms on depolarization, recovery being accelerated with higher external Ca2+. The Ca2+ and voltage dependence of the light-induced current is virtually eliminated in the trp mutant. In inaC, hyperpolarizing voltage steps induced transient currents that appeared similar to those in WT during early phases of the light response. However, 200 ms after the onset of light, the currents induced by voltage steps inactivated more rapidly with time constants similar to those of the RDC. It is suggested that the Ca(2+)-dependent inactivation of the light-sensitive channels first occurs at some concentration of Ca2+ not normally reached during the moderate illumination regimes used, but that the defect in inaC allows this level to be reached (Hardie, 1994b).

INAF, a protein required for transient receptor potential Ca(2+) channel function

The trp gene of Drosophila encodes a subunit of a class of Ca(2+)-selective light-activated channels that carry the bulk of the phototransduction current. Transient receptor potential (TRP) homologs have been identified throughout animal phylogeny. In vertebrates, TRP-related channels have been suggested to mediate 'store-operated Ca(2+) entry', which is important in Ca(2+) homeostasis in a wide variety of cell types. However, the mechanisms of activation and regulation of the TRP channel are not known. Drosophila inaF gene encodes a highly eye-enriched protein, INAF, that appears to be required for TRP channel function. INAF is a novel protein without known vertebrate homologs. A null mutation in this gene significantly reduces the amount of the TRP protein and, in addition, specifically affects the TRP channel function so as to nearly shut down its activity. The inaF mutation also dramatically suppresses the severe degeneration caused by a constitutively active mutation in the trp gene. Although the reduction in the amount of the TRP protein may contribute to these phenotypes, several lines of evidence support the view that inaF mutations also more directly affect the TRP channel function, suggesting that the INAF protein may have a regulatory role in the channel function (Li, 1999).

Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo

Drosophila transient receptor potential (TRP) is a prototypical member of a novel family of channel proteins underlying phosphoinositide-mediated Ca(2+) entry. Although the initial stages of this signaling cascade are well known, downstream events leading to the opening of the TRP channels are still obscure. In the present study patch-clamp whole-cell recordings were applied and measurements of Ca(2+) concentration were made by ion-selective microelectrodes in eyes of normal and mutant Drosophila to isolate the TRP and TRP-like (TRPL)-dependent currents. Anoxia rapidly and reversibly depolarizes the photoreceptors and induces Ca(2+) influx into these cells in the dark. Openings of the light-sensitive channels, which mediate these effects, can be obtained by mitochondrial uncouplers or by depletion of ATP in photoreceptor cells, whereas the effects of illumination and all forms of metabolic stress are additive. Effects similar to those found in wild-type flies were also found in mutants with strong defects in rhodopsin, Gq-protein, or phospholipase C, thus indicating that the metabolic stress operates at a late stage of the phototransduction cascade. Genetic elimination of both TRP and TRPL channels prevents the effects of anoxia, mitochondrial uncouplers, and depletion of ATP, thus demonstrating that the TRP and TRPL channels are specific targets of metabolic stress. These results shed new light on the properties of the TRP and TRPL channels by showing that a constitutive ATP-dependent process is required to keep these channels closed in the dark, a requirement that would make them sensitive to metabolic stress (Agam, 2000).

Calcium influx via TRP channels is required to maintain PIP2 levels in Drosophila photoreceptors

The trp gene encodes a Ca2+ channel responsible for the major component of the phospholipase C (PLC) mediated light response in Drosophila. In trp mutants, maintained light leads to response decay and temporary total loss of sensitivity (inactivation). Using genetically targeted PIP2-sensitive inward rectifier channels (Kir2.1) as biosensors, evidence is provided that trp decay reflects depletion of PIP2. Two independent mutations in the PIP2 recycling pathway (rdgB and cds) prevent recovery from inactivation. Abolishing Ca2+ influx in wild-type photoreceptors mimics inactivation, while raising Ca2+ by blocking Na+/Ca2+ exchange prevents inactivation in trp. The results suggest that Ca2+ influx prevents PIP2 depletion by inhibiting PLC activity and facilitating PIP2 recycling. Without this feedback one photon appears sufficient to deplete the phosphoinositide pool of approximately 4 microvilli (Hardie, 2001).

Ca²⁺ overload is responsible for a form of retinal degeneration resulting from defects in the Trp channel

In sensory neurons, Ca(2+) entry is crucial for both activation and subsequent attenuation of signaling. Influx of Ca(2+) is counterbalanced by Ca(2+) extrusion, and Na(+)/Ca(2+) exchange is the primary mode for rapid Ca(2+) removal during and after sensory stimulation. However, the consequences on sensory signaling resulting from mutations in Na(+)/Ca(2+) exchangers have not been described. This reports that mutations in the Drosophila Na(+)/Ca(2+) exchanger calx have a profound effect on activity-dependent survival of photoreceptor cells. Loss of CalX activity results in a transient response to light, a dramatic decrease in signal amplification, and unusually rapid adaptation. Conversely, overexpression of CalX has reciprocal effects and greatly suppresses the retinal degeneration caused by constitutive activity of the Trp channel. These results illustrate the critical role of Ca(2+) for proper signaling and provide genetic evidence that Ca(2+) overload is responsible for a form of retinal degeneration resulting from defects in the Trp channel (T. Wang, 2005a).

Mechanisms of light adaptation in Drosophila photoreceptors -- light adaptation is primarily mediated downstream of PLC and independently of PKC by Ca(2+)-dependent inhibition of TRP channels

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. However, PLC activity monitored with genetically targeted PIP(2)-sensitive ion channels (Kir2.1) is first inhibited by much higher (≥ approximately 50 microM) Ca(2+) levels, which occur only transiently in vivo. Ca(2+)-dependent inhibition of PLC, but not the LIC, is impaired in mutants (inaC) of protein kinase C (PKC). The results indicate that light adaptation is primarily mediated downstream of PLC and independently of PKC 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).

Photoreceptors in both vertebrates and invertebrates generate discrete electrical events, known as quantum bumps, in response to absorption of single photons. In Drosophila, quantum bumps represent the concerted opening of ~10-20 Ca²⁺ permeable TRP channels, most probably localized within a single microvillus. The channels, which are encoded by trp and trp-like (trpl) genes, are activated downstream of PLC without involvement of InsP₃ receptors, most likely via diacylglycerol (DAG) or one of its lipid metabolites. Whereas vertebrate rods saturate with photon fluxes of ~10³ photons per photoreceptor per second, as in most invertebrate microvillar photoreceptors, Drosophila continues light adapting up to the brightest daylight intensities, approaching 10⁶ photons. Attempts have been made to identify the molecular mechanisms responsible for light adaptation by exploring the Ca²⁺ dependence of different components of the transduction cascade (Gu, 2005).

The high Ca²⁺ permeability of the light-sensitive channels in Drosophila results in a massive Ca²⁺ influx into the microvilli during the light response. From resting levels in the dark of ~160 nM, Ca²⁺ concentrations in the microvilli are believed to increase transiently to ~1 mM before relaxing to values of maximally ~10 μM during steady-state adaptation. The major homeostatic mechanism involved in controlling this Ca²⁺ influx is an electrogenic Na⁺/Ca²⁺ exchanger encoded by the calX gene, strongly expressed in the photoreceptor microvillar membrane. Assuming a stoichiometry of 3 Na⁺:1 Ca²⁺, and in the absence of other fluxes, the Na⁺/Ca²⁺exchanger should generate an equilibrium internal Ca²⁺ concentration (Ca_i) determined by the external Ca²⁺ concentration (Ca_o), the Na⁺ gradient (Na_i/Na_o), and E, the membrane voltage (Gu, 2005).

This behavior was exploited to manipulate cytosolic Ca²⁺ during whole-cell patch clamping of dissociated photoreceptors. For example, with 20 mM internal Na⁺ in the patch pipette, Na⁺/Ca²⁺ exchange can be driven in reverse mode by partial substitution of external Na⁺ for Li⁺; this generates an outward exchange current and raises internal Ca²⁺ into the micromolar range. Responses to test flashes were rapidly inhibited by such solution changes but recovered on Na⁺ reperfusion, which results in forward Na⁺/Ca²⁺ exchange extruding the accumulated Ca²⁺ at the expense of Na⁺ influx, now generating a transient inward exchange current. In theory, virtually any internal Ca²⁺ concentration can be achieved by varying external and internal Na⁺, allowing the Ca²⁺ dependence of the light response to be explored over a wide range (Gu, 2005).

In wt photoreceptors recorded with 20 mM Na_i and 110 mM Na_o (predicted Ca_i at -70 mV, 564 nM), the amplitude of the peak LIC in comparison to controls with 10 mM internal Na⁺ (Ca_i 70 nM) was reduced by ~25%, indicative of inhibition of the LIC by even submicromolar Ca²⁺ concentrations. Sensitivity was further progressively reduced as Ca_i was raised by perfusion with decreasing external Na⁺ concentrations. Assuming the predicted Ca_i equilibrium values (Equation 1), the estimated IC₅₀ was ~1 μM, with ≥90% inhibition being achieved with [Ca_i] ≥ 10 μM (Gu, 2005).

The profound inhibition of the light-induced current (LIC) suggested that raising Ca²⁺ in this range also effectively mimics light adaptation. To confirm this, the effects of background light adaptation were compared with manipulation of Ca²⁺ by the exchanger. As in most photoreceptors, although background illumination suppresses the response to flashes of a given intensity, large responses can still be elicited by further increasing the intensity. Raising Ca²⁺ in the dark closely mimics this behavior, with both reverse Na⁺/Ca²⁺ exchange and background illumination resulting in a shift in response-intensity function, best described by a simple multiplicative reduction in response amplitude across the range of currents that could be accurately voltage clamped. Under physiological conditions, when the currents are transformed into voltages, this should result in approximately parallel shifts of the response-intensity function along the intensity axis. Reverse Na⁺/Ca²⁺ exchange also accurately mimics another major feature of light adaptation, namely the acceleration of response kinetics. These results show that raising Ca²⁺ in the dark accurately mimics the major features of light adaptation, indicating that Ca²⁺ is not only required, but is also sufficient for light adaptation (Gu, 2005).

Recent evidence indicates that Ca²⁺ influx via TRP channels inhibits PLC activity, thereby preventing the near total PIP₂ depletion that occurs when Ca²⁺ influx is compromised (Hardie, 2001). Nevertheless, evidence presented suggests that light adaptation is mediated primarily downstream of PLC (Gu, 2005).

The InsP₃ receptor is not involved in excitation or adaptation in Drosophila; however, both TRP and TRPL channels are sensitive to Ca²⁺-dependent inactivation (Hardie, 1994b; Scott, 1994 and Reuss, 1997), raising the surprising possibility that the major features of light adaptation might be mediated at the level of the channels. To test this further, the Ca²⁺ dependence of inhibition of the light-sensitive channels themselves was quantified. To measure this, the so-called rundown current (RDC) was exploited that develops after a few minutes of whole-cell recording with pipette solutions containing no ATP, and which represents spontaneous activity of the light-sensitive TRP channels dissociated from the transduction cascade (Hardie, 1994a and Agam, 2000). The RDC was rapidly and reversibly inhibited by reverse Na⁺/Ca²⁺ regimes with a Ca²⁺ dependence indistinguishable from that of the LIC itself. This is entirely consistent with the suggestion that light adaptation is primarily mediated by Ca²⁺-dependent inhibition of the light-sensitive channels, although the contribution of additional mechanisms downstream of PLC with a similar Ca²⁺ dependence cannot be excluded (Gu, 2005).

The results indicate that PKC is required for the effective Ca²⁺-dependent inhibition of PLC and termination of the response but is not directly required for the major features of light adaptation, manifest in the Ca²⁺-dependent inhibition and acceleration of the LIC. Instead, the failure to inhibit PLC in inaC results in light-induced PIP₂ depletion and a collapse of excitation so that adaptation is masked indirectly as a consequence. The results also suggest a simple interpretation of the deactivation defect in inaC, namely the additional response to the continued PLC activity, which otherwise terminates as soon as Ca²⁺ enters (Gu, 2005).

Although these results redefine PKC's role in Drosophila phototransduction, it is not clear whether the conspicuously high Ca²⁺ levels required to inactivate PLC are needed to activate PKC or whether PKC is required to enable PLC to be inhibited by such Ca²⁺ levels. The relevant PKC phosphorylation target also remains uncertain: PLC is not known as a substrate for PKC in Drosophila; however, PLC is organized into a multimolecular signaling complex with PKC and the TRP channel protein by the PDZ domain scaffolding protein INAD. INAD itself has been reported to be a PKC substrate, raising the possibility that phosphorylation of INAD could indirectly modulate the activity of the PLC or its susceptibility to Ca²⁺-dependent inhibition (Gu, 2005).

Detailed theoretical considerations as well as in vivo Ca²⁺ indicator measurements in a related dipteran fly (Calliphora) indicate that in dark-adapted photoreceptors, Ca²⁺ influx transiently raises microvillar Ca²⁺ from ~160 nM in the dark to ~1 mM after illumination, but Ca²⁺ then rapidly returns to resting levels by diffusion into the cell body and extrusion by Na⁺/Ca²⁺ exchange. During light adaptation, continued Ca²⁺ influx results in a progressive increase in the steady-state Ca²⁺ concentration in the cell body and microvilli, reaching saturating values of ~10 μM under bright adaptation. Under light-adapted conditions, incremental flashes generate greatly reduced inward currents so that the Ca²⁺ transients in the microvilli now reach only ~50 μM. Strikingly, these global steady-state (160 nM-10 μM) and localized transient (~1 mM-50 μM) Ca²⁺ ranges map closely onto the estimated Ca²⁺-dependent operating ranges of the LIC and PLC, respectively. In particular, the steady-state light-adapted Ca²⁺ levels closely match the inhibitory range of the LIC, which also closely mimic the major features of light adaptation, including gain reduction and acceleration in response kinetics. These results suggest that these features of adaptation are primarily mediated by the Ca²⁺-dependent inhibition of the light-sensitive TRP channels, independently of PKC. Both TRP and TRPL are reported to have one or more calmodulin binding sites (CBS) (Montell, 1989; Chevesich, 1997) and indeed, calmodulin mutants and mutants of the TRPL CBS have both been reported to have defects in Ca²⁺-dependent inactivation (Scott, 1997). The surprising conclusion that adaptation is mediated primarily at the level of the channels is not without precedent; a similar situation has been reported in olfactory receptors, where Ca-calmodulin-dependent modulation of cAMP-gated channels has been proposed (Kurahashi, 1997) as the primary mechanism of olfactory adaptation (Gu, 2005).

In contrast, PLC activity was unaffected by the steady-state Ca²⁺ concentrations reached during light adaptation, but it was inhibited over the range of concentrations experienced during the Ca²⁺ transients. As a functional rationale, it is proposed that it is important to maintain high PLC activity during light adaptation in order to ensure rapid responses. Macroscopic responses represent the linear summation of quantum bumps, and, hence, their kinetics are jointly determined by quantum-bump duration and bump latency. Bump latency represents the time taken for second messenger concentration to exceed threshold for channel activation and is therefore critically dependent on the rate of PLC activity. Why then inhibit PLC at all? (1) The deactivation defect in inaC mutants suggests that PKC-dependent inhibition of PLC by Ca²⁺ transients is required to terminate the light response. (2) Light-activated PLC activity in Drosophila is exceptionally high, and without feedback by Ca²⁺, a single effectively absorbed photon depletes all the PIP₂ of at least one microvillus within less than a second. Under normal conditions, Ca²⁺ influx via the TRP channels appears essential to rapidly inhibit PLC, thereby preventing this precipitous loss of PIP₂ (Gu, 2005).

Lipid products activate TRP channels in vivo

The TRP family of ion channels mediates a wide range of calcium-influx phenomena in eukaryotic cells. Many members of this family are activated downstream of phosphoinositide hydrolysis but the subsequent steps that lead to TRP channel activation in vivo remain unclear. Recently, the lipid products of phosphoinositide hydrolysis (such as diacylglycerol and its metabolites) have been implicated in activating TRP channels in both Drosophila and mammals. In Drosophila photoreceptors, lack of diacylglycerol kinase (DGK) activity (encoded by rdgA) leads to both constitutive TRP-channel activity and retinal degeneration. In this study, using a novel forward-genetic screen, InaD, a multivalent PDZ domain protein, was identified as a suppresser of retinal degeneration in rdgA mutants. InaD suppresses rdgA, and the rescue is correlated with reduced levels of phospholipase Cß (PLCß), a key enzyme for TRP channel activation. Furthermore, it was shown that light, Gq and PLCß all modulate retinal degeneration in rdgA. The results demonstrate a previously unknown requirement for a balance of PLCß and DGK activity for retinal degeneration in rdgA. They also suggest a key role for the lipid products of phosphoinositide hydrolysis in the activation of TRP channels in vivo (Georgiev, 2005).

Transmembrane signalling cascades initiated by G-protein-coupled receptors are a widely used mechanism for signalling the detection of many sensory modalities. These cascades end with the activation of plasma-membrane ion channels whose activity alters membrane potential and initiates synaptic transmission of a signal to the central nervous system. Several different families of ion channels have been implicated in this process. Historically, the oldest and best characterized are cyclic-nucleotide-gated channels, whose role in vertebrate visual and olfactory transduction is well established. More recently, members of the TRP family of ion channels have been implicated in the transduction of several sensory modalities in both vertebrate and invertebrate systems. These include light (Drosophila TRPC), pheromones (rodent TRPC2), taste (rodent TRPM), physical stimuli and temperature (Drosophila and mammal TRPV, TRPA and TRPN). Currently, a crucial factor limiting the understanding of how TRP channels encode sensory modalities is the lack of information about how these channels are activated. In several cases, only a few transduction components have been identified and the inability to perform in vivo analysis of channel activation has been a major obstacle in revealing how TRP channels are activated (Georgiev, 2005).

The Drosophila phototransduction cascade is historically the oldest and to date the best understood model for the analysis of a TRP channel involved in sensory transduction. In the fly eye, rhodopsin, a seven-transmembrane-span G-protein-coupled receptor, activates phospholipase Cß (PLCß) via Gq. This initiates a biochemical cascade that ends with the opening of two classes of calcium- and cation-selective TRPC channels, TRP and TRPL. Several key elements of the transduction cascade have been identified including Gq, PLCß and protein-kinase C. Several of these components, along with the TRP channel, are clustered into a macromolecular signalling complex by the multivalent PDZ-domain protein INAD. The INAD complex is thought to increase the speed and specificity of the light response. However, despite this wealth of detail about the components of the transduction cascade, the mechanism of activation of TRP and TRPL remains poorly understood, and is one of the outstanding problems in both sensory neurobiology and intracellular calcium signalling (Georgiev, 2005),

Although the essential role of PLCß in the activation of TRP and TRPL is well established, the biochemical events initiated by this enzyme that lead to channel activation remain unclear. Inositol-1,4,5-trisphosphate (IP₃), the best-understood second messenger generated from phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] hydrolysis by PLCß was originally postulated to be the second messenger that leads to TRP and TRPL activation. However, several recent lines of evidence strongly indicate that IP₃-induced calcium (Ca²⁺) release, or indeed a physical interaction between the IP₃ receptor (IP₃R) and the light-activated channels, is unlikely to underlie the mechanism of TRP and TRPL activation. More recently, lipid second messengers derived from PI(4,5)P₂ have been implicated in the activation of TRP and TRPL as well as their vertebrate homologues. Polyunsaturated fatty acids, potential metabolites of diacylglycerol (DAG), the primary lipid generated by PI(4,5)P₂ hydrolysis, have been shown to activate TRP and TRPL in situ, as well as in inside-out patches of TRPL channels expressed in S2 cells. In addition, both DAG and PI(4,5)P₂ have been shown to modulate TRPL channel activity in cell culture models. Analysis of TRPC2 activation in the rodent vomeronasal organs shows considerable parallels to the current understanding of the mechanism of Drosophila TRP and TRPL activation. However, despite these findings, the physiological relevance of PI(4,5)P₂-derived lipids as activators of Drosophila TRP channels in vivo remains to be established and the precise identity of the phospholipid species that is involved is unknown (Georgiev, 2005),

Recently, genetic evidence of a role for lipid messengers in the activation of TRPC channels in vivo has been obtained in Drosophila photoreceptors from an analysis of the retinal degeneration A (rdgA) mutant. The rdgA mutant was first isolated because it failed to respond to light in a behavioural assay. Analysis of retinal ultrastructure revealed that all alleles show varying degrees of photoreceptor degeneration. Biochemical analysis showed impaired diacylglycerol kinase (DGK) activity and reduced levels of light induced phosphatidic acid (PA) formation in head extracts of rdgA mutants. The gene that is defective in rdgA mutants has been cloned and found to encode an eye-enriched isoform of DGK, the principal enzyme that inactivates DAG by phosphorylation to PA. However, and most significantly, under voltage-clamp conditions, several alleles including rdgA¹, rdgA³, rdgA⁶ and rdgA^KS60 all show a small constitutively active inward current, which, on the basis of its biophysical characteristics, genetics and pharmacology, has been shown to be composed largely of TRP channels. The retinal degeneration phenotype of rdgA can be rescued by genetically removing TRP channels (i.e., the double mutant rdgA;trp), whose photoreceptors now lack their principal plasma-membrane calcium-influx channels. These results suggested a model in which excessive calcium influx through constitutively active TRP channels results in retinal degeneration in rdgA. The light response of rdgA;trp photoreceptors shows defects in deactivation suggesting that DGK might play a role in terminating the light response and recent evidence suggests that DGK plays a role in regulating signal amplification during the response to light (Hardie, 2002). Despite these recent observations that suggest a direct role for rdgA in phototransduction, previous studies have suggested a distinct mechanism underlying the retinal degeneration phenotype of rdgA. (1) Unlike most other phototransduction mutants, the retinal degeneration of rdgA is reported to be light independent; (2) norpA mutants, which lack the PLC activity essential for TRP channel activation, were reported not to suppress the retinal degeneration of rdgA; (3) several studies have suggested that a failure of rhabdomere biogenesis and protein trafficking underlies the rdgA phenotype (Georgiev, 2005),

To address these apparently conflicting results and to understand the mechanism of degeneration in rdgA, a genome-wide forward-genetic screen was undertaken for mutants that suppress or enhance the retinal-degeneration phenotype of rdgA. The goal is to identify molecules whose function might help gain an understanding of basis of the constitutive TRP-channel activity that is associated with the rdgA phenotype. This study describes the isolation and characterization of two mutants identified in the screen. Experiments are described that address the requirement for the light response in the degeneration phenotype of rdgA (Georgiev, 2005),

Therefore, although most studies agree that the hydrolysis of PI(4,5)P₂ by receptor-regulated PLCß is required for TRPC (TRP and TRPL) activation, there is little agreement about the downstream biochemical mechanisms that result in TRPC channel opening. For some members, such as TRPC3, equally compelling studies have been published showing roles in activation for IP₃/IP₃R mediated store depletion and for the lipid products of PI(4,5)P₂ hydrolysis. Recently, it has become clear that many of these conflicting results arise from several experimental factors, including the level of overexpression of the channel, the presence of endogenous TRPC members in the cell lines used and the relative promiscuity of pharmacological agents used in manipulating their activation. By contrast, in Drosophila photoreceptors, the detection of light by rhodopsin activates a signalling cascade that ends with endogenous TRPC activation. In this model, too, current debate centers around the identity of the products of PI(4,5)P₂ hydrolysis that are crucial for channel activation. Although there is substantial evidence to suggest that IP₃-mediated signalling is not essential, recent evidence suggests that the lipid products of PI(4,5)P₂ hydrolysis might be involved in activation. Analysis of photoreceptors lacking DGK activity (rdgA) has provided the first genetic evidence that suggests a role for lipid second messengers in activating TRP and TRPL in vivo. However, questions remain about the biochemical basis of the rdgA phenotype and its relevance to the normal phototransduction cascade; for example, is the constitutive channel activity the cause or the consequence of the degeneration? In addition, how can one reconcile recent findings suggesting a role for rdgA in phototransduction with long-standing observations that imply a phototransduction-independent basis for the rdgA phenotype (Georgiev, 2005)?

To address this issue, a forward-genetic screen was carried out to identify suppressors of the retinal degeneration phenotype of rdgA mutants. Such an approach is unbiased and makes no assumptions about the mechanisms underlying the degeneration process. Using a combination of deficiency mapping and bioinformatic analysis, su(1) and su(100) were identified as new alleles of InaD, a PDZ-domain protein required for the assembly of signalling complexes in Drosophila photoreceptors that is suggested to have a role in the regulation of signalling specificity and speed. InaD has not previously been reported to interact with rdgA. Trp³⁶⁵ contains a point mutation at the cytoplasmic end of S5 in the TRP channel and shows constitutive channel activity and degeneration. The finding that InaD is largely ineffective at suppressing the degeneration of Trp³⁶⁵ suggests that the mechanism of suppression is at or above the level of the channel in the transduction cascade rather than by blocking events downstream of excessive calcium influx through constitutively active TRP channels. InaD clusters several key molecules required for phototransduction, including the TRP channel; this strongly suggests that the constitutive channel activity and degeneration in rdgA are a consequence of altered phototransduction (Georgiev, 2005),

To identify the specific known (or perhaps undiscovered) protein-protein interactions of InaD that contribute to the rescue of rdgA, the InaD complex was manipulated in a manner that allowed its interaction with specific transduction components such as the TRP channel and NORPA to be individually disrupted. Wild-type TRP protein levels were found in InaD on eclosion and by analysing the effect of InaD^P215 and TRP^Delta1272 on rdgA it was found that TRP channels that could not be recruited to the INAD complex were able to mediate degeneration in rdgA just as well as wild-type channels. Thus, loss of the TRP-INAD interaction is unlikely to be a significant mechanism by which InaD rescues rdgA and the function of TRP channels within the INAD complex is not crucial to retinal degeneration (Georgiev, 2005),

By contrast, it was found that NORPA (the PLC activity) levels in su(1) and su(100) mutants are reduced on eclosion, as has been reported for InaD¹, suggesting that a reduction in PLCß activity might underlie the mechanism of suppression. InaD¹, a protein-null allele, and InaD², an allele known to disrupt the INAD-NORPA interaction, produce equivalent levels of rescue of rdgA¹, and that the levels of NORPA are inversely correlated to the extent of rescue. Thus, a major mechanism by which InaD¹, su(1) and su(100) suppress rdgA¹ is likely to involve the reduced levels of NORPA in these alleles. However, the possibility cannot be excluded that additional, unknown protein-protein interactions of PDZ5 in INAD that might also be disrupted in InaD² might also contribute to the rescue of rdgA. Given the essential role of PLCß in the activation of TRP channels, this finding implies a key role for the balance of PLCß and DGK activity in the degeneration of rdgA (Georgiev, 2005),

Although the degeneration phenotype of rdgA has been previously reported to be light independent and not suppressed by norpA mutants, the finding that InaD suppresses rdgA and that it does so by reducing levels of NORPA suggests that defects in light-induced phosphoinositide turnover might underlie the degeneration phenotype of rdgA. In the light of these findings on the suppression of rdgA by InaD, the effect was re-examined of three key elements of the phototransduction cascade that are required for activation, namely light, Galphaq and PLC. Although the absence of light could not completely suppress the degeneration of rdgA³, there was substantial suppression of degeneration in rdgA³ flies grown in complete darkness compared with those grown on a 12 hour light/12 hour dark cycle. Degeneration could also be partially suppressed but not blocked by a strong hypomorph that reduced Galphaq levels to <5% of the wild-type levels. However, most importantly, it was found that norpA mutants that lack PLCß could suppress the degeneration of rdgA in several allelic combinations for both genes. These results demonstrate a key role for activation of the phototransduction cascade in the degeneration phenotype of rdgA (Georgiev, 2005),

Although this study shows a requirement for light, Galphaq and PLC activity in the degeneration phenotype of rdgA, it was not possible to completely suppress the degeneration of even the weakest allele, rdgA³, by rearing flies in complete darkness. Indeed, reducing levels of Galphaq using the strong hypomorph Galphaq¹ (which has <5% of the wild-type Galphaq levels) was able only to slow the rate of degeneration of rdgA³. However mutants in norpA^P24 were able completely to suppress the degeneration of both rdgA³ and rdgA¹. In a recent study that measured basal PLCß activity in photoreceptors (Hardie, 2004), it was found that, similar to wild-type photoreceptors, rdgA mutants showed reduced but still substantial basal PLCß activity. This implies that, even in the dark, there is a basal turnover of PI(4,5)P₂ in rdgA photoreceptors. Thus, basal PI(4,5)P₂ hydrolysis could lead to the build up of a lipid metabolite of PI(4,5)P₂ that triggers constitutive TRP channel activity and retinal degeneration (Georgiev, 2005),

Although several studies have demonstrated the importance of INAD in targeting and stabilizing members of the phototransduction cascade to the rhabdomere, there is little agreement about the requirement, if any, for intact INAD complexes once assembled and transported to the rhabdomere to activate TRP channels. Although some studies have suggested that an intact INAD complex is crucial for generating the channel activity that underlies a quantum bump (the response to a single photon of light) others have suggested that this might not be the case. In this analysis of the mechanism by which InaD suppresses rdgA, it was found that that TRP channels not included within the INAD complex but still present in the rhabdomere are able to mediate retinal degeneration. These results support the idea that presence within the INAD macromolecular complex is not necessary for the constitutive activity of TRP channels seen in rdgA (Georgiev, 2005),

Although these data support the hypothesis that a principal mechanism by which InaD suppresses rdgA is via reduction in the levels of PLCß, they do not exclude the possibility that the disruption of INAD interactions with currently undiscovered proteins that function downstream of NORPA might play a role in constitutive TRP channel activation and degeneration in rdgA. Testing this would require the generation of an InaD allele in which the INAD-NORPA interaction is intact while disrupting the function of the other protein-protein interactions of INAD. No such allele exists, but the use of such an allele in conjunction with the rdgA mutant could be an useful approach to identifying currently undiscovered members of the INAD complex as well as the phototransduction cascade (Georgiev, 2005),

TRP channels appear to be key components of signalling cascades for the detection and coding of several sensory modalities. However, a limiting factor in advancing their role in sensory transduction is the poor understanding of their mechanism of activation. In the case of TRPC channels, this is limited by the lack of genetic model systems in which relevant components of the activation cascade can be identified. In the present study, a novel modifier screen has been described that should provide a powerful method for identifying the relevant transduction components in vivo. Starting with the rdgA mutant in which TRP channels are constitutively active and result in retinal degeneration, two new alleles of INAD, a known component of the phototransduction cascade, have been identified as suppressors of rdgA. This approach is likely to be a powerful tool to identify further components of the transduction cascade that are relevant in vivo (Georgiev, 2005),

TRP, TRPL and cacophony channels mediate Ca(2+) influx and exocytosis in photoreceptors axons in Drosophila

In Drosophila photoreceptors Ca(2+)-permeable channels TRP and TRPL are the targets of phototransduction, occurring in photosensitive microvilli and mediated by a phospholipase C (PLC) pathway. Using a novel Drosophila brain slice preparation, the distribution and physiological properties were studied of TRP and TRPL in the lamina of the visual system. Immunohistochemical images revealed considerable expression in photoreceptors axons at the lamina. Other phototransduction proteins are also present, mainly PLC and protein kinase C, while rhodopsin is absent. The voltage-dependent Ca(2+) channel Cacophony is also present there. Measurements in the lamina with the Ca(2+) fluorescent protein G-CaMP ectopically expressed in photoreceptors, revealed depolarization-induced Ca(2+) increments mediated by Cacophony. Additional Ca(2+) influx depends on TRP and TRPL, apparently functioning as store-operated channels. Single synaptic boutons resolved in the lamina by FM4-64 fluorescence revealed that vesicle exocytosis depends on Cacophony, TRP and TRPL. In the PLC mutant norpA bouton labeling was also impaired, implicating an additional modulation by this enzyme. Internal Ca(2+) also contributes to exocytosis, since this process was reduced after Ca(2+)-store depletion. Therefore, several Ca(2+) pathways participate in photoreceptor neurotransmitter release: one is activated by depolarization and involves Cacophony; this is complemented by internal Ca(2+) release and the activation of TRP and TRPL coupled to Ca(2+) depletion of internal reservoirs. PLC may regulate the last two processes. TRP and TRPL would participate in two different functions in distant cellular regions, where they are opened by different mechanisms. This work sheds new light on the mechanism of neurotransmitter release in tonic synapses of non-spiking neurons (Astorga, 2012).

Light transduction in Drosophila occurs in retinal microvillar arrangements running along the photoreceptor soma, termed rhabdomere (see Drosophila visual system and brain slices). The axon of this non-spiking neuron releases histamine in a tonic manner. It presents a T-bar ribbon synapse, a particular structure of the active zones specialized for fast and sustained multivesicular neurotransmitter release in response to graded membrane depolarizations. R1-R6 photoreceptors make multiple axo-axonic synaptic contacts with large monopolar (LI-L3) and amacrine cells in the lamina. Cell somata are located in the outermost part of this neuropile, leading to a particular situation where axonal arrays (named cartridges) are the predominant components of the lamina. The axons of centrifugal medullar neurons (C2-C3), a T-shaped centripetal neuron (T1) and a wide field tangential neuron (Tan) are also found in the lamina. In the rhabdomere, photon absorption triggers rhodopsin isomerization into an active state which, upon interaction with a G_q-protein, activates phospholipase C (PLC_β4). This enzyme, encoded by norpA, hydrolyses phosphatidylinositol biphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG). This signaling cascade has been widely implicated in the activation of TRP and TRPL. Although the mechanism of channel gating remains undetermined, there is evidence that under experimental conditions, DAG, polyunsaturated fatty acids (PUFAs), PIP₂ and protons are involved in opening TRP and TRPL, whereas IP₃ receptor does not. Interestingly, TRP and TRPL expressed in heterologous systems are activated by Ca²⁺ depletion of the endoplasmic reticulum (ER). This study confirmed the presence of TRP in the lamina, where TRPL is also expressed. For the first time, evidence is provided that these channels are implicated in neurotransmitter release in the lamina, where they apparently allow Ca²⁺ influx via a store-operated channel (SOC) mechanism and could also be regulated by a PLC-mediated cascade. Furthermore, it was shown that the voltage-dependent Ca²⁺ channel Cacophony, the only fly homologue of vertebrate N-, P/Q- and R-type, is present in the lamina where it plays an important role in photoreceptor synaptic transmission, probably as a first step in a complex cascade involving both intracellular and extracellular Ca²⁺ signalling (Astorga, 2012).

TRP and TRPL are the targets of Drosophila phototransduction in the rhabdomere, gated by an as yet undetermined PLC-dependent mechanism independent of internal membrane systems, which are absent in the microvilli. This study provides the first evidence that both channels additionally participate in exocytosis in photoreceptor synaptic terminals, where they can be activated by depletion of Ca²⁺ stores. It is also demonstrated that the voltage-dependent Ca²⁺ channel, Cacophony, plays a critical role in exocytosis (Astorga, 2012).

This study confirmed that, in addition to the rhabdomere, TRP localizes to the lamina and the medulla. Additionally, TRPL was found in these two neuropiles, where photoreceptors synapse with secondary neurons. The lamina, where most photoreceptors make synaptic connections into well-defined structures, was studied (Astorga, 2012).

A Drosophila slice preparation suitable for immunohistochemistry and functional experiments in the lamina was studied. In addition to TRP and TRPL, PLC and PKC exhibited high expression levels, while G_q and INAD were scarce and rhodopsin was absent. The four former proteins colocalized with ectopically expressed GFP, used as photoreceptor marker, whereas G_q and INAD colocalization with GFP was low. While TRP, TRPL and PLC were not restricted to photoreceptors, the relevant conclusion is that their presence in photoreceptors axons in the lamina suggests a participation in presynaptic events (Astorga, 2012).

The prominent cacophony immunostaining in the lamina is relevant. This Ca²⁺ channel is involved in synaptic transmission in Drosophila neuromuscular junction, brain and retina, suggesting a role in synaptic transmission in the lamina. A role of cacophony in photoreceptor synaptic transmission is supported by the observation that inhibition of this channel by PLTX-II affected bouton labeling. Although the possibility that PLTX-II could also affect other Ca²⁺ channels cannot be ruled out, the role of cacophony in vesicle release was further strengthened by the substantial reduction in FM4-64 fluorescence in the thermosensitive cacophony mutant cac^TS at non-permissive temperature. In agreement with this, a mutation in the dα₂δ-3 gene encoding a cacophony subunit abolishes the ERG 'on' transient. In contrast depolarization-induced G-CaMP Ca²⁺ fluorescence changes in the photoreceptors were significantly decremented by PLTX-II, providing additional evidence involving cacophony in the synaptic events (Astorga, 2012).

The observations that TRP and TRPL are also in the photoreceptors axons and are considerably Ca²⁺-permeable (P_Ca:P_Na ~100:1 and ~4:1, respectively) suggested a synaptic role. Accordingly, vesicle release was drastically impaired in the double mutant. Opening a Ca²⁺ pathway with the ionophore induced exocytosis in this mutant, an observation that opposes to a generalized degeneration of synaptic machinery. This evidence shows that TRP and TRPL are involved in exocytosis. Only one of these channels was sufficient for sustaining exocytosis (Astorga, 2012).

FM4-64 is presumably incorporated by all lamina neurons and therefore not only photoreceptor boutons should be labeled. However, it is expected that the dramatic changes in release observed include photoreceptor terminals, which represent the most numerous synaptic contacts in the lamina. Altogether, these results support the participation of TRP, TRPL and cacophony in synaptic transmission in photoreceptor terminals (Astorga, 2012).

What is PLC doing in photoreceptors synaptic terminals? Depolarization-induced exocytosis was markedly reduced in norpA mutant, suggesting a role of PLC in neurotransmitter release. An obvious possibility is that it mediates TRP/TRPL activation. In principle, PLC may act by either DAG or IP₃. PUFAs can activate the light-dependent channels when added to intact ommatidia, as well as to excised rhabdomeric membrane patches, in which DAG can do the same. Thus, it is conceivable that these lipids may also activate TRP/TRPL channels in the lamina. Nevertheless, there is no evidence that PUFAs are generated in these photoreceptors (Astorga, 2012).

How is PLC activated? In Drosophila photoreceptors, a level of PLC activity has been observed both in vitro and in vivo. This basal activity is probably a property of the PLC molecule itself, as it is not affected by mutation of G_q-protein. In addition, a positive modulation of PLC activity by micromolar Ca²⁺ has been reported in Drosophila head membranes. Therefore, basal PLC activity could be boosted by Ca²⁺ influx through cacophony (and additional Ca²⁺ pathways described in this study) during depolarization-induced vesicle exocytosis, representing a feed-forward mechanism in this graded synapse. Alternatively, PLC activation may be a consequence of a direct activation of G_q by depolarization, as reported in other insects. In contrast, the substantial PKC expression in the terminals suggests that it may down-regulate PLC, as in the rhabdomere (Astorga, 2012).

Calcium reservoirs appear to be involved in exocytosis, since inhibition of SERCA with Thg deeply affected vesicle release. Moreover, exposure of serca^TS to the non-permissive temperature considerably decreased bouton labeling compared to permissive temperature, and this study shows that this decrease cannot be explained exclusively by a temperature effect. These results strongly implicate ER Ca²⁺ release in photoreceptors exocytosis (Astorga, 2012).

The robust Ca²⁺ signals in the lamina after Ca²⁺ depletion implicated TRP/TRPL, as it was absent in trpl;trp animals. This supports the function of TRP/TRPL as SOCs in the synaptic terminals, allowing Ca²⁺ influx. This mechanism drives exocytosis, as indicated by the Ca²⁺-depletion protocol, where bouton labeling was significant. Interestingly, TRP and TRPL function as SOCs in heterologous expression systems, but not in the rhabdomere (Astorga, 2012).

Mammalian homologues of Drosophila TRP, TRPC1, 2, 4 and 6, are proposed to function as SOCs in different cell types. Moreover, TRPC1 operating as SOC regulates Ca²⁺ influx related to neurotransmission in rods and cones. The Drosophila genome has one gene encoding STIMh, an ER Ca²⁺ sensor protein that forms functional SOCs in association with TRPC1. It remains to be determined whether TRP/TRPL could form equivalent presynaptic macromolecular complexes in photoreceptors (Astorga, 2012).

This study showed that it is improbable that in the Ca²⁺-depletion experiments TRPL/TRP opening could be induced by a PLC-dependent mechanism mediated by phospholipase activation by a cytoplasmic Ca²⁺ increase due to altered reticular release/uptake balance during Thg treatment. In these experiments PLC contribution to exocytosis was possibly by-passed. In normal conditions, this enzyme may elicit Ca²⁺ elevation in the synaptic terminals by DAG-mediated activation of TRP/TRPL and/or by inducing Ca²⁺ release (Astorga, 2012).

Photoreceptors synaptic transmission must accurately follow the fast photoresponses generated in the rhabdomere. As graded synapses support rapid changes in neurotransmitter release, they should undergo fast variations in internal free Ca²⁺. Small and fast Ca²⁺ increments induce correspondent changes in release, something that would be implausible if a threshold were involved, as in non-graded synapses (Astorga, 2012).

Besides cacophony contribution to exocytosis, the presence of the ryanodine receptor (RyR) in the lamina suggests the participation of Ca²⁺-induced Ca²⁺ release (CICR), but direct evidence for this is lacking. CICR regulates exocytosis in rods allowing high rates of neurotransmitter release. A reasonable expectation is that Drosophila photoreceptors use all available Ca²⁺ pathways (cacophony; TRP/TRPL; the IP₃ receptor, IP₃R and RyR) to satisfy the synaptic demands required by their extremely fast photoresponse. It has been speculated that the IP₃R might reinforce transmitter release, but no direct evidence for it has been shown. This possibility is supported by the current results implicating PLC. Moreover, the observation that Ca²⁺ from the ER contributes to depolarization-induced exocytosis strengthens the possibility of internal release via IP₃R and/or RyR (Astorga, 2012).

Bouton labeling experiments were conducted under prolonged depolarization, implying that vesicle exocytosis was at steady-state. Thg experiments under such conditions show that released Ca²⁺ plays an essential role in neurotransmission. In tonic synapses, this mechanism may be crucial to sustain synaptic transmission for extensive periods of time (Astorga, 2012).

The following model is proposed for the synaptic events at the axon terminals (see Model for photoreceptor synaptic events in the lamina): the receptor potential activates cacophony in the axon, allowing its propagation towards the axonal terminal, where Ca²⁺ enters through cacophony inducing vesicle release, perhaps enhanced by CICR. Additionally, PLC activated by an unknown mechanism which may be Ca²⁺ itself or depolarization, generates IP₃, triggering Ca²⁺ release through IP₃Rs. ER Ca²⁺ depletion in turn opens TRP/TRPL by a SOC mechanism, incrementing the Ca²⁺ supply. These channels may also be opened by lipid and pH changes resulting of PLC activity. This multi-source transient Ca²⁺ increment guarantees efficient, rapid and sustained neurotransmitter release. After depolarization, resting Ca²⁺ levels would be restored by extrusion by the Na⁺/Ca²⁺ exchanger and uptake by the ER (Astorga, 2012).

It is important to integrate the data into a plausible working model that could be helpful for designing further experiments. Although the model accounts for the data, it is by no means the only possible one. Accordingly, some aspects of it may be interpreted differently or given a different weight. For example, the relative contributions of cacophony, CICR, IP₃-induced Ca²⁺ depletion and TRP/TRPL to presynaptic Ca²⁺ for vesicle release can vary widely. Also, the activation of TRP/TRPL may rely on ER depletion and/or lipids associated to PLC activity. It may be thought that the Ca²⁺ influx through cacophony should be sufficient to account for exocytosis, making Ca²⁺ release redundant and rather unnecessary. However, in this graded synapse the level of cacophony activation will follow the graded depolarization. The amplitude attained by the receptor potential are most likely within a small voltage range above the threshold for cacophony activation (-20 or -40 mV), inconsistent with a massive cacophony-dependent Ca²⁺ influx. Therefore, additional Ca²⁺ sources amplifying this initial signal are likely to be required for light-induced synaptic transmission (Astorga, 2012).

This study has provide novel evidence for TRP/TRPL function in Drosophila photoreceptors. For the first time, it was shown that these channels have dual roles in separate regions of the same cell, namely the rhabdomere and the synapse, apparently involving different mechanisms. More generally, the observations reported herein shed light on the mechanism controlling presynaptic events in graded synapses (Astorga, 2012).

Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids.

Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved this study predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, the study used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38% to ~5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ~10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ~2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade (Randall, 2015).

The ability to manipulate fatty acid composition of phospholipids in flies by diet has been reported previously. However, there are no convincing reports of physiological consequences. This study found that the proportionof polyunsaturated phospholipids in flies reared on yeast was reduced ~7-fold, and that this was associated with pronounced effects on visual performance, with photoreceptor responses slowed 2- to 3-fold,without significantly affecting quantum bump waveform, Na⁺/Ca²⁺ exchange, or PLC activity. Although absolute sensitivity was little affected by diet in wild-type photoreceptors, this could be attributed to compensation by Ca²⁺-dependent positive feedback acting on TRP channels. In the absence of Ca²⁺ influx or in trp mutants expressing only TRPL channels, sensitivity was reduced up to ~10-fold, and essentially abolished in hypomorphic Gαq¹ mutants. The results indicate that these effects, all of which were rescued by the addition of a single species of PUFA tothe yeast diet, were mediated predominantly downstream of PLC and were associated with slowing of photomechanical responses (Randall, 2015).

The macroscopic light response in Drosophila reflects the summation of quantum bumps, each arising from activation of most of the ~20 TRP channels in a single microvillus. Briefly, a single activated rhodopsin is believed to activate ~5 or so G_q-proteins by random diffusional encounters, and each released G_q α-subunit diffuses further before binding and activating PLC. Each PLC molecule rapidly hydrolyzes PIP₂, building up sufficient excitatory 'messenger' to overcome a finite threshold required to activate the first TRP channel with a stochastically variable latency of ~15-100 ms. The resulting Ca²⁺ influx raises Ca²⁺ throughout the microvillus into the micromolar range within milliseconds, facilitating activation of the remaining channels, and generating an 'all-or-none' quantum bump localized to a single microvillus. This raises Ca²⁺ within the affected microvillus to ~1 μm, terminating the bump by Ca²⁺-dependent inactivation of the channels and preceding steps of the cascade. This highly nonlinear positive and negative feedback cycle, which shapes the quantum bump waveform, was apparently not influenced by diet. However, bump latency, i.e., the time taken to activate the first channel, was clearly profoundly delayed in flies reared on the YF diet (Randall, 2015).

Which products of PLC activity are responsible for this initial gating remains controversial. InsP₃ and Ca²⁺ stores apparently play no role, because mutants of the InsP₃ receptor have normal phototransduction. Alternative candidates include DAG or PUFAs, which might be released from DAG by an appropriate lipase. Of these, DAG has generally proved ineffective as an agonist when exogenously applied; however, there is genetic evidence, based on mutants of DAG kinase (rdgA), for DAG as an excitatory messenger. One lab has also reported that DAG can activate TRP channels in excised patches from dissociated rhabdomeres; however, activation was very sluggish with delays of up to 60 s, in a preparation that is in a physiologically severely compromised state (Randall, 2015).

In contrast, there is universal consensus that PUFAs are very effective agonists when exogenously applied to both native channels in the photoreceptors and heterologously expressed TRPL channels. Apparent support for endogenous PUFAs came from the reduced light response found in a Drosophila DAG lipase mutant, inaE. However, inaE encodes an sn-1 DAG lipase, which rather than PUFAs, releases mono-acyl glycerols (MAGs), which are at best weak and slowly acting channel agonists when applied exogenously (Hardie, unpublished results). For PUFA generation, either an sn-2 DAG lipase or an additional enzyme (MAG lipase) would be required, but there is no evidence for either in photoreceptors. Furthermore, the inaE gene product immunolocalizes to the cell body with, at most, occasional puncta in the rhabdomere, and there is no evidence that PUFAs are generated in response to illumination (Randall, 2015).

An alternative hypothesis comes from evidence showing that the channels can be activated by the strict combination of two further consequences of PLC action, namely PIP₂ depletion and proton release. Furthermore it was suggested that PIP₂'s role is mediated not by ligand binding/unbinding but by the physical effects of PIP₂ depletion on the lipid bilayer. Thus, removal of PIP₂'s inositol headgroup from the inner leaflet effectively reduces membrane area, and it was proposed that the resulting mechanical effects (on, e.g., membrane tension, curvature, or lateral pressure) lead to mechanical gating of the channels, in combination with protons. Evidence supporting this included: (1) the ability of light to activate ectopic mechanosensitive channels (gramicidin); (2) facilitation of light responses by hypotonic solutions; and (3) rapid contractions of the photoreceptors in response to light, interpreted as the concerted contraction of microvilli due to PIP₂ hydrolysis in the inner leaflet (Randall, 2015).

The present study used diet to increase the degree of saturation of the phospholipids, which should make membranes stiffer and less flexible, and it ws predicted that this might suppress mechanosensitive channel activity. Lipidomic analysis confirmed a drastic sevenfold reduction in PUFA content across all phospholipid species in YF-reared flies. This was associated with a 2- to 3-fold longer latency and a marked reduction in open probability once Ca²⁺-dependent positive feedback was eliminated. In principle PLC activity might be suppressed by the YF diet, because membrane fluidity, and hence diffusion (e.g., of G-proteins, or PIP₂), might be slower in a membrane dominated by saturated phospholipids. However, molecular dynamic simulations predict that lateral diffusion coefficients in fact depend only weakly on the degree of phospholipid saturation. Importantly, the rate of PIP₂ hydrolysis monitored by proton release appeared unaffected by diet suggesting that diet was acting primarily downstream of PLC. That dietary manipulation had indeed resulted in alteration to the mechanical properties of the membrane was supported by AFM measurements of the light-induced contractions and responses mediated by ectopic mechanosensitive gramicidin channels. These indicated that, despite the lack of effect on PLC activity, YF-reared flies generated slower photomechanical responses (Randall, 2015).

Although consistent with the mechanical gating hypothesis, by themselves these data do not exclude the alternative suggestions, that the excitatory messenger is DAG or a PUFA. According to a recent study (Delgado, 2014), DAG generated by light-induced PIP₂ hydrolysis in Drosophila photoreceptor membrane includes both monounsaturated and polyunsaturated species (32:1, 32:2, 34:1, 34:2, 36:3, and 36:4) suggesting at least some derive from polyunsaturated PIP₂ species. In turn, should any fatty acids be released from DAG, they would most likely include PUFAs. However, the near absence of polyunsaturated PIP₂ in flies reared on YF diets implies that DAG generated in response to light would now be predominantly saturated or monounsaturated. If different species of DAG and/or PUFA had different potencies for activating the channels, then in principle this might also explain the results. In this respect, the otherwise equivocal evidence for DAG and PUFAs as messengers notwithstanding, it should be noted that heterologously expressed TRPL channels are activated more potently by linolenic than by oleic acid and that polyunsaturated DAG species have been reported to be more effective activators of PKC in mammalian cells (Randall, 2015).

In conclusion, these results confirm that fatty acid composition of phospholipids is strongly influenced by diet in Drosophila. Increasing the degree of saturation leads to pronounced slowing of the electrical light response, which is associated with slower photomechanical responses. These findings are consistent with the suggestion that TRP and TRPL channel activation in Drosophila photoreceptors are mediated, together with protons, by mechanical forces in the membrane induced by PIP₂ hydrolysis by PLC. To what extent visual performance in flies might be affected by dietary fatty acids under natural conditions is unknown. Although Drosophila thrive on a yeast diet in the laboratory, their typical food is rotting plant material, which will contain PUFAs to varying degrees. Interestingly, in food-preference studies, Drosophila larvae show preference for unsaturated over saturated fatty acids, but this preference is reversed in adult flies. The fatty acid composition of mammalian phospholipids is also influenced by diet, and the effects of fatty acid intak on health and disease are extensively documented. Certain PUFAs such as ω-3 (e.g., α-linolenic acid) and ω-6 (e.g., linoleic acid) can only be supplied by dietary intake in mammals. Of these, α-linolenic acid is also precursor for docosahexaenoic acid (22:6), which is the most abundant ω-3 fatty acid in mammalian brain and retina (though absent in flies). ω-3 and ω-6 deficiency is associated with cardiovascular disease and diabetes, can lead to cognitive defects in rodents, and within the mammalian retina leads to a reduced a-wave in the ERG. However, the underlying mechanisms are poorly understood, and in this respect, the current results provide a striking and novel example of how dietary fatty acids can profoundly and specifically influence in vivo performance and behavior via a defined step within the context of a classical G-protein-coupled signaling cascade. Interestingly, the importance of the mechanical properties of membranes containing polyunsaturated phospholipids has also been highlighted in recent studies of mechanosensation and rapid endocytosis (Randall, 2015).

The phosphorylation state of the Drosophila TRP channel modulates the frequency response to oscillating light in vivo

Drosophila photoreceptors respond to oscillating light of high frequency (approximately 100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. This study reports that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca2+-dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC). Electroretinogram measurements of the frequency response to oscillating lights in vivo revealed that dark-reared flies expressing wild-type TRP exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation. Strikingly, preventing phosphorylation of the S936-TRP site by alanine substitution in transgenic Drosophila (trpS936A) abolished the difference in frequency response between dark-adapted and light-adapted flies, resulting in high-frequency response also in dark-adapted flies. In contrast, inserting a phosphomimetic mutation by substituting the S936-TRP site to aspartic acid (trpS936D) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies. Light-adapted rdgC mutant flies showed relatively high S936-TRP phosphorylation levels and light-dark phosphorylation dynamics. These findings suggest that RDGC is one but not the only phosphatase involved in pS936-TRP dephosphorylation. Together, this study indicates that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions (Voolstra, 2017).

The latency of the light response is modulated by the phosphorylation state of Drosophila TRP at a specific site

Drosophila photoreceptors respond to oscillating light of high frequency (approximately 100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca2+-dependent process. It was further found that this process affects the detection limit of high frequency oscillating light. Accordingly, transgenic Drosophila, which do not undergo phosphorylation at the S936-TRP site (trpS936A), revealed a short time-interval before following the high stimulus frequency (oscillation-lock response) in both dark- and light-adapted flies. In contrast, the trpS936D transgenic flies, which mimic constant phosphorylation, showed a long-time interval to oscillation-lock response in both dark- and light-adapted flies. This study extends these findings by showing that dark-adapted trpS936A flies reveal light-induced current (LIC) with short latency relative to trpWT or trpS936D flies, indicating that the channels are a limiting factor of response kinetics. The results indicate that properties of the light-activated channels together with the dynamic light-dependent process of TRP phosphorylation at the S936 site determine response kinetics (Katz, 2017).

Protein Interactions

Interaction between TRP channels

Drosophila phototransduction is a G protein-coupled, calcium-regulated signaling cascade that serves as a model system for the dissection of phospholipase C (PLC) signaling in vivo. The Drosophila light-activated conductance is constituted in part by the Transient receptor potential ion channel, yet trp mutants still display a robust response demonstrating the presence of additional channels. The trpl gene encodes a protein displaying 40% amino acid identity with TRP. Mammalian homologs of TRP and TRPL recently have been isolated and postulated to encode components of the elusive I(crac) conductance. TRP and TRPL localize to the membrane of the transducing organelle, together with rhodopsin and PLC, consistent with a role in PLC signaling during phototransduction. To determine the function of TRPL in vivo, trpl mutants were characterized physiologically and genetically. The light-activated conductance is composed of TRP and TRPL ion channels and each can be activated on its own. Tenetic and electrophysiological tools were used to study the contribution of each channel type to the light response and show that TRP and TRPL can serve partially overlapping functions (Niemeyer, 1996).

The Drosophila retinal-specific protein TRP is the founding member of a family of store-operated channels (SOCs) conserved from C. elegans to humans. In vitro studies indicate that TRP is a SOC, but that the related retinal protein, TRPL, is constitutively active. In the current work, coexpression of TRP and TRPL is shown to lead to a store-operated, outwardly rectifying current distinct from that owing to either TRP or TRPL alone. TRP and TRPL interact directly, indicating that the TRP-TRPL-dependent current is mediated by heteromultimeric association between the two subunits. It is proposed that the light-activated current in photoreceptor cells is produced by a combination of TRP homo- and TRP-TRPL heteromultimers (Xu, 1997).

This study tested the proposal that the light-sensitive conductance in Drosophila is composed of two independent components by comparing the wild-type conductance with that in mutants lacking one or the other of the putative light-sensitive channel subunits, TRP and TRPL. For a wide range of cations, ionic permeability ratios in wild type were always intermediate between those of trp and trpl mutants. Effective channel conductances derived by noise analysis in wild type were again intermediate and also showed a complex voltage dependence, which was quantitatively explained by the summation of TRPL and TRP channels after taking their different reversal potentials into account. Although La3+ partially blocks the light response in wild-type photoreceptors, it increases the effective single channel conductance. The results indicate that the wild-type light-activated conductance is composed of two separate channels, with the properties of TRP- and TRPL-dependent channels as determined in the respective mutants (Reuss, 1997).

The trp and trpl genes are thought to encode two classes of light-activated ion channels in Drosophila. The properties of Trpl photoreceptor responses have been studied by using electroretinogram (ERG) and intracellular recording techniques in combination with light stimuli of relatively long durations. Distinct mutant phenotypes were detectable under these conditions. These consisted of a reduced sustained component, oscillations superimposed on the response, a poststimulus hyperpolarization, and altered adaptation properties to dim background light. Comparison of photoreceptor responses obtained from wild type, trp, and trpl showed that the responses obtained from the trp and trpl null mutants did not sum up to that of the wild-type response. To explain the nonlinear summation at the peak of the response, it has been proposed that Ca(2+) ions entering through the TRP channel modulate TRP and TRPL channel activities differentially. However, nonlinear summation was present not only at the peak but throughout the duration of response. Two lines of evidence are presented to suggest that, in addition to these interactions, there are other forms of interactions between TRP and TRPL channels, probably involving the channel proteins themselves (Leung, 2000).

Interaction of InaD with TRP

Drosophila vision involves a G protein-coupled phospholipase C-mediated signaling pathway that leads to membrane depolarization through activation of Na+ and Ca2+ channels. inaD mutant flies have a M442K point mutation and display a slow recovery of the Ca2+ dependent current. Anti-InaD antibodies coimmunoprecipitate Trp, which is identified by its electrophoretic mobility, cross reactivity with anti-Trp antibody, and absence in a null allele trp mutant. This interaction is abolished by the inaD point mutation, both in vitro and in vivo. Interaction is localized to the 19 amino acid C-terminus of Trp by overlay assays, and to the PDZ domain of InaD, encompassing the point mutation. Flies homozygous for the InaD and trp mutations were generated and their phenotype analyzed by electroretinogram (ERG). In this extracellular recording of the compound eye, light triggers a depolarizing receptor potential and the Inad and trp mutants display characteristic responses: trp shows a receptor potential that lacks the maintained component, and Inad lacks the prolonged depolarizing afterpotential. While the InaD and trp ERG phenotypes are similar when stimulated by a pulse of intense blue light, they are distinguished using low intensity stimulation. When stimulated with a 10 second pulse of orange light, InaD displays a sustained response, whereas trp flies show the transient receptor potential response. Under the same conditions, double mutants exhibit a phenotype similar to that of trp. The finding that the response of the double mutants is qualitatively similar to that of trp alone is consistent with the interpretation that Trp and InaD act in the same sequential pathway. In the absence of Trp, InaD is not able to effect its modulatory activity, and thus, the double mutant phenotype corresponds to that of trp. Given the impaired electrophysiology of the inaD mutant, this novel interaction suggests that InaD functions as a regulatory subunit of the Trp Ca2+ channel (Shieh, 1996).

INAD, a novel protein mutated in the inactivation no afterpotential D mutation in Drosophila, is a PDZ domain protein, sharing a protein interaction domain with Drosophila proteins Discs large, Dishevelled and Canoe. InaD photoreceptor cells show a slow deactivation of light-induced current and an increased sensitivity to dim light. The store-operated Ca2+ channel, TRP, is required in photoreceptor cells for a sustained response to light. TRP forms a complex with phospholipase C-ß (No receptor potential A), rhodopsin (RH1) Calmodulin, and INAD. The current model for Drosophila phototransduction is that IP3 generated through activation of NORPA binds to the IP3 receptor, resulting in release of Ca2+ from internal Ca2+ stores. The Calmodulin binding site of TRP bears no similarity to that of TRPL, the non specific cation channel that otherwise shares sequence homology with TRP. In InaD mutant flies, TRP is no longer spatially restricted to its normal subcellular compartment, the rhabdomere. In inaD^P215 mutant flies, TRP is no longer restricted to the rhabdomeres. Instead, some TRP is detected in the cell bodies, and a large proportion of TRP is found in the extracellular central matrix. The mislocalization of TRP in the central matrix might have occurred during the normal turnover of the photoreceptor cell membrane, which involves shedding of the microvillar rhabdomeral membrane into the central matrix. The alteration in localization of TRP in inaD^P215 is specific since all other rhabdomere-specific proteins examined displayed indistinguishable expression patterns in wild type and inaD^P215. Although a significant proportion of TRP is mislocalized in inaD^P215, some TRP remains in the rhabdomeres. Since the only existing inaD allele, inaD^P215, is caused by a point mutation in the second PDZ domain, it is possible that there remains some weak interaction between TRP and INAD^P215 protein that cannot be detected in vitro. If so, it is possible that no proportion of TRP would be detected in the rhabdomeres of null inaD flies. However, the dramatic change in localization of TRP detected in inaD^P215 provides evidence that a PDZ domain protein is required in vivo for targeting or anchoring an ion channel to its normal subcellular localization. These results provide evidence that a PDZ domain protein is required, in vivo, for the anchoring of an ion channel to a signaling complex. Furthermore, disruption of this interaction results in retinal degeneration. It is proposed that the TRP channel is linked to RORPA and RH1 to facilitate feedback regulation of these upstream signaling molecules. It is suggested that TRP may be inactivated through a Ca2+-dependent mechanism mediated by Calmodulin (Chevesich, 1997).

Phototransduction in invertebrate microvillar photoreceptors is thought to be mediated by the activation of phospholipase C (PLC), but how this leads to gating of the light-sensitive channels is unknown. Most attention has focused on inositol-1,4,5-trisphosphate, a second messenger produced by PLC from phosphatidylinositol-4,5-bisphosphate; however, PLC also generates diacylglycerol, a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Both of these fatty acids reversibly activate native light-sensitive channels [transient receptor potential (TRP) and TRP-like (TRPL)] in Drosophila photoreceptors, as well as recombinant TRPL channels expressed in Drosophila S2 cells. Recombinant channels are activated rapidly in both whole-cell recordings and inside-out patches, with a half-maximal effector concentration for linolenic acid of approximately 10 microM. Four different lipoxygenase inhibitors, which might be expected to lead to build-up of endogenous fatty acids, also activate native TRP and TRPL channels in intact photoreceptors. As arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999).

The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies have shown that the PDZ domain containing protein INAD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The INAD signaling complex also includes rhodopsin, protein kinase C (PKC), and calmodulin, though it is not known whether these proteins bind to INAD. The current work shows that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to INAD. A second ion channel, TRPL, binds to INAD. Thus, most of the proteins involved directly in phototransduction appear to bind to INAD. Furthermore, INAD formed homopolymers and the homomultimerization occurs through two PDZ domains. Thus, it is propose that the INAD supramolecular complex is a higher order signaling web consisting of an extended network of INAD molecules through which a G protein-coupled cascade is tethered (Xu, 1998).

The light response in Drosophila photoreceptor cells is mediated by a series of proteins that assemble into a macromolecular complex referred to as the signalplex. The central player in the signalplex is Inactivation no afterpotential D (InaD), a protein consisting of a tandem array of five PDZ domains. At least seven proteins bind InaD, including the Transient receptor potential (Trp) channel, which depends on InaD for localization to the phototransducing organelle, the rhabdomere. However, the determinants required for localization of InaD are not known. InaD is required for retention rather than targeting of Trp to the rhabdomeres. In addition, Trp binds to InaD through the COOH terminus, and this interaction is required for localization of InaD. Two other proteins that depend on InaD for localization, phospholipase C and protein kinase C, also mislocalize. However, elimination of any other member of the signalplex has no impact on the spatial distribution of InaD. A direct interaction between Trp and InaD does not appear to have a role in the photoresponse independent of localization of multiple signaling components. Rather, the primary function of the Trp/InaD complex is to form the core unit required for localization of the signalplex to the rhabdomeres (Li, 2000).

Trp is initially localized to the rhabdomeres in young InaD^P215 and trp^delta1272 flies, whereas in older flies, the spatial distribution of Trp is severely disrupted. These data suggest that InaD may be required for retention rather than targeting of Trp to the rhabdomeres. However, an alternative interpretation of these data is that those Trp molecules synthesized in young flies are targeted through an InaD-independent mechanism, whereas InaD is required for targeting of Trp synthesized in older flies. In support of the proposal that the InaD/Trp interaction is required for retention is the observation that Trp is long lived in vivo. Trp molecules synthesized before day 1.5 decline only ~25% in concentration during the next 8 d. Thus, it appears that Trp is initially targeted to the rhabdomeres, and is subsequently mislocalized in the absence of a direct link to InaD (Li, 2000).

An intriguing question concerns the identification of proteins required for localization of InaD. The NinaC myosin III would appear to be an excellent candidate, since it binds InaD and is a putative molecular motor expressed in the photoreceptor cells. Nevertheless, NINAC is not required for localization of InaD. Other InaD-interacting proteins that function in phototransduction, such as rhodopsin, PLC, PKC, and TrpL, are also dispensable for rhabdomeral distribution of InaD. In contrast to these proteins, Trp is specifically required for proper localization of InaD. Moreover, in trp mutant flies and in transgenic flies, trp^delta1272, in which the InaD binding site is deleted, the spatial distribution of InaD is disrupted in an age-dependent manner. These data, in combination with the findings that the half-life of InaD is ~5.5 d, suggest that the mislocalization of InaD in trp mutant flies is due to a defect in retention (Li, 2000).

In addition to a requirement for the Trp/InaD interaction for localization of Trp and InaD, elimination of trp or mutation of the InaD binding site in Trp leads to an alteration in the spatial distribution of other proteins that require InaD for rhabdomere localization. These include PLC and PKC. Moreover, the mislocalization of PLC and PKC appeared to be more pronounced than that of InaD in young trp flies. One possibility is that these signaling proteins may also interact with Trp and depend on both Trp and InaD for localization. PKC appears to interact at least transiently with Trp, since Trp is a substrate for PKC. Thus, Trp and InaD appear to form the core unit that is required for localization of many of the signalplex components in the rhabdomeres (Li, 2000).

The putative tetrameric structure of Trp may contribute to the stability of the Trp/InaD core unit, since each channel would have the potential to bind four InaD proteins. Although InaD is mislocalized in trp^delta1272, there is no major impact on the localization of InaD in InaD^P215, suggesting the existence of residual interaction between Trp and InaD^P215 in vivo. Consistent with this proposal, Trp is more unstable in trp^delta1272 than in InaD^P215. The presumed tetrameric structure of Trp could enhance a weak interaction between InaD^P215 and Trp in vivo, which is not observed in pull-down assays using a monomeric Trp tail, as noted above because each channel would have the potential to bind four InaD proteins. The data presented here raise the possibility that other PDZ-containing scaffold proteins form similar core complexes in vivo with tetrameric ion channels. In support of this proposal are recent in vitro experiments indicating that there is a reciprocal requirement for localization of PSD-95 and the K⁺ channel, Kv1.4 (Li, 2000 and references therein).

A separate question that awaits further investigation concerns the mechanism underlying targeting of the signalplex to the rhabdomeres. Evidence has been presented that another PDZ-containing scaffold protein, PSD-95, is trafficked to the postsynaptic compartment after assembling on vesicles. Thus, it is plausible that the components of the signalplex may get trafficked to the rhabdomeres via vesicular transport and require the Trp/InaD core unit for retention (Li, 2000 and references therein).

The finding that Trp and InaD are long lived is surprising considering that there is very active turnover of the rhabdomeric membrane. Such turnover results in shedding of rhabdomeral membrane into the central matrix and blebbing of membrane from the base of the microvilli into the cell bodies. The association between Trp and InaD may serve to prevent removal of these proteins into the central matrix and cell bodies during membrane turnover. Interestingly, the mutual requirement for the Trp/InaD interaction for retention in the rhabdomeres is less critical in trp^delta1272 flies maintained in the dark. It is suggested that a greater proportion of Trp and InaD is retained in the dark because of less turnover of the rhabdomeral membrane in the absence of light (Li, 2000).

In contrast to InaD^P215, the electroretinogram (ERG) response in young trp^delta1272 is similar to wild-type. The only significant ERG phenotype in trp^delta1272 flies was an age-dependent decrease in the amplitude of the maintained component. This defect was presumably due to mislocalization of Trp and InaD, since the amplitude of the maintained component gradually decreases in parallel with the mislocalization of Trp and InaD in older flies. Moreover, the termination of the photoresponse appears normal even in old trp^delta1272 flies. This latter result is surprising, because PKC is mislocalized in old trp^delta1272 photoreceptor cells, and PKC is required for termination of the photoresponse. However, the rhabdomeric concentration of two substrates for PKC (Trp and InaD) is also reduced in trp^delta1272. Given that PKC, Trp, and InaD have been reported to be present in about equimolar concentrations, it is proposed that the relative stoichiometry of PKC and its substrates is important for normal termination of the photoresponse. Thus, the delay in termination resulting from a reduction in PKC concentration may be suppressed by a concomitant decrease in the levels of InaD and Trp (Li, 2000).

The defect in termination associated with InaD^P215 may not be due to perturbation of the Trp/InaD interaction, because the mutation in PDZ3 may also affect binding to other target proteins. The observation that the termination defect does not became more severe in old InaD^P215 flies suggests that the phenotype is not due to the disruption of the spatial distribution of Trp, since the mislocalization of Trp is more severe in older InaD^P215 flies (Li, 2000).

To address the specific role of the Trp/InaD interaction, the InaD binding site was mapped and transgenic flies were generated expressing a Trp derivative that does not associate with InaD. PDZ domains typically recognize COOH-terminal sequences consisting of an S/T-X-V/I motif or hydrophobic or aromatic residues. As is the case with most PDZ target proteins, it was found that the critical binding motif is at the COOH terminus. Specifically, it was found that deletion of the last four amino acids (SGWL) completely disrupts Trp/InaD binding. Furthermore, Trp^delta1272 obtained from transgenic fly head extracts fails to associate with InaD in pull-down assays. Mutation of an internal S-X-V motif (V1266D), which abolishes interaction between Trp and InaD in an overlay assay, has only minor effects. An additional deletion (amino acids 1257-1264), which eliminates the first residue of the S-X-V motif within the context of the short Trp tail (1252-1275), also retains binding with InaD in vitro (Li, 2000).

It is concluded that the primary role of the direct interaction between Trp and InaD is not to facilitate rapid signaling. The apparently normal ERG in young trp^delta1272 suggests that there is no defect in any aspect of the photoresponse. Rather, binding of Trp to InaD is critical for forming the core unit of the signalplex, which is necessary for retention of multiple signaling proteins in the rhabdomeres. This conclusion contrasts with previous reports, which have concluded that InaD functions as a regulatory subunit of the Trp channel. These analyses of trp^delta1272 indicate that the delayed termination associated with InaD^P215 is not due to disruption of the interaction with Trp. Instead, it appears that the phenotype is due to disruption of the interaction of InaD with another signaling protein that is required for proper response termination. Thus, contrary to expectations, a direct association between Trp and InaD appears to be dispensable for rapid termination (Li, 2000).

It appears that there are at least three classes of InaD binding proteins. The first class consists exclusively of Trp, because it is the only known InaD binding partner that is required for retention of InaD as well as of those InaD targets that depend on the signalplex for localization. However, there may be additional proteins that along with Trp and InaD comprise the core unit. The second group includes two proteins, PLC and PKC, which rely on InaD for localization and stability. However, there is no reciprocal requirement for these proteins for retention of any other protein in the rhabdomeres. Mutation of the InaD binding sites in PLC have been reported to cause defects in the photoresponse. However, these effects may reflect mislocalization or instability of these InaD targets rather than a direct requirement for coupling to InaD (Li, 2000).

The third class of InaD target proteins includes proteins such as rhodopsin, NINAC, and TrpL that are not dependent on InaD for localization in the rhabdomeres. It is proposed that the class I and II proteins, which depend on interaction with InaD for retention in the rhabdomeres, are constitutively bound to InaD, whereas the class III proteins may interact dynamically with InaD. As a consequence, only a subset of the class III proteins may bind to InaD at any given time. The observation that class III proteins do not depend on InaD for localization suggests that these InaD/target protein interactions have an alternative function, such as a direct role in the photoresponse. In support of this proposal, it was found that mutation of the InaD binding site in NINAC results in a pronounced delay in termination of the photoresponse. Thus, proteins that do not depend on InaD for localization may participate in the rapid activation and/or termination of the photoresponse (Li, 2000).

In Drosophila photoreceptors the multivalent PDZ protein InaD organizes the phototransduction cascade into a macromolecular signaling complex containing the effector PLC, the light-activated TRP channels, and a regulatory PKC. The subcellular localization of InaD signaling complexes is critical for signaling. How InaD complexes are anchored and assembled in photoreceptor cells has been examined. trp mutants, or transgenic flies expressing inaD alleles that disrupt the interaction between InaD and TRP, cause the mislocalization of the entire transduction complex. The InaD-TRP interaction is not required for targeting but rather for anchoring of complexes, because InaD and TRP can be targeted independently of each other. In addition to its scaffold role, InaD functions to preassemble transduction complexes. Thus the interaction of InaD with the TRP channel is required for anchoring signaling complexes in the rhabdomeres. TRP then may interact with the cytoskeleton, securing InaD and the whole complex to the membrane; ankyrin repeats on the N terminus of TRP could play a role in linking TRP to the cytoskeleton. Another possibility is that the InaD-TRP interaction reveals, or unmasks, sites on TRP or InaD that are important for membrane anchoring. Preassembly of signaling complexes helps to ensure that transduction complexes with the appropriate composition end up in the proper location. This may be a general mechanism used by cells to target different signaling machinery to the pertinent subcellular location (Tsunoda, 2001).

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).

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. Deletion of the COOH-terminal four residues in Trp destroys the PDZ binding site and results in mislocalization of INAD. 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, 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. 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. 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 (Wang, 2005 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). Currently, there are no loss-of-function mutations in Trpgamma and elimination of Trpl has only subtle effects on the photoresponse. Nevertheless, Trpl contributes to phototransduction since flies that are missing both Trp and Trpl are blind (Wang, 2005 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. 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 (Wang, 2005 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. 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 (Wang, 2005 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).

Phosphorylation of TRP

Photoreceptors that use a phospholipase C-mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cbeta (PLCbeta), the light-activated Ca2+ channel TRP, and an eye-specific protein kinase C (ePKC) are clustered by the PDZ domain protein InaD. The function of ePKC was investigated by cloning the Calliphora homolog of Drosophila ePKC, by precipitating the TRP signaling complex with anti-ePKC antibodies, and by performing phosphorylation assays in isolated signaling complexes and in intact photoreceptor cells. The deduced amino acid sequence of Calliphora ePKC comprises 685 amino acids and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti-ePKC antibodies leads to the coprecipitation of PLCbeta, TRP, InaD and ePKC but not of rhodopsin. Phorbolester- and Ca2+-dependent protein phosphorylation reveals that, apart from the PDZ domain protein InaD, the Ca2+ channel TRP is a substrate of ePKC. TRP becomes phosphorylated in isolated signaling complexes. TRP phosphorylation in intact photoreceptor cells requires the presence of extracellular Ca2+ in micromolar concentrations. It is proposed that ePKC-mediated phosphorylation of TRP is part of a negative feedback loop that regulates Ca2+ influx through the TRP channel (Huber, 1998).

Mutations in the Drosophila retinal degeneration A (rdgA) gene, which encodes diacylglycerol kinase (DGK), result in early onset retinal degeneration and blindness. Whole-cell recordings revealed that light-sensitive Ca2+ channels encoded by the trp gene were constitutively active in rdgA photoreceptors. Early degeneration was rescued in rdgA;trp double mutants, lacking TRP channels; however, the less Ca2+-permeable light-sensitive channels (TRPL) are constitutively active instead. No constitutive activity is seen in rdgA;trpI;trp mutants lacking both classes of channel, although, like rdgA;trp, these still show a residual slow degeneration. Responses to light are restored in rdgA;trp but deactivate abnormally slowly, indicating that DGK is required for response termination. The findings suggest that early degeneration in rdgA is caused by uncontrolled Ca2+ influx and support the proposal that diacylglycerol or its metabolites are messengers of excitation in Drosophila photoreceptors (Raghu, 2000).

Scaffolding protein INAD regulates deactivation of vision by promoting phosphorylation of transient receptor potential by eye protein kinase C in Drosophila

Drosophila visual signaling is one of the fastest G-protein-coupled transduction cascades, because effector and modulatory proteins are organized into a macromolecular complex ('transducisome'). Assembly of the complex is orchestrated by inactivation no afterpotential D (INAD), which colocalizes the transient receptor potential (TRP) Ca²⁺ channel, phospholipase Cβ, and eye protein kinase C (eye-PKC), for more efficient signal transduction. Eye-PKC is critical for deactivation of vision. Moreover, deactivation is regulated by the interaction between INAD and TRP, because abrogation of this interaction in InaD^p215 results in slow deactivation similar to that of inaC^p209 lacking eye-PKC. To elucidate the mechanisms whereby eye-PKC modulates deactivation, this study demonstrates that eye-PKC, via tethering to INAD, phosphorylates TRP in vitro. Ser⁹⁸² of TRP is phosphorylated by eye-PKC in vitro and, importantly, in the fly eye, as shown by mass spectrometry. Furthermore, transgenic expression of modified TRP bearing an Ala substitution leads to slow deactivation of the visual response similar to that of InaD^p215. These results suggest that the INAD macromolecular complex plays an essential role in termination of the light response by promoting efficient phosphorylation at Ser⁹⁸² of TRP for fast deactivation of the visual signaling (Popescu, 2006; full text of paper).

Drosophila visual transduction is a G-protein-coupled signaling pathway that provides a model system for understanding the molecular basis of signal transduction in the vertebrate nervous systems. Drosophila visual signaling is initiated with the activation of rhodopsin by light. Activated rhodopsin, via a Gq heterotrimeric protein, stimulates phospholipase Cβ (PLCβ) named no-receptor potential A (NORPA). NORPA hydrolyzes PIP₂ (phosphatidylinositol 4,5-bisphosphate) to inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG), which leads to opening of the transient receptor potential (TRP) Ca²⁺ and TRP-like channels, and depolarization of photoreceptors. The key second messenger that activates the TRP Ca²⁺ channel is thought to be either DAG or its lipid metabolites, whereas IP₃ does not appear to play a role. DAG may have a dual function, because it also activates the eye-specific protein kinase C (eye-PKC; InaC or Inactivation no afterpotential C) essential for deactivation of the light response (Popescu, 2006 and references therein).

Reversible phosphorylation modulates the dynamics of signal transduction by transiently altering activities of signaling proteins. Members of the conventional PKC family, which are activated by Ca²⁺ and DAG, are capable of phosphorylating a wide variety of protein substrates for temporal and spatial regulation of signaling processes. In Drosophila, eye-PKC is involved in the negative regulation of visual signaling, because inaC^p209 flies lacking eye-PKC display abnormal desensitization, slow deactivation, and defects in light adaptation. Eye-PKC is anchored to a macromolecular complex by tethering to INAD. Interaction with INAD enhances the stability of eye-PKC as well as targets eye-PKC to the rhabdomeres of photoreceptors, in which visual signaling occurs. Importantly, the in vivo function of eye-PKC is regulated by interaction with INAD. Previously, it was shown that eye-PKC phosphorylates TRP in vitro. The present study investigated the molecular basis of TRP phosphorylation by eye-PKC (Popescu, 2006).

To mimic eye-PKC phosphorylation of TRP in vitro, a complex-dependent kinase assay was designed. The in vitro complex-specific phosphorylation of TRP is regulated by the presence of the INAD-interacting domain in TRP, as well as the existence of INAD in the fly extracts. Extracts lacking either eye-PKC or INAD fail to support TRP phosphorylation. Similarly, extracts prepared from InaD^p215 (a modified INAD devoid of the TRP binding) are not able to promote TRP phosphorylation. Together, these findings indicate that INAD targets eye-PKC to its substrates, similar to RACK (receptor for activated C kinase). By the complex-dependent kinase assay, Ser⁹⁸² of TRP was identified as an eye-PKC phosphorylation site. Moreover, TRP isolated from flies by LC-MS was analyzed and it was found that Ser⁹⁸² of TRP is indeed phosphorylated in vivo by eye-PKC, because phosphorylated peptides encompassing Ser⁹⁸² of TRP are present in wild-type, but absent in inaC^p209 flies (Popescu, 2006).

Next, the in vivo functional contribution of phosphorylation was investigated by characterizing transgenic flies expressing a modified TRP bearing an Ala substitution at Ser⁹⁸² (trp^S982A). Remarkably, these transgenic flies displayed prolonged deactivation kinetics in response to bright light stimuli, indicating that phosphorylation of TRP at Ser⁹⁸² by eye-PKC is involved in inactivation of TRP, leading to fast deactivation. A model of the TRP regulation by eye-PKC is proposed. TRP is an integral part of the INAD complex and is opened by light. After light termination, the visual response is rapidly deactivated. Although molecular mechanisms underlying deactivation remain elusive, Ca²⁺ is known to play a vital role in response termination. The increased intracellular Ca²⁺ (primarily mediated by TRP) and DAG activate eye-PKC, which, in turn, phosphorylates TRP at Ser⁹⁸². Phosphorylation of TRP leads to a rapid inactivation of the channel on cessation of the light stimulation, without affecting the interaction between TRP and INAD. How does phosphorylation influence the TRP channel activity? Ser⁹⁸² is located within the Lys-Pro-rich region of TRP, which may function in TRP gating. It is speculated that phosphorylation at Ser⁹⁸² may induce a conformational change in the pore domain, which in turn leads to a rapid closure and inactivation of TRP. Phosphorylation has been linked directly to conformational changes that play key roles in the regulation of ion channels. It is also possible that phosphorylation of TRP at Ser⁹⁸² affects the interaction with some yet-unidentified proteins that may be important for the modulation of the TRP channel activity (Popescu, 2006).

In the absence of eye-PKC-mediated phosphorylation of TRP, deactivation of visual signaling is slower as observed in inaC^p209 or trp^S982A. It was found that inaC^p209 displays a more complex deactivation defect, whereas trp^S982A exhibits prolonged deactivation only in response to bright light. These findings suggest that, in addition to TRP, eye-PKC phosphorylates other substrates for efficient termination of the light response. Indeed, eye-PKC has been shown to phosphorylate INAD, but the functional relevance of this phosphorylation is not known. Furthermore, eye-PKC is required for the Ca²⁺-dependent inhibition of NORPA. NORPA is part of the INAD complex; however, it is not known to be phosphorylated by eye-PKC. The Ca²⁺-dependent inactivation of the light-induced current is unaltered in inaC^p209. This finding suggests the existence of a parallel Ca²⁺-dependent mechanism in inaC^p209 by which TRP is inactivated or of an upregulation of a Ca²⁺-dependent mechanism that activates other kinases to compensate for the loss of eye-PKC in inaC^p209 (Popescu, 2006).

Importantly, trp^S982A displays slow deactivation kinetics similar to that of InaD^p215. InaD^p215 was isolated based on the ina (inactivation no afterpotential) phenotype elicited by ERG. By whole-cell recordings, it was shown that InaD^p215 exhibits slow deactivation kinetics. However, a delay in latency of the quantum bump has been proposed and that activation is affected in the InaD^p215 mutant. To resolve this discrepancy, the mutant was reexamined and it was concluded that the primary defect in InaD^p215 is prolonged deactivation and not slow activation. InaD^p215 expresses INAD^M442K, which fails to associate with TRP. How does a loss of INAD–TRP interaction lead to abnormal deactivation of visual signaling? It is likely that the lack of the INAD–TRP interaction prevents the recruitment of TRP to the INAD complex and, consequently, eye-PKC-mediated regulation. Indeed, both trp^S982A and InaD^p215 exhibit similar deactivation defects, indicating that the molecular basis underlying the slow deactivation defect in InaD^p215 is attributable to a lack of negative regulation of the TRP channel by eye-PKC. Together, these findings suggest that formation of the INAD complex is essential for fast deactivation of the visual response by promoting phosphorylation of TRP by eye-PKC. Moreover, Ser⁹⁸² may be the sole eye-PKC phosphorylation site in TRP, because trp^S982A and InaD^p215 display similar deactivation defects. A loss of INAD–TRP interaction has been investigated in transgenic flies expressing modified TRP in which the INAD-interacting domain was deleted (trp^Δ1272). A reduced light response with normal deactivation kinetics in trp^Δ1272 has been reported. It has been proposed that the suppression of the delayed termination, which is attributable to a reduced eye-PKC level in trp^Δ1272 is probably masked by a concomitant decrease in TRP and INAD levels (Popescu, 2006).

To date, many proteins related to Drosophila TRP have been discovered in both invertebrates and vertebrates. These TRP ion channels are subdivided into seven subfamilies (TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, and TRPML). Drosophila TRP belongs to the TRPC subfamily. Members of the TRPC subfamily are also activated by receptor-induced activation of phospholipase C and therefore may be regulated by PKC. Indeed, phosphorylation of the TRPC channels by PKC appears important for modulating the channel activity. For example, the PKC-mediated phosphorylation of TRPC1 was shown to contribute to its SOC (store operated channel) activation, triggering Ca²⁺ entry into endothelial cells. In contrast, PKC-mediated phosphorylation was demonstrated to inhibit the activity of TRPC3 in HEK 293 cells and of TRPC6 in PC12D neuronal cells. In both cases, TRPC3 and TRPC6 are activated by DAG, whereas DAG also turns on PKC. It has been proposed that timing is important because the channels are activated by DAG more rapidly than they are inhibited by DAG-activated PKC. Heterologously expressed TRPC7 was also shown to be regulated by PKC: inhibition of PKC prolonged inactivation of the channel. Moreover, PKC phosphorylation of heterologously expressed TRPC5 resulted in desensitization of this channel, a process that was dependent on both extracellular and intracellular Ca²⁺ concentrations (Popescu, 2006).

In conclusion, this study has uncovered the molecular mechanism underlying the complex-dependent phosphorylation of TRP by eye-PKC and its role in fast deactivation of vision. Specifically, it was shown that eye-PKC phosphorylates TRP at Ser⁹⁸² in vitro and in vivo. Importantly, phosphorylation of TRP facilitates rapid inactivation of the channel because transgenic flies bearing an Ala substitution at Ser⁹⁸² display prolonged deactivation kinetics of the light response. Significantly, this slow deactivation defect is similar to that observed in InaD^p215 in which TRP fails to associate with INAD. These findings provide insights into the mechanistic basis of slow deactivation in InaD^p215, suggesting that INAD plays a critical role in targeting eye-PKC to TRP for rapid deactivation of the visual signaling. Together, these data indicate that the INAD macromolecular complex is important for deactivation of the visual response by directing eye-PKC to TRP. Furthermore, PKC-mediated phosphorylation of TRP at Ser⁹⁸² leads to fast deactivation of vision by promoting inactivation of the TRP channel (Popescu, 2006).

Calmodulin regulation of TRP channels

The characterization of Drosophila Calmodulin mutants and the role of CAM in photoreceptor cell function have been described. In Drosophila photoreceptor neurons, light activation of rhodopsin activates a heterotrimeric G protein, which in turn activates phospholipase C (PLC). PLC catalyzes the hydrolysis of the minor membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) into the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG). Activation of PLC then leads to the opening of cation-selective membrane channels encoded by the transient receptor potential (trp) and trp-like (trpl) genes. It has been hypothesized that calcium release from internal stores is required for activation of the phototransduction cascade and that the TRP channel functions as a store-operated channel gated by the light-induced emptying of the internal stores (Scott, 1997 and references).

Contrary to current models of excitation and TRP channel function, the transient phenotype of trp mutants can be explained by CAM regulation of the TRPL channel rather than by the loss of a store-operated conductance leading to depletion of the internal stores. In fact, introduction of calcium intracellularly in trp mutants does not restore responsiveness. The finding that trp mutants can maintain responsiveness in the absence of calcium suggests that there is calcium-dependent inactivation of light-induced currents in the trp mutant. Light responses were analyzed in a variety of mutant and transgenic backgrounds. The transient respone of trp mutants reflects TRPL channel function. Deletion of either of the two CAM binding sites of TRPL results in a prolonged current suggesting that CAM binding functions to inactivate TRPL. Thus, Calmodulin is essential for calcium-dependent negative regulation of phototransduction. Mutants for cam display dramatic defects in deactivation kinetics, displaying greatly prolonged deactivation times. In the absence of extracellular calcium, mutant and wild-type responses are not significantly different from each other, demonstrating that calcium entry is required to reveal the cam mutant phenotype and highlighting the absolute requirement for calcium for the rapid deactivation of the phototransduction cascade. CAM also regulates the catalytic lifetime of activated rhodopsin by regulating the binding of arrestin to rhodopsin. Thus CAM coordinates termination of the light response by modulating receptor and ion channel activity (Scott, 1997).

Light-regulated interaction of Dmoesin with Trp and Trpl channels is required for maintenance of photoreceptors

Recent studies in Drosophila retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. Since ezrin-radixin-moesin (ERM) proteins are known to regulate actin-membrane interactions in a signal-dependent manner, the role of Dmoesin, the unique D. melanogaster ERM, in response to light was analyzed. The illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (Trp) and Trp-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, these data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells (Chorna-Ornan, 2005).

Actin has been reported to undergo light-induced reorganization in both squid and Drosophila photoreceptors, thus showing that light-sensitive cytoskeletal rearrangements are a common phenomenon. However, it remains unclear how illumination can modify the intracellular distribution of both signaling and cytoskeletal molecules. As a step toward understanding the molecular mechanisms that underlie these aspects of the light response in Drosophila photoreceptors, the potential role of Dmoesin in this process was analyzed (Chorna-Ornan, 2005).

In dark-raised flies, Dmoesin interacts with both the Trp and Trpl channels, as evidenced by reciprocal coimmunoprecipitation experiments. In contrast, virtually no Dmoesin-Trp and -Trpl complexes are coimmunoprecipitated from illuminated eyes, thus providing strong evidence for Dmoesin binding to the photoreceptor-specific channels primarily in the dark. Furthermore, the results show that light induces dissociation of Dmoesin from Trp and Trpl channels followed by movement of Dmoesin from the rhabdomere membranes to the cytoplasm. Since there is increasing evidence to suggest that functions of invertebrate Trps are conserved in their mammalian counterparts, these findings might provide new insights for characterizing vertebrate Trp functions. Interestingly, TrpC3 is part of a multimolecular signaling complex containing Ezrin, PLCß1, and Galphaq/11 that is involved in Ca²⁺-mediated regulation of channel activity and cytoskeletal reorganization (Lockwich, 2001). In addition, it has been shown that the ERM adaptor EBP50/Na+/H+ exchanger regulatory factor associates with PLCß, TrpC4, and TrpC5 and regulates channel activity and subcellular localization (Tang, 2000; Mery, 2002; Obukhov, 2004). Altogether, these data strongly suggest that Trp-ERM interactions are an evolutionarily conserved mechanism with important functional properties. The ability to modify Dmoesin binding to Trps in vivo using illumination should constitute an invaluable tool for investigating the molecular mechanisms regulating this interaction (Chorna-Ornan, 2005).

In this study, the critical role of T559 phosphorylation on Dmoesin activation (Polesello, 2002: Speck, 2003) was extended through the demonstration that dissociation of the Dmoesin from the channel proteins upon illumination depends on T559 dephosphorylation. Accordingly, specific antibodies for the phosphorylated T559 form of Dmoesin immunoprecipitate the Trp channel of dark-raised flies, but not of illuminated flies. Moreover, monospecific Trp antibody immunoprecipitates the phosphorylated form of Dmoesin only in dark-raised flies. These results strongly suggest that only the phosphorylated form of Dmoesin binds Trp. This finding further suggests that light induces dephosphorylation of Dmoesin, leading to dissociation of Dmoesin from the channel proteins, followed by its movement to the cell body (Chorna-Ornan, 2005).

Using WT and transgenic flies that express Dmoesin-GFP fusion proteins, the light-induced movement of Dmoesin from the rhabdomere to the cell body was directly visualized, through confocal imaging of fixed and living retinae. The critical role of T559 phosphorylation on light-induced Dmoesin movement in vivo was further demonstrated through the use of two mutant forms of Dmoesin, in which T559 was replaced by alanine or aspartate residues. The fact that light-activated movement of Dmoesin is blocked in the T559A mutant that remains localized primarily to the soluble fraction of the cell body strongly supports the conclusion that phosphorylation of T559 is crucial for binding of Dmoesin to the channel proteins. Although the T559A mutation keeps Dmoesin in its inactive cytosolic state, the T559D phosphomimetic mutation is expected to keep Dmoesin constitutively active. Although some T559D Dmoesin was also found in the cytosol, a significant fraction of T559D Dmoesin was indeed found in the membrane fraction that remains associated with the rhabdomeres after illumination. In addition, both T559A and T559D mutations block the light-dependent movement of Dmoesin (Chorna-Ornan, 2005).

How could nontrafficking forms of Dmoesin (Dmoesin T559A and T559D) lead to light-induced retinal degeneration when expressed in an otherwise WT background? T559 phosphomutants of the Dmoesin protein have been shown to perturb the role of endogenous Dmoesin in actin organization and Oskar localization during oogenesis (Polesello, 2002). Expression of DmoesinT559A-GFP and DmoesinT559D-GFP consistently impairs the ability of endogenous Dmoesin to move upon illumination. Since ERM proteins are capable of homotypic interaction (usually as dimers, Dmoesin T559A and Dmoesin T559D can titrate either WT Dmoesin or other functional partners. Therefore, the light-induced degeneration observed upon Dmoesin T559A and Dmoesin T559D expression can be explained by this reduction of endogenous Dmoesin traffic (Chorna-Ornan, 2005).

Altogether, these findings indicate that the rhabdomeric localization of Dmoesin requires its open (active) state, which is achieved either by phosphorylation or by the T559D phosphomimetic mutation. The results, further, support that light-induced dephosphorylation triggers the movement of Dmoesin to the cytosol, and when this reaction is impaired by mutations, the light dependent movement of Dmoesin is blocked (Chorna-Ornan, 2005).

Recent studies have demonstrated reversible light-induced reorganization of the actin cytoskeleton of the microvilli and translocation of the Trpl channel (Bähner, 2002) from the rhabdomere to the cell body in time scales comparable to that of light-induced Dmoesin movement. Therefore, the light-induced movement of Dmoesin is likely involved in the control of the aforementioned processes (Chorna-Ornan, 2005).

Interestingly, genetic elimination of either signaling protein PLCß (norpA) or Trp prevents the light-induced movement of Dmoesin. These mutations are known to either block (norpA), or to strongly reduce (trp), the light-induced Ca²⁺ entry into the photoreceptor cells. The effect of light on Dmoesin movement could thus be mediated via Ca²⁺-induced dephosphorylation of Dmoesin; e.g., by activation of a Ca²⁺-dependent phosphatase. PLCß-mediated hydrolysis of PIP₂ (which is highly enriched in rhabdomere membranes) might also participate in the release of Dmoesin into the cytoplasm upon illumination, since its positive effect of PIP₂ binding on ERM protein activation, membrane localization, and binding to their partners has been demonstrated. The data accumulated in this study indicate the existence of a tight link between light reception and the Dmoesin-mediated reorganization of the rhabdomere cytoarchitecture (Chorna-Ornan, 2005).

Although the elucidation of the full spectrum of the physiological functions of the light-induced Dmoesin movement now awaits further works, it is suggested that these light-induced changes are necessary for the functional maintenance of photoreceptor cells. Photoreceptors are vulnerable cells because of their prolonged interaction with light. The peculiar organization of the rhabdomere in the form of very long (and tightly packed) microvilli makes it difficult for housekeeping mechanisms to operate in the rhabdomere. Light-activated reorganization of actin, along with cytoarchitectural changes, may allow the housekeeping function to operate and/or to participate in the down-regulation of signaling mechanisms triggered by light reception (Chorna-Ornan, 2005).

Activation of TRP channels by protons and phosphoinositide depletion in Drosophila photoreceptors

Phototransduction in microvillar photoreceptors is mediated via G protein-coupled phospholipase C (PLC), but how PLC activation leads to the opening of the light-sensitive TRPC channels (TRP and TRPL) remains unresolved. In Drosophila, InsP(3) appears not to be involved, and recent studies have implicated lipid products of PLC activity, e.g., diacylglycerol, its metabolites, or the reduction in PIP₂. The fact that hydrolysis of the phosphodiester bond in PIP₂ by PLC also releases a proton is seldom recognized and has neither been measured in vivo nor implicated previously in a signaling context. In this study, following depletion of PIP₂ and other phosphoinositides by a variety of experimental manipulations, the light-sensitive channels in Drosophila photoreceptors become remarkably sensitive to rapid and reversible activation by the lipophilic protonophore 2-4 dinitrophenol in a pH-dependent manner. It was further shown that light induces a rapid (<10 ms) acidification originating in the microvilli, which is eliminated in mutants of PLC, and that heterologously expressed TRPL channels are activated by acidification of the cytosolic surface of inside-out patches. These results indicate that a combination of phosphoinositide depletion and acidification of the membrane/boundary layer is sufficient to activate the light-sensitive channels. Together with the demonstration of light-induced, PLC-dependent acidification, this suggests that excitation in Drosophila photoreceptors may be mediated by PLC's dual action of phosphoinositide depletion and proton release (Huang, 2010).

These measurements of rapid, light-induced and PLC-dependent acidification represent the first evidence for in vivo, PLC-induced proton release in any system. Although additional sources from reactions operating downstream of PLC cannot be excluded, calculation suggests that the protons released by hydrolysis of PIP₂ are sufficient to account for the magnitude of the observed pH shift. It was also found that the lipophilic protonophore DNP rapidly and reversibly activates both TRP and TRPL channels, following phosphoinositide depletion in situ, whereas heterologously expressed TRPL channels can be activated not only by DNP but also by direct acidification of excised inside-out patches. Together, these results suggest an unexpected novel mechanism for microvillar phototransduction, namely that the channels might be gated by a combination of phosphoinositide depletion and protons (Huang, 2010).

As well as DNP, a variety of 'conventional' weak acids, which also cause cytosolic acidification, was tested. Propionic (20 mM) and benzoic (10 mM) acid failed to activate channels; however, octanoic acid (5 mM), which was the most lipophilic weak acid tested, induced modest TRPL channel activation following phosphoinositide depletion in trp mutants. Significantly, it was not possible to show activation of recombinant TRPL channels by protons without recourse to pharmacological agents in inside-out patches from S2 cells. However, the pH required to achieve robust activation was more acidic than the bulk pH changes induced by DNP or light in the photoreceptors. To reconcile these observations, it is suggested that a putative pH-sensitive site on the channels or associated proteins is close to the site of PIP₂ hydrolysis (and proton release) within the membrane or boundary layer and is relatively protected from the bulk pH of the aqueous phase. It is suggested that the particular efficacy of DNP relates to its lipophilic nature and to the fact that even the anionic (deprotonated) form is lipid soluble and adsorbs to the bilayer. This means that protonated DNP can ionize and donate protons (possibly by direct charge transfer) in the membrane or the boundary layer, whereas conventional weak acids only ionize and release protons in the aqueous phase. Consistent with this interpretation, carbonyl cyanide m-chlorophenylhydrazone, which is another lipophilic weak acid protonophore with broadly similar mode of action to DNP, also rapidly and potently activatea TRPL channels following phosphoinositide depletion. It is also noted that although the bulk light-induced pH shift was only 0.1-0.2 pH units, the results clearly show a pH gradient from the microvilli to cytosol. It seems likely that the immediate pH change in the membrane or boundary layer where PIP₂ is hydrolyzed may be yet greater. A further consequence of PIP₂ hydrolysis suggested by the results is a transient local increase in positive charge density due to negatively charged InsP3 being cleaved from the membrane. The possibility cannot be excluded that this might also contribute to excitation (Huang, 2010).

A range of manipulations that deplete PIP₂ and other phosphoinositides resulted in profound sensitization of the channels to activation by DNP. However, the mechanism by which phosphoinositide depletion results in sensitization to protonophores requires further study. Although TRPL channels have been reported to be inhibited by exogenous PIP₂, in the current experiments diC8PIP₂ robustly activated heterologously expressed TRPL channels in excised patches. At first sight, this seems difficult to reconcile with the in vivo data; however, the EC50 (1-2 mM PIP₂) probably corresponds to only ~5%-10% of endogenous resting PIP₂ levels and might reflect a constitutive requirement for PIP₂ unrelated to any role in activation. It is also noted that depletion of PIP₂ inevitably depletes the upstream phosphoinositide reserve pool (PI and PI₄P) and that diC8 analogs of these were effective inhibitors of heterologously expressed TRPL channels in inside-out patches. This raises the possibility that the knockon depletion of PI and PI(4)P by PI and PIP kinases, rather than PIP₂, may be a key event in channel activation. Another possibility is that the effects of PIP₂ and/or other lipids on channel activity might be mediated, not by ligand binding but by altering the properties of the lipid bilayer. In this respect, there are major differences between the lipid environment of TRP channels in situ (in signaling complexes within microvilli) and heterologously expressed channels in cell lines. For example, the depletion and resynthesis of phosphoinositides in the inner leaflet of the highly curved microvillar membrane may affect bilayer properties such as membrane stress curvature in a manner that cannot be readily mimicked in an expression system or by exogenous application of lipids (Huang, 2010).

A role for PIP₂ depletion in invertebrate phototransduction has been discussed previously, but the dominant hypothesis is that activation is mediated by DAG or downstream metabolites (PUFAs), such as arachidonic acid (AA), which are potent activators of TRP and TRPL channels. Recently, mutants in a DAG lipase (inaE) were found to have reduced sensitivity to light. However, INAE is an sn-1 lipase, which generates 2-arachidonoyl glycerol rather than AA, and it remains unclear whether it is directly involved in phototransduction. An excitatory role for DAG has been strongly suggested by the phenotypes of mutants in DAG kinase (rdgA), which include constitutive channel activity and the rescue of light responses in hypomorphic mutants of PLC or Gq, both of which could be interpreted in terms of increased production of DAG. Exogenous DAG has also recently been reported to activate channels, albeit slowly, in patches excised from rhabdomeres. However, in other studies DAG had little, if any, effect either in situ or on heterologously expressed TRPL channels. It is also noted that the product of DAG kinase, namely phosphatidic acid, is both a precursor for phosphoinositide resynthesis and an allosteric activator of PI₄)P-5-kinase, i.e., rdgA mutants may also be expected to have defects in the (rapid) resynthesis of PIP₂ . Nevertheless, given the emerging consensus that TRP channels are polymodally regulated, the possibility should also be considered that protons, phosphoinositide depletion, DAG, and/or PUFAs may all contribute to excitation (Huang, 2010).

These results suggest an unexpected new paradigm for phototransduction in Drosophila, namely that the channels may be activated combinatorially by the simultaneous reduction in PIP₂ and/or other phosphoinositides, combined with localized PLC-dependent proton release in the membrane boundary layer. Drosophila TRP and TRPL channels are the prototypical members of the TRPC family, widely expressed throughout the body. As is the case in Drosophila, TRPCs are gated downstream of PLC, but by unknown mechanisms. Although DAG can activate a subset of mammalian TRPCs, whether this action is direct is unclear, and even less is understood about the mechanism of activation of the DAG-insensitive TRPC4 and TRPC5. Interestingly, TRPC6, TRPC4, and TRPC5 have all recently been reported to be inhibited by PIP₂. The effects of cytosolic pH on any TRPC channels have yet to be investigated - though TRPC4 and TRPC5 were reported to be potentiated by extracellular acidification. Extracellular pH has also been reported to upregulate several members of the broader TRP channel family, including TRPV1, TRPV4, TRPP2, and TRPA1, but the only TRP channels previously reported to be regulated by cytosolic pH are TRPM7 and TRPM2, which were both inhibited by protons. It will be interesting to see whether any mammalian TRPC channels are coregulated by phosphoinositides and pH in a manner similar to that suggested in this study (Huang, 2010).

INAF, a protein required for transient receptor potential Ca(2+) channel function

In both vertebrates and invertebrates, ion channels of the TRP superfamily are known to be influenced by a variety of accessory factors, but the list of interacting proteins is acknowledged to be incomplete. Although previous work showed that Drosophila TRP function is disrupted by mutations in the inaF locus (Li, 1999), the mechanism of this effect has remained obscure. This study shows that a previously overlooked small protein, INAF-B, is encoded by the locus and fulfills its critical role in retinal physiology. The 81-aa INAF-B gene product is an integral membrane protein that colocalizes to rhabdomeres along with TRP channels. Immunoprecipitation experiments demonstrate that the two proteins participate in a complex, and blotting experiments show that neither protein survives in the absence of the other. Both proteins are normally part of a large supramolecular assembly, the signalplex, but their interaction persists even in the absence of the scaffold for this structure. The inaF locus encodes three other proteins, each of which has diverged from INAF-B except for a 32-aa block of residues that encompasses a transmembrane domain. This conserved sequence defines an inaF motif, representatives of which are found in proteins from organisms as diverse as nematodes, fish, and humans. Given the role of INAF-B, these proteins are good candidates for interacting partners of other members of the TRP superfamily (Cheng, 2007).

Translocation of the Drosophila transient receptor potential-like (TRPL) channel requires both the N- and C-terminal regions together with sustained Ca2+ entry

In Drosophila photoreceptors the transient receptor potential-like (TRPL), but not the TRP channels undergo light-dependent translocation between the rhabdomere and cell body. This study addressed which of the TRPL channel segments are essential for translocation and why the TRP channels are required for inducing TRPL translocation. Transgenic flies were generated expressing chimeric TRP and TRPL proteins that formed functional light-activated channels. Translocation was induced only in chimera containing both the N- and C-terminal segments of TRPL. Using an inactive trp mutation and overexpressing the Na(+)/Ca(2+) exchanger revealed that the essential function of the TRP channels in TRPL translocation is to enhance Ca(2+)-influx. These results indicate that motifs present at both the N and C termini as well as sustained Ca(2+) entry are required for proper channel translocation (Richter, 2011).

The physiological properties of cells are largely determined by a specific set of ion channels at the plasma membrane. Besides regulation at the gene expression level, trafficking of ion channels into and out of the plasma membrane has been established as an important mechanism for manipulating the number of channels at a specific cellular site. For instance, the translocation of AMPA-type glutamate receptors from endosomal membranes into the synapses of hippocampal and lateral amygdala neurons underlies the formation of long-term potentiation and is thus involved in associative learning. Regulation by controlled insertion and internalization of ion channels has also been studied for a number of vertebrate TRP channels and for the Drosophila TRPL channel. TRP channels function in sensory systems such as invertebrate photoreceptors, mechano-receptors, pheromone receptors, taste receptors, pain receptors or receptors for detection of hot and cold temperature, but also as regulators of ion homeostasis in non-neuronal cells. The TRP channel superfamily is classified into seven related subfamilies designated TRPC (canonical or classical), in which the Drosophila TRP and TRPL are members, TRPM (Melastatin), TRPN (NompC), TRPV (Vanilloid receptor), TRPA (ANKTM1), TRPP (Polycystin) and TRPML (Mucolipin). Regulated subcellular translocation has been reported for TRPV1 and TRPV2 that are translocated from an internal compartment to the plasma membrane upon hormonal stimulation with nerve growth factor, insulin-like growth factor-I, or neuropeptide head activator. The cell surface delivery of another TRPV channel, TRPV5, is stimulated by the serine protease tissue kallikrein in a protein kinase C-dependent manner and it is, in addition, dynamically controlled by extracellular pH. In the case of TRPC channels, epidermal growth factor induces rapid insertion of TRPC4 and TRPC5 into the plasma membrane. Despite these numerous examples, the mechanisms and the molecular determinants of vertebrate TRP channels translocation are only beginning to be understood (Richter, 2011 and references therein).

Another prominent example for ion channel translocation is the Drosophila TRPL channel. Together with TRP, TRPL is expressed in the photoreceptor cells of the compound eye where it becomes activated by a G-protein coupled, phospholipase C-mediated visual transduction cascade. In dark-raised flies TRPL is located, together with other components of the phototransduction cascade, in the microvillar photoreceptor membrane, which forms the rhabdomere along one side of the photoreceptor cell. Upon illumination, TRPL translocates from the rhabdomere into a storage compartment in the cell body at a time scale of hours. This translocation is a two-stage process in which TRPL is first transported to the base of the rhabdomeral membrane and to the adjacent stalk membrane followed by internalization into the cell body. This is performed by a transport pathway utilizing vesicular structures that also contain internalized rhodopsin. Subsequent dark adaptation of the flies results in redistribution of the stored TRPL channels back to the rhabdomeric membrane. The light-triggered internalization of TRPL depends on the activation of the phototransduction cascade and requires the presence of the second light-activated ion channel, TRP (Cronin, 2006; Meyer, 2006). In contrast to TRPL, TRP is located in the rhabdomere irrespective of the light conditions. Besides their translocation behavior, TRP and TRPL also differ in their electrophysiological properties. Studies have revealed that TRP is a highly Ca2+ selective ion channel with a reversal potential of about +13 mV at 1.5 mM external Ca2+ while TRPL is a nonselective cation channel with a reversal potential of about -4 mV. TRP and TRPL also differ in their single channel conductance, which was estimated from noise analysis to be about 4 pS for TRP and 35 pS for TRPL in physiological Ringer’s solution. These channels also differ in their susceptibility to the channel blocker La3+ that blocks TRP but not TRPL at micromolar concentrations. Therefore, the light-dependent translocation of TRPL alters the properties of the light-response of the photoreceptor cells (Richter, 2011).

The current study addressed the question: which segments of the TRPL protein are responsible for the observed light-triggered internalization of the ion channel. To this end, chimeric eGFP-tagged ion channels were generated composed of segments from TRP and TRPL. The light-dependent translocation of TRPL was found to require both the N- and C-terminal segments, indicating that this process is not mediated by a simple single internalization motif. To further establish the role of Ca2+ entry into photoreceptor cells for initiating TRPL translocation, mutants were used that affect cellular Ca2+ concentrations. TRPL translocation was found to be inhibited in these mutants, indicating that sustained Ca2+-influx is required for TRPL translocation. Electrophysiological characterization of the chimeric channels expressed on trpl³⁰²; trp^P343 double null background revealed functional channels with pore properties of either TRP or TRPL that were solely determined by the transmembrane region. All chimeras revealed a decline of the light response towards baseline during prolonged intense light, characteristic of the trpP343 mutant phenotype, thus limiting Ca2+-influx required for the normal translocation process (Richter, 2011).

DEVELOPMENTAL BIOLOGY / EFFECTS OF MUTATION

Recent studies suggest that the fly uses the inositol lipid signaling system for visual excitation and that the Drosophila transient receptor potential (trp) mutation disrupts this process subsequent to the production of IP3. trp is shown to encode a novel 1275 amino acid protein with eight putative transmembrane segments. Immunolocalization indicates that the Trp protein is expressed predominantly in the rhabdomeric membranes of the photoreceptor cells (Montell, 1989).

Phototransduction in Drosophila depends on Ca(2+)-release mediated signalling and TRP is essential for the normal function of this process

Invertebrate phototransduction is an important model system for studying the ubiquitous inositol-lipid signaling system. In the transient receptor potential (trp) mutant, one of the most intensively studied transduction mutants of Drosophila, the light response quickly declines to baseline during prolonged intense light. Using whole-cell recordings from Drosophila photoreceptors, the wild-type response is shown to be mediated by at least two functionally distinct classes of light-sensitive channels and both the trp mutation and a Ca2+ channel blocker (La3+) selectively abolish one class of channel with high Ca2+ permeability. Evidence is also presented that Ca2+ is necessary for excitation and that Ca2+ depletion mimics the trp phenotype. It is concluded that the recently sequenced Trp protein represents a class of light-sensitive channels required for inositide-mediated Ca2+ entry, and it is suggested that this process is necessary for maintained excitation during intense illumination in fly photoreceptors (Hardie, 1992).

Phototransduction in Drosophila is mediated by the ubiquitous phosphoinositide cascade, leading to opening of the TRP and TRPL channels, which are prototypical members of a novel class of membrane proteins. Drosophila mutants lacking the TRP protein display a response to light that declines to the dark level during illumination. It has recently been suggested that this response inactivation results from a negative feedback by calcium-calmodulin, leading to closure of the TRPL channels. It is also suggested that in contrast to other phosphoinositide-mediated systems, Ca2+ release from internal stores is neither involved in channel activation nor in phototransduction in general. This study shows that inactivation of the light response in Trp photoreceptors is enhanced upon reduction of the intracellular Ca2+ concentration. Furthermore, in Ca(2+)-free medium, when there is no Ca2+ influx into the photoreceptors, a significant elevation of intracellular Ca2+ is seen upon illumination. This elevation correlates with ability of the cells to respond to light. Accordingly, malfunctioning of Ca2+ stores, either by Ca2+ deprivation or by application of the Ca2+ pump inhibitor, thapsigargin, confers a trp phenotype on wild type flies. The results indicate that the response inactivation in trp cells results from Ca2+ deficiency rather than from Ca(2+)-dependent negative feedback. The results also indicate that there is light-induced release of Ca2+ from intracellular stores. Furthermore, the response to light is correlated to Ca2+ release, and normal function of the stores is required for prolonged excitation. It is suggested that phototransduction in Drosophila depends on Ca(2+)-release mediated signalling and that TRP is essential for the normal function of this process (Cook, 1999).

Photoreceptor degeneration caused by mutations of trp

The Drosophila trp gene encodes a light-activated Ca(2+) channel subunit that is a prototypical member of a novel class of channel proteins. Previously identified trp mutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. A new mutant, Trp(P365), is described that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TRP protein and rapid, semi-dominant degeneration of photoreceptors. In spite of its unusual phenotypes, Trp(P365) is a trp allele because a Trp(P365) transgene induces the mutant phenotype in a wild-type background, and a wild-type trp transgene in a Trp(P365) background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause the Trp(P365) phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels of Trp(P365) are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration in Drosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors in Drosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain (Yoon, 2000).

Single amino acid change in the fifth transmembrane segment of the TRP Ca2+ channel causes massive degeneration of photoreceptors

The trp gene encodes subunits of a highly Ca(2+)-permeable class of light-activated channels of Drosophila photoreceptors. The recently characterized mutation in this gene, Trp(P365), is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. A single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of Trp(P365). Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel. The following are some tentative conclusions from this study: (1) Trp(P365) is important for the regulation of TRP channel opening; (2) The particular amino acid residue preferred for this position appears to be different for different species (Phe for Drosophila and Leu for mammals) perhaps because of slightly different channel environments; (3) Ile appears to be particularly poorly tolerated at this position. It is concluded that the residues at positions 550 or in its immediate vicinity may be in a position to critically affect channel gating (Hong, 2002).

Dissecting independent channel and scaffolding roles of the Drosophila Trp: elimination of the anchoring function alone has minor effects on retinal morphology whereas disruption of channel function causes profound light-induced cell death

To identify new alleles of the trp locus, a screen was performed for the recently isolated collection of chemically induced third chromosome mutations, which display defects in the electroretinogram (ERG) recording (for details see T. Wang, 2005a). Exposure of wild-type flies to light results in two discriminable components in the ERG. These include a sustained corneal negative maintained component arising from responses of all retinal cells (photoreceptor cells and pigment cells) and on- and off-transients emanating from activity in the second-order neurons in the optic lobes. The classic trp phenotype is characterized by a transient response to light, resulting from rapid light-dependent inactivation of the remaining Trpl cation channel (T. Wang, 2005b).

Each of the third chromosome mutations was crossed to the strong trp^P343 allele and five were identified that failed to complement the recessive Trp phenotype: therefore, they represented new trp alleles. Four of the new trp alleles exhibited a transient ERG phenotype indistinguishable from trp^P343, whereas the phenotype of the fifth (trp¹⁴) was distinct in that the decline in the light response was much slower than in trp^P343 or other alleles isolated in this or previous studies. The trp¹⁴ phenotype was due to an autonomous defect in the photoreceptor cells, rather than the pigment cells, since the slower decline in the receptor potential was evident in single photoreceptor cells assayed by performing intracellular recordings (T. Wang, 2005b).

Currently, all of the existing loss-of-function mutations cause large reductions in protein levels, although the molecular lesions have not been defined. Among the extant trp alleles, the one with the strongest phenotype is trp^P343. The trp^P343 genomic region was sequenced and it was found that the Trp protein coding region was identical to wild type. Rather, there was a mutation in a conserved 5' splice site that was essential for mRNA splicing. The mutation presumably results in instability of the mRNA, since no trp^P343 mRNA is detected (Montell, 1989). Given the strong phenotype and lack of mRNA or Trp protein expressed in trp^P343 (Montell, 1989), this allele would appear to represent a null (T. Wang, 2005b).

To examine the levels of Trp protein expressed in the new alleles described in this study, Western blots were performed. Among those alleles that displayed a phenotype typical of trp^P343, three did not express any detectable Trp protein (trp³⁸, trp⁷⁴, and trp⁹²), whereas a fourth expressed very low levels of Trp (trp⁴⁷). This was in contrast to trp¹⁴ flies in which Trp was expressed at ~60% the level as in wild type. Other rhabdomeral proteins including INAD, INAC, NORPA, and Rh1 (rhodopsin 1) were expressed at comparable levels in trp¹⁴, trp^P343, and wild-type flies (T. Wang, 2005b).

To determine the molecular defects associated with the five new trp alleles, the genomic DNA was sequenced and the sequences were compared to that of the original isogenized stock used to conduct the mutagenesis. Among the four alleles that expressed very low or no detectable Trp, one had a frameshift mutation resulting in premature translation termination (trp⁹²), two had missense mutations (trp⁴⁷ and trp⁷⁴), and one had no mutation in the transcribed region and therefore may contain a mutation affecting the trp promoter. The allele (trp¹⁴) expressing Trp at 60% wild-type levels, and which exhibited a phenotype distinct from other trp alleles, had two missense mutations. One of these mutations changed an amino acid in the pore loop between transmembrane domains five and six (residue 612; leucine to phenylalanine), whereas the other was situated between the sixth transmembrane segment and a highly conserved sequence referred to as Trp box 1 (residue 671; arginine to glutamine) (T. Wang, 2005b).

Given that Trp has dual functions as a molecular anchor and as a cation channel (Li, 2000; Tsunoda, 2001), whether the Trp phenotype in trp¹⁴ was a consequence of perturbation of the anchoring role was considered. Drosophila compound eyes consist of ~800 repetitive units, referred to as ommatidia, each of which includes six outer photoreceptor cells (R1-6) and a central R7 or R8 cell in the distal region of the retina. Each photoreceptor cell contains a microvillar segment, the rhabdomere, where most of the proteins that function in phototransduction, such as the core members of the signalplex, are concentrated. These include Trp, protein kinase C (INAC), phospholipase C (NORPA), and INAD. Mutations that eliminate or disrupt the anchoring role of Trp result in mislocalization of these core members such that they are present in both the rhabdomeres and cell bodies (Chevesich, 1997; Tsunoda, 1997; Li, 2000; Tsunoda, 2001). However, other INAD binding partners, such as Rh1, do not depend on Trp for normal localization (Li, 2000). In trp¹⁴ photoreceptor cells, each of the core and other rhabdomeral proteins examined displayed a rhabdomere localization pattern indistinguishable from wild type. Consistent with these data, it was found that INAD coimmunoprecipitated effectively with the Trp¹⁴ protein. These data indicate that the Trp phenotype in trp¹⁴ flies is not due to an alteration in the Trp anchoring function. Rather, they raise the possibility that the phenotype is due to a defect in Trp channel function (T. Wang, 2005b).

In trp flies such as trp^P343 the transient potential ERG phenotype is a consequence of inactivation of the Trpl channel during constant light stimulation. However, in Trpl mutant flies, which express Trp but not Trpl, the ERG response is maintained during a typical 5-10-s light pulse. To examine Trp¹⁴ channel function independent of Trpl, the trp¹⁴ allele was introduced into a Trpl-null mutant (Trpl³⁰²) background. As previously shown, Trpl³⁰²;trp^P343 flies are blind, since they do not express Trpl or Trp (Niemeyer, 1996; Reuss, 1997). Flies harboring just the Trpl³⁰² mutation show a response to 10 s of light similar to wild type, because these flies express wild-type Trp. In contrast, Trpl³⁰²;trp¹⁴ flies displayed a Trp phenotype similar to trp¹⁴. These data indicate that the transient light response in trp¹⁴ flies is due to disruption of Trp channel function (T. Wang, 2005b).

To exclude the possibility that the trp¹⁴ phenotype is a consequence of mislocalization of Trp¹⁴, the mutant protein was spatially localized. Immunostaining experiments were performed and it was found that Trp¹⁴ was detected exclusively in the rhabdomeres, as is the case for wild-type Trp. Therefore, the transient light response in trp¹⁴ is not due to mislocalization of the Trp protein (T. Wang, 2005b).

Given that trp¹⁴ flies express a 40% lower concentration of Trp, whether a transient light response could be induced by expression of low levels of Trp was tested. Therefore, transgenic flies that expressed varying levels of wild-type Trp under the control of the heat-shock promoter (hs-trp) were generated and the transgene was placed in a Trpl³⁰²;trp^P343 genetic background. Even though use of the heat shock protein 70 (hsp70) promoter typically results in widespread expression, the Trp protein expressed under the control of the hsp70 promoter was found exclusively in the retina and not in the optic lobes or elsewhere in the adult head. To induce different low levels of Trp, the hs-trp flies were exposed to 30, 60, and 120 min heat-shock treatments, which resulted in the production of ~4%, 7%, and 10% of the wild-type levels of Trp, respectively. Expression of only ~10% the normal concentration of Trp restored an ERG in Trpl³⁰²;trp^P343 flies, which was not transient. Even 4% the normal levels of Trp did not cause a transient light response similar to trp¹⁴, although the amplitude of the ERG was reduced. These data indicate that the trp¹⁴ mutant phenotype was due to the mutation of the Trp¹⁴ protein, rather than simply due to expression of low levels or mislocalized Trp protein (T. Wang, 2005b).

Strong loss-of-function mutations in trp, such as in trp^P343, result in light dependent retinal degeneration. Considering Trp has dual channel and nonchannel roles, disruption of either function could potentially cause retinal degeneration. To address which of these two roles is more critical to prevent retinal cell death, the morphology was examined of trp^Delta1272 (Li, 2000: and trp¹⁴ ommatidia, which display specific defects in the scaffold and Trp channel functions, respectively. Wild-type ommatidia contain eight photoreceptor cell rhabdomeres, seven of which are present in any given plane regardless of their age or whether the flies were maintained in the dark or under a light-dark cycle (T. Wang, 2005b).

The retinal degeneration in trp¹⁴ and trp^P343 was much more severe than that in trp^Delta1272 flies. In both trp^P343 and trp¹⁴ flies, the rhabdomeres began to disappear between 7 and 10 d after eclosion and almost no rhabdomeres remained after 14 d of exposure to a 12-h light-12-h dark cycle. By contrast most trp^Delta1272 flies maintain a full complement of seven rhabdomeres after 14 d of a light-dark cycle. Nevertheless, the size of the rhabdomeres was typically smaller than in similarly aged wild-type and large intracellular vacuoles were present inside the cell bodies indicating that retinal degeneration had initiated. By 30 d after eclosion, most of the rhabdomeres in these flies had degenerated. Thus, the retinal degeneration is much more severe in trp^P343 and trp¹⁴ than trp^Delta1272, indicating that the Trp channel function rather than the scaffold function is more critical to prevent the retinal cell death (T. Wang, 2005b).

The retinal degeneration resulting from defects in Trp function was suppressed by maintaining the flies in the dark; this keeps the Trp channels in an inactive state. To assess the extent of suppression, the flies were maintained in the dark for 30 d, which was more than twice as long the 14-d light-dark cycle that caused elimination of almost all rhabdomeres in trp¹⁴ or trp^P343 flies. In dark-maintained trp^Delta1272 flies, seven rhabdomeres were present in ommatidia, although the size of the rhabdomeres was reduced. Indistinguishable results were obtained with trp^P343. The suppression of retinal degeneration was even more complete with trp¹⁴, as all ommatidia contained a full set of seven rhabdomeres of normal size. This result was striking since the retinal degeneration occurring under a light-dark cycle is significantly more rapid in trp¹⁴ than in trp^Delta1272 (T. Wang, 2005b).

To explore the mechanism underlying the retinal degeneration in trp mutants, a genetic approach was used. The combination of results described above indicates that a defect in Trp channel function underlies the retinal degeneration in trp flies. If the basis of the retinal degeneration in the trp mutant was due to diminished Ca²⁺ influx during light stimulation, then the cell death might be reduced by mutations in the Na⁺/Ca²⁺ exchanger, CalX, which functions in Ca²⁺ extrusion in photoreceptor cells (T. Wang, 2005b).

To test whether calx can suppress the retinal degeneration in trp^P343 and trp¹⁴, the morphology of calx^B,trp^P343, and calx^B,trp¹⁴ compound eyes was examined. trp¹⁴ or trp^P343 flies maintained under a light-dark cycle for 14 d displayed nearly complete loss of the rhabdomeres. The retinal degeneration in calx^B flies was even more severe; there were few rhabdomeres left after a 7 d light-dark cycle and almost no rhabdomeres left after a 14-d light-dark cycle. In contrast, most ommatidia from either calx^B,trp^P343 or calx^B,trp¹⁴ double mutant flies contained all the rhabdomeres after 14 d under a light-dark cycle. Moreover, the core signalplex proteins, NORPA, INAC, and INAD were mislocalized in calx^B,trp^P343 flies, indicating that introducing the calx mutation does not prevent loss of the Trp scaffold function in trp^P343. The effect of calx on trp was specific because the calx^B mutation did not suppress the cell death resulting from mutations in other phototransduction genes such as inaC, which encodes an eye-enriched PKC. These results indicated that the retinal degeneration in trp¹⁴ or trp^P343 is a consequence of decreased intracellular Ca²⁺ levels, whereas the photoreceptor cell death in the calx mutant results from Ca²⁺ overload (T. Wang, 2005b).

To explore further the mechanism of the retinal degeneration in trp¹⁴ and trp^P343, tests were made to see whether it could be suppressed by mutations in the gene encoding the major arrestin (arrestin2 -- arr2). Elimination of Arr2 reduces the retinal degeneration associated with certain mutations, such as norpA (disrupts phospholipase C), which prevents light-dependent activation of Trp channels. The retinal degeneration in norpA results from formation of stable rhodopsin-arrestin complexes and subsequent endocytosis of rhodopsin (T. Wang, 2005b). It was found that strong mutations in arr2 partially suppress the retinal degeneration in trp¹⁴ and trp^P343 flies. Whereas a 14-d exposure to a light-dark cycle results in extensive loss of rhabdomeres in arr2⁵,trp^P343 or trp¹⁴ eyes, most ommatidia in arr2⁵,trp^P343 or arr2⁵,trp¹⁴ double mutants contained seven rhabdomeres. However, the sizes of the rhabdomeres were significantly reduced (T. Wang, 2005b).

The two mutations in trp¹⁴ alter residues in the pore loop or immediately NH₂ terminal to Trp box 1 (residues 612 and 671, respectively). Given the potential effects of a pore-loop mutation on ion selectivity and the highly conserved nature of the region between the sixth transmembrane segment (TM6) and the Trp domain (Montell, 2005b), both of these mutations were in intriguing positions that could potentially be responsible for the trp¹⁴ phenotype (T. Wang, 2005b).

To determine whether one or both mutations were responsible for the transient light response phenotype in trp¹⁴, transgenic flies expressing Trp isoforms with just the L612F or R671Q amino acid substitution (trp^612F and trp^671Q, respectively) were generated and tested. The wild-type or mutant trp cDNAs were fused to the ninaE (neither inactivation nor afterpotential E [encodes Rh1]) promoter and introduced into the Trpl³⁰²;trp^P343 double mutant background. Subsequently Western blots were performed on the transgenic flies demonstrating that wild-type Trp, Trp^612F, and Trp^671Q were all expressed at similar levels (T. Wang, 2005b).

It was found that the missense mutation juxtaposed to the Trp domain was responsible for the phenotype in trp¹⁴. The Trpl;ninaE-trp^612F,trp^P343 flies displayed a wild-type ERG response indistinguishable from Trpl;ninaE-trp^wt,trp^P343. Conversely, the Trpl;ninaE-trp^671Q,trp^P343 flies showed a transient ERG phenotype. Moreover, the trp^671Q flies exhibited an ERG phenotype with a more rapid decline typical of trp^P343, suggesting that the 612F mutation resulted in a slight suppression of the Trp phenotype. These data demonstrate that the missense mutation at residue 671 situated between the sixth transmembrane domain and Trp box 1 (Montell, 2005b) was the key mutation responsible for the trp¹⁴ phenotype (T. Wang, 2005b).

The residues in Trpl and Trpgamma, corresponding to the required arginine 671 in Trp, are also basic amino acids (histidine 678 and arginine 662, respectively) suggesting that a basic residue at this position flanking the Trp domain is essential in the Drosophila TrpC channels. Therefore, transgenic flies were generated expressing derivatives of Trpl in which histidine 678 was replaced either with a conservative arginine substitution (Trpl^678R) or with an uncharged glutamine (Trpl^678Q). The mutant and wild-type Trpl cDNAs were fused to the ninaE promoter and introduced into a Trpl³⁰²;trp^P343 background. The transgenic Trpl proteins were all expressed at similar levels, though at an approximate sevenfold higher level than in wild-type due to the strong ninaE promoter (T. Wang, 2005b).

To determine the consequences of the mutations in Trpl, ERGs were performed after introducing the transgenes in a Trpl³⁰²;trp^P343 background. Whereas the double Trpl³⁰²;trp^P343 mutant was blind, overexpression of the wild-type Trpl in this genetic background (Trpl^wt) restored a transient response to light indistinguishable from trp^P343. Furthermore, introduction of the Trpl^678R transgene into the genome of Trpl³⁰²;trp^P343 flies resulted in an ERG response similar to Trpl^wt. Thus, replacing histidine 678 with an arginine did not disrupt Trpl function. However, the amplitude of the ERG was significantly reduced in Trpl³⁰²;trp^P343 flies expressing the Trpl^678Q transgene with the histidine to glutamine substitution in residue 678. The combination of these results demonstrates a critical role of a basic residue at the corresponding positions in Trp and Trpl, flanking the highly conserved Trp box 1 (T. Wang, 2005b).

Subcellular translocation of the eGFP-tagged TRPL channel in Drosophila photoreceptors requires activation of the phototransduction cascade

Signal-mediated translocation of transient receptor potential (TRP) channels is a novel mechanism to fine tune a variety of signaling pathways including neuronal path finding and Drosophila photoreception. In Drosophila phototransduction the cation channels TRP and TRP-like (TRPL) are the targets of a prototypical G protein-coupled signaling pathway. The TRPL channel translocates between the rhabdomere and the cell body in a light-dependent manner. This translocation modifies the ion channel composition of the signaling membrane and induces long-term adaptation. However, the molecular mechanism underlying TRPL translocation remains unclear. This study reports that eGFP-tagged TRPL expressed in the photoreceptor cells formed functional ion channels with properties of the native channels, allowing TRPL-eGFP translocation to be directly visualized in intact eyes. TRPL-eGFP failed to translocate to the cell body in flies carrying severe mutations in essential phototransduction proteins, including rhodopsin, Gαq, phospholipase Cß and the TRP ion channel, or in proteins required for TRP function. These data, furthermore, show that the activation of a small fraction of rhodopsin and of residual amounts of the Gq protein is sufficient to trigger TRPL-eGFP internalization. In addition, it was found that endocytosis of TRPL-eGFP occurs independently of dynamin, whereas a mutation of the unconventional myosin III, NinaC, hinders complete translocation of TRPL-eGFP to the cell body. Altogether, this study revealed that activation of the phototransduction cascade is mandatory for TRPL internalization, suggesting a critical role for the light induced conductance increase and the ensuing Ca²⁺-influx in the translocation process. The critical role of Ca²⁺ influx was directly demonstrated when the light-induced TRPL-eGFP translocation was blocked by removing extracellular Ca²⁺ (Meyer, 2006; full text of article).

Besides TRPL, at least two other proteins mediating Drosophila phototransduction, Arrestin 2 and the visual Gαq, undergo light-dependent translocation between the rhabdomere and the cell body. Likewise, in vertebrate photoreceptors arrestin and the visual G protein transducin translocate between the inner and outer segment in a light-dependent way. In both visual systems, arrestin and G protein movements occur in opposite directions, that is, in the light arrestin accumulates whereas the G protein is depleted in the photoreceptive membrane and vice versa in the dark. Accordingly, these light-dependent relocations of visual signaling proteins make the photoreceptor more sensitive in the dark and less sensitive in the light and mediate long-term adaptation of the Drosophila and vertebrate visual systems. A third protein that translocates in vertebrate photoreceptors is the Ca²⁺ binding protein recoverin (Meyer, 2006).

The mechanisms underlying these protein translocations have been elucidated in part for Drosophila arrestin, the Gαq subunit and for vertebrate transducin. Translocation of Drosophila arrestin from the cell body to the rhabdomere has been reported to require the ninaC-encoded myosin III which may actively transport arrestin along the actin cytoskeleton of the photoreceptor microvilli through PIP₃-enriched vesicles, to which arrestin binds. However, the requirement of the myosin III NINAC for arrestin translocation has been challenged in a more recent publication. Removal of arrestin from the rhabdomeral membranes in the dark does not require cytoskeletal elements and may thus occur passively. Likewise, Gαq translocation into the rhabdomere, but not its removal, is facilitated by the myosin III NINAC. Translocation of vertebrate transducin is aided by phosducin, an abundant photoreceptor-specific protein that binds to the ßγ subunits of transducin. Phosducin increases the solubility of the G protein subunits and may thereby facilitate transducin translocation (Meyer, 2006).

These mechanisms are markedly different from the mechanism underlying TRPL translocation because TRPL is a transmembrane protein that cannot enter the soluble fraction and needs to be removed from the rhabdomere by an endocytotic pathway, whereas arrestin and the visual G protein change from a membrane attached state to a soluble state. Therefore, elucidating the triggering mechanism of TRPL translocation reported in the present study is the first step for unraveling the mechanism underlying an important cellular process (Meyer, 2006).

DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses (Leung, 2008).

Visual transduction in Drosophila utilizes a G protein-coupled, phospholipase C-mediated signaling cascade. Phospholipase C, upon activation via rhodopsin and G protein, Gq, catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) into two potential second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP₃). A body of evidence suggests that IP₃ is not involved in Drosophila phototransduction, leaving the DAG branch as a likely source of messenger(s) of activation for the phototransduction channels, transient receptor potential (TRP) and TRP-like (TRPL). The mechanism by which the diacylglycerol (DAG) branch might activate the TRP/TRPL channels is still unresolved. The first indication that a lipid messenger might be involved was provided by Chyb (1999), who showed that polyunsaturated fatty acids (PUFAs) could activate both TRP and TRPL channels either in intact photoreceptors or heterologous expression systems. Later, evidence was provided that DAG is required for photoreceptor excitation using DAG kinase mutants, rdgA. Because the conversion of DAG to phosphatidic acid is blocked in these mutants, they should have an elevated DAG basal level. TRP/TRPL channels are constitutively active in rdgA, and diminished responses of hypomorphic PLC (norpA) mutants can be greatly enhanced by rdgA mutations, in support of the contention that DAG might be excitatory to the channels. However, rdgA mutations are expected to raise the basal levels of not only DAG but also its metabolites. In addition to these two molecules, phosphatidylinositol 4,5-bisphosphate (PIP₂) has also been suggested to play a role in channel excitation. Currently, no consensus exists as to which, if any, of these might be the excitatory agent for TRP/TRPL channels (Leung, 2008).

Drosophila TRP is the founding member of a superfamily of TRP channel proteins. There are now nearly 30 mammalian members of this superfamily comprising seven subfamilies. Although these channels are heterogeneous in their modes of activation, at least four mammalian TRP channels have been reported to be activated by DAG: TRPC2, -3, -6, and -7. While there may be variations in the mechanisms of activation of these channels, elucidation of Drosophila TRP/TRPL channel activation could provide insight into activation of these channels as well (Leung, 2008).

Because both DAG and its potential metabolite, PUFA, have been implicated in the activation of TRP/TRPL channels, a key enzyme in this process is likely to be DAG lipase, which catalyzes the hydrolysis of DAG. Little is known about DAG lipases. Two mammalian DAG lipase genes, DAGLα and -β, have been identified by a bioinformatics approach and characterized both biochemically and molecularly), and many proteins homologous to DAGα and -β have been identified across species. In the case of Drosophila, , rolling blackout (rbo) has been suggested to be in a DAG lipase gene. The protein encoded by the rbo gene, however, shows little homology to the known mammalian DAG lipases. Moreover, conditional loss of the RBO protein leads to rapid depletion of DAG, the opposite of what one would expect if RBO catalyzes the hydrolysis of DAG. Furthermore, in the absence of previous activity, the receptor potential is normal in rbo mutants, making it unlikely that RBO has any direct involvement in the activation of TRP/TRPL channels. Other than rbo, no candidate DAG lipase that might function in phototransduction has been reported in any species (Leung, 2008).

This work reports on a Drosophila DAG lipase (DAGL) gene, inaE, identified from mutants that are defective in photoreceptor responses to light. The protein isoforms encoded by this gene show high sequence similarity to the two known mammalian DAGLs, exhibit DAGL activity in vitro, are highly expressed in photoreceptors, and have access to rhabdomeres. Genetic evidence suggests that the inaE-encoded DAGLs interact in vivo with the DAG generated in the phototransduction cascade. Analysis of mutants generated by imprecise excision of P element insertion in inaE show that no physiologically meaningful photoreceptor responses can be generated if inaE gene is severely impaired (Leung, 2008).

The inaE gene was identified by two ethylmethane sulfonate (EMS)-induced allelic mutants: N125 and P19. These mutants are characterized by their 'ina' (inactivation, no afterpotential) electroretinogram (ERG) phenotype. Wild-type flies, when placed on a white-eye (w) background, respond to a bright blue stimulus with a large response during light stimulus followed by a prolonged depolarizing afterpotential (PDA) after the light is turned off. A second blue stimulus elicits only a small response, originating from R7/8 photoreceptors, superposed on the PDA. By contrast, in ina mutants, the response begins to decay during stimulus (inactivation), and the decay continues after the stimulus. As a result, the PDA is greatly diminished in amplitude (no afterpotential). This phenotype can also be viewed as a mild form of the 'trp' phenotype displayed by strong mutants of the TRP channel gene. In trp mutants, the response to the first blue stimulus decays nearly to baseline during stimulus, and there is no PDA (Leung, 2008).

Moreover, responses of inaE mutants resemble those of trp in that they both display refractory properties. Following a response to the first stimulus, only very small responses can be elicited from trp until they recover over a period of minutes, while wild-type responses recover almost immediately. Likewise, inaE mutants exhibit a similar refractory period, although the degree and duration of response suppression are not as pronounced or prolonged as in trp (Leung, 2008).

In addition to the above similarities, inaE^N125 acts as a genetic enhancer of Trp^P365. Trp^P365 is a semidominant allele of trp, which causes constitutive activation of TRP channels and, as a result, massive photoreceptor degeneration from excessive Ca²⁺ influx. In Trp^P365 homozygotes, degeneration is already so advanced in 1- to 2-day-old flies that essentially no ERGs can be elicited. Trp^P365 heterozygotes exhibit a much milder phenotype and elicit ERGs of substantial amplitude at the same age. However, if inaE^N125 is introduced into the Trp^P365/+ background, the resulting phenotype is as severe as that of Trp^P365 homozygotes. The genetic enhancement of Trp^P365/+ by inaE^N125 and the basic similarity of ERG phenotypes between inaE and trp mutants led to the hypothesis that the protein products of these two genes may interact and/or subserve closely related functions (Leung, 2008).

The CG33174 gene had not been characterized previously, and its function was electronically inferred as 'triacylglycerol lipase (TAGL) activity'. However, the possibility is considered that the above annotation could simply reflect a dearth of information on DAGLs. The first two human DAGLs, DAGLα and -β, were cloned and characterized by a bioinformatic approach and shown to be sn-1 type DAGLs. Multiple alignment of INAE-A and INAE-D with DAGLα and -β revealed extensive sequence and domain conservations. All four proteins are predicted to have four transmembrane segments near the N-terminal region, and they all have a lipase_3 domain with a highly conserved serine active site. Overall sequence homology between INAE-D and the two human DAGLs is 39% identity and 56% similarity for DAGLα and 30% identity and 50% similarity for DAGLβ, respectively. In the lipase_3 domain, the sequence homology between INAE-A/D and the mammalian proteins rises to 60% and 45% identity and 73% and 63% similarity for DAGLα and DAGLβ, respectively (Leung, 2008).

To demonstrate that the INAE proteins have DAG lipase activity, the INAE-A and -D protein isoforms were expressed in E. coli and purified to >95% purity to carry out DAG lipase assays using 1-stearoyl-2-arachidonoyl-sn-glycerol as substrate, and the lipase assay products were analyzed by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) for identification of hydrolysis products and kinetic studies (Leung, 2008).

LC-MS detected four products from the analysis of both INAE isoforms: two primary products, stearic acid (18:0) and 2-arachidonoyl glycerol (2-AG), and two minor products, arachidonic acid (20:4) and 1-stearoyl glycerol (1-SG), eluting at 6.3, 9.3, 5.8, and 8.7 min, respectively. Two primary products corresponded to hydrolysis at the sn-1 position of DAG substrate, and the two minor products corresponded to hydrolysis at the sn-2 position. Thus, in vitro, the two recombinant INAE isoforms are both DAG lipases highly preferential for hydrolysis at the sn-1 position, with the D form having much higher activity than the A form (Leung, 2008).

A growing body of evidence suggests that Drosophila phototransduction utilizes the DAG branch of the G protein-coupled, PLC_β-mediated signaling pathway). Although DAG lipase is expected to play a critical role in this pathway, no DAG lipase that could play such a role had been identified previously in Drosophila. This study reports on a DAG lipase identified from the Drosophila mutants, inaE. The inaE gene was found to encode two protein isoforms, INAE-A and INAE-D, by alternative splicing. Both of these proteins are highly homologous to the two previously identified mammalian sn-1 type DAG lipases, and in vitro DAG lipase assays of recombinant INAE-A and INAE-D showed that both are DAG lipases highly preferential for hydrolysis at the sn-1 position. Expression of the INAE protein is not restricted to the eye but occurs throughout the head, consistent with the finding that strong mutations in this gene are homozygous lethal. In photoreceptors, anti-INAE antibody labeling occurs as punctate staining scattered throughout the photoreceptor cytoplasm. Occasionally, some of the puncta are found within the rhabdomeres, indicating that some DAGL enters the rhabdomeres. Results of the norpA inaE double mutant study provide strong functional support for the above observation. In this study, the receptor potential disappears in an inaE allele-dependent manner -- the stronger the inaE allele in the double mutant, the more severe the double mutant phenotype. The allele dependence strongly suggests that the action of inaE-encoded DAGL is responsible for the observed double mutant phenotype. Furthermore, to affect the receptor potential phenotype, DAGL must act on the DAG generated by norpA-encoded PLC_β, and, for that to occur, DAGL must enter the rhabdomeres (Leung, 2008).

Because inaE mutations already available were all relatively mild, severe mutations were generated by imprecise excisions of a P element insertion in the inaE gene. These imprecise excision alleles were homozygous lethal and had to be studied as eye mosaics. Quantitative RT-PCR results showed that even the severest of these imprecise excision mutants, inaE^xl18, is not a null mutant and expresses RNA at ~25% of the normal level. This mutation profoundly affects the photoreceptor responses to light. If xl18 is placed on a norpA^H43 background to reduce the amount of DAG generated, the light stimulus generates no response at all. In xl18 flies themselves, a bright prolonged stimulus generates only a small response of slow kinetics that decays to baseline completely during the stimulus. This response most likely represents the residual DAGL activity in this severely affected mutant. As the severity of mutation progressively decreases in the xl series of mutants, the receptor potential phenotype returns to normal in an allele-dependent manner. Again, the inaE allele dependence strongly argues that the action of inaE-encoded DAGL is responsible for the observed change in the receptor potential phenotype. These results, taken together, suggest (1) that the production of DAG metabolite(s) through the action of the inaE-encoded DAGL is required for the generation of photoreceptor responses to light and (2) that, in the absence of the metabolite, DAG plays little direct role in the activation of channels. However, the identity of the excitatory molecule cannot be specified from this work. It could be one or more of the products generated by INAE, such as monoacylglycerol (2-AG) or stearic acid or even DAGL (INAE) itself (Leung, 2008).

While DAG may not have a direct role in channel activation, evidence was found suggesting that it may be important in regulating the action of the DAG metabolite that acts as an excitatory agent, although the evidence is still largely indirect. The ability of inaE^N125 to act as an enhancer of Trp^P365/+ seems to present a quandary when considered in relation to the results summarized above. If DAG has little or no direct role in channel activation, how does one explain the disappearance of the small response present in P365/+ when N125 is added to this background? A simple explanation for the phenomenon would be that DAG is excitatory to the channels and that adding N125 to the P365/+ background raised the level of DAG to make more channels to become constitutively active in N125;P365/+ than in P365/+. However, results of the experiment replacing N125 with a stronger inaE allele, xl18, in the N125;P365/+ double mutant run counter to this simple explanation. Replacing N125 with xl18 should have sharply raised the basal DAG level further in the double mutant. If DAG were excitatory, the resting potential should have depolarized even more than before the N125 replacement, and no receptor potential at all should have been obtained. Just the opposite results were obtained. A small but distinct receptor potential could be recorded from xl18;P365/+, and the resting potential has returned to the level in P365/+. These results are incompatible with the hypothesis that DAG is excitatory to the channel and instead provide another line of evidence for the conclusions summarized earlier (Leung, 2008).

However, the fact that a much more severe phenotype is obtained in N125;P365/+ than in P365/+ or xl18;P365/+ suggests that DAG may have a role in facilitating, enhancing, and orchestrating the action of the DAG metabolite that serves as the excitatory agent. This action of DAG would be more noticeable under conditions in which a sufficient amount of the excitatory product is produced, as in N125;P365/+ rather than in xl18;P365/+. A similar action of DAG can also be inferred from the norpA inaE double mutant studies. In this series of experiments, hypomorphic norpA mutation, H43, was used to restrict the amount of DAG generated both in the single and double mutants. The response obtained from H43 N125 is short in duration but has nearly the same maximum amplitude as the H43 response and much faster time course of rise than the H43 response. The shortness of response duration may be due to the fact that, under the conditions of this experiment (restricted DAG generation), the response cannot be sustained during a bright prolonged stimulus. This response arises as a result of DAGL activity because further reducing the DAGL activity (xl18 mutation) abolishes the response. However, the fact that adding N125 to the H43 background resulted in a response of faster time course may also be a manifestation of the enhancing and facilitatory effects of DAG on the excitatory agent. Speculating further, the facilitatory action of DAG might be in place to ensure that the excitation of channels is light regulated, because DAGL activity is not light regulated while the generation of DAG is (Leung, 2008).

Distinct TRP channels are required for warm and cool avoidance in Drosophila melanogaster

The ability to sense and respond to subtle variations in environmental temperature is critical for animal survival. Animals avoid temperatures that are too cold or too warm and seek out temperatures favorable for their survival. At the molecular level, members of the transient receptor potential (TRP) family of cation channels contribute to thermosensory behaviors in animals from flies to humans. In Drosophila larvae, avoidance of excessively warm temperatures is known to require the TRP protein dTRPA1. Whether larval avoidance of excessively cool temperatures also requires TRP channel function, and whether warm and cool avoidance use the same or distinct TRP channels has been unknown. This study identified two TRP channels required for cool avoidance, TRPL and TRP. Although TRPL and TRP have previously characterized roles in phototransduction, their function in cool avoidance appears to be distinct, as neither photoreceptor neurons nor the phototransduction regulators NORPA and INAF are required for cool avoidance. TRPL and TRP are required for cool avoidance; however they are dispensable for warm avoidance. Furthermore, cold-activated neurons in the larvae are required for cool but not warm avoidance. Conversely, dTRPA1 is essential for warm avoidance, but not cool avoidance. Taken together, these data demonstrate that warm and cool avoidance in the Drosophila larva involves distinct TRP channels and circuits (Rosenzweig, 2008).

As has been reported using RNAi, this study confirmed, using classical genetic mutations, that dTRPA1 is essential for larval warm avoidance. These findings were extended to show that cool avoidance does not require dTRPA1. Rather, it was discovered that cool avoidance depends on the TRPC family members TRPL and TRP. Although these two TRP channels also have critical functions in phototransduction, larval cool avoidance and phototransduction are distinct, since neither larval photoreceptors nor the phototransduction molecules NORPA and INAF are required for cool avoidance (Rosenzweig, 2008).

TRPL and TRP belong to the TRPC family of TRP channels. The TRPC family is evolutionarily conserved, with seven members in mammals. Although individual members of the TRPV, TRPM, and TRPA families are known to be temperature-activated ion channels, an involvement for TRPC proteins in mediating temperature perception has not been previously demonstrated, and it will be interesting to learn whether mammalian TRPCs also contribute to thermosensation. This study demonstrates a clear requirement for the Drosophila TRPCs TRPL and, to a lesser extent, TRP in cool avoidance. However, in contrast to classic thermoTRPs such as dTRPA1, which exhibits strong warmth activation when ectopically expressed in oocytes, neither TRPL nor TRP showed detectable cool activation in oocytes. In addition, whereas expression of TRPL and TRP was readily detected in the larval photoreceptors using RNA in situ hybridization, expression could not be detected in the putative cold receptor neurons of the terminal organ. Thus, whether these TRPC proteins participate directly in thermotransduction or affect thermosensory behavior by acting at a downstream step remains to be determined. Although the mechanism by which TRPL and TRP mediate cool avoidance is not clear, it appears distinct from the mechanisms by which TRPL and TRP channels mediate visual system signal transduction, since the latter rely on NORPA and INAF, which are dispensable for cool avoidance (Rosenzweig, 2008).

It was also found that that the neural pathways for cool and warm avoidance are distinct. Whereas the larval cold sensors, located in the terminal organ, are essential for larval cool avoidance, they are not necessary for warm avoidance. As for the larval warm sensors, a set of dTRPA1-expressing neurons in the brain has been implicated in warm avoidance in third instar larvae. Unfortunately, it is not yet technically possible to assess the function of these cells in cool avoidance, since available promoters for manipulating these dTRPA1-expressing neurons are expressed too late to effectively manipulate neuronal function in first and second instar larvae, the stages at which cold avoidance is most robust (Rosenzweig, 2008).

Together these data indicate that Drosophila use distinct TRP channels and neurons to respond to different, discrete ranges of temperature. The channels TRPL and TRP and the neurons of the terminal organ are specifically involved in the avoidance of cool temperatures, whereas dTRPA1- and dTRPA1-expressing neurons are required for the avoidance of moderately warm temperatures. At even higher temperatures, Painless mediates avoidance by acting in multiple-dendritic neurons, whereas Pyrexia has a potentially general neuroprotective effect possibly reflecting its broad neuronal expression. Thus, Drosophila possesses a suite of thermosensory detection pathways, each of which responds at specific temperatures and promotes a characteristic set of behavioral responses, ranging from gradual migration away from moderately warm or cool temperatures to immediate withdrawal from extreme temperatures that cause rapid tissue damage. As mammals also use distinct sensors for detecting different portions of the thermal spectrum, these studies support a fundamental similarity in the logic of thermosensation in both mammals and insects, with both types of animals sensing the range of temperatures they encounter using a series of TRPs and thermosensory cells, with different sensors tuned to different portions of the temperature spectrum (Rosenzweig, 2008).

Regulation of arrestin translocation by Ca2+ and myosin III in Drosophila photoreceptors

Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown. Using real-time fluorescent imaging of Arr2-green fluorescent protein translocation in dissociated ommatidia, this study shows that translocation is profoundly slowed in Ca(2+)-free solutions. Conversely, in a blind PLC mutant with ~100-fold slower translocation, rapid translocation was rescued by the Ca(2+) ionophore, ionomycin. In mutants lacking NINAC (calmodulin [CaM] binding myosin III) in the cell body, translocation remained rapid even in Ca(2+)-free solutions. Immunolabelling revealed that Arr2 in the cell body colocalizes with NINAC in the dark. In intact eyes, the impaired translocation found in trp mutants was rescued in ninaC;trp double mutants. Nevertheless, translocation following prolonged dark adaptation was significantly slower in ninaC mutants, than in wild type: a difference that was reflected in the slow decay of the electroretinogram. The results suggest that cytosolic NINAC is a Ca(2+)-dependent binding target for Arr2, which protects Arr2 from immobilization by a second potential sink that sequesters and releases arrestin on a much slower timescale. It is proposed that rapid Ca(2+)/CaM-dependent release of Arr2 from NINAC upon Ca(2+) influx accounts for the acceleration of translocation by phototransduction (Hardie, 2012a).

Photoreceptors are highly polarized cells with membrane-rich photosensory compartments separated from the rest of the cell body. It has recently become widely recognized that several phototransduction proteins translocate between these compartments in response to light, representing a form of long-term light and dark adaptation. One of the best-studied examples is arrestin, which terminates the light response by binding to photo-isomerized rhodopsin (metarhodopsin). In dark-adapted photoreceptors most arrestin localizes to the cell body in both vertebrate and insect photoreceptors, but on illumination translocates to the photosensory compartmen. In fly photoreceptors the photosensory compartment is represented by the rhabdomere, a light-guiding, rod-like stack of ~30,000 densely packed apical microvilli loaded with rhodopsin and proteins of a phototransduction cascade mediated by heterotrimeric Gq protein, phospholipase C (PLC), and Ca²⁺-permeable 'transient receptor potential' (TRP) channels (Hardie, 2012a).

Although the possible role of active transport by molecular motors remains debated, recent evidence in both vertebrate rods and Drosophila microvillar photoreceptors supports an essentially passive diffusional model of arrestin translocation, down gradients established by light-regulated 'sinks'. Recent studoes provided evidence that metarhodopsin (M) is the major light-activated sink in fly rhabdomeres by showing that the dominant arrestin isoform (Arr2) translocated in a 1:1 stoichiometric relationship to the number of rhodopsin photo-isomerizations (Satoh, 2010). This study also showed that Arr2 translocation was very rapid (τ ~10 s), but profoundly slowed in mutants of various phototransduction proteins including Gq, phospholipase C (PLC) (norpA), and the major Ca²⁺-permeable TRP channel (Satoh, 2010). The evidence suggested that Ca²⁺ influx via the light-sensitive channels was required to accelerate Arr2 translocation, possibly by releasing Arr2 from a Ca²⁺-dependent cytosolic sink (Satoh, 2010). However, direct evidence for the role of Ca²⁺ was lacking, while the identity of the putative cytosolic sink and the mechanism(s) mediating the acceleration remained unresolved (Hardie, 2012a).

This study shows directly that Ca²⁺ is both necessary and sufficient to accelerate Arr2 translocation and provides evidence that the Ca²⁺-regulated cytosolic sink is the cytosolic isoform of NINAC, a calmodulin (CaM) binding myosin III. The evidence also suggests the existence of another potential Ca²⁺ dependent cytosolic sink, which sequesters and releases arrestin on a much slower timescale, and that NINAC protects Arr2 from sequestration and immobilization by this site. The data support a mechanism for the Ca²⁺-dependent translocation of Arr2 that is remarkably similar to a previously proposed disinhibitory mechanism of Ca²⁺-dependent inactivation of M (Liu, 2008) required for rapid termination of the light response (Hardie, 2012a).

Previously studies have proposed that Ca²⁺ influx via the light-sensitive TRP channels is required for rapid Arr2 translocation, because the slow translocation in trp mutants could be rescued by genetic elimination of Na⁺/Ca²⁺ exchanger activity (Satoh, 2010). The present study confirmed the role of Ca²⁺ directly by imaging Arr2-GFP translocation in dissociated ommatidia, and showing that extracellular Ca²⁺ is required for rapid Arr2 translocation. Ca²⁺ was not only required, but also sufficient to enable rapid translocation without any products of PLC activity, since the Ca²⁺ ionophore, ionomycin, fully rescued translocation in blind norpA mutants lacking PLC. Significantly, it was found that the requirement of Ca²⁺ for rapid translocation was obviated in null mutants of NINAC (CaM binding MyoIII), with the cytosolic p132 isoform of NINAC alone being sufficient to slow down translocation in Ca²⁺-free conditions. This suggests that cytosolic NINAC p132 acts as a Ca²⁺/CaM-dependent 'brake' on translocation by binding Arr2, releasing it in response to Ca²⁺ influx associated with the photoresponse. This conclusion was further supported by finding that the ninaC mutation rescued rapid translocation in trp mutants (in ninaC;trp double mutants). However, the failure to rescue translocation in norpA;ninaC mutants, except under special conditions, and the demonstration of significant slowing of translocation following prolonged dark adaptation in ninaC mutants also indicated the existence of a second Ca²⁺-dependent cytosolic sink (Hardie, 2012a).

Despite an earlier study reporting that Arr2 translocation was impaired in ninaC mutants, as shown here and previously (Satoh, 2005; Satoh, 2010), Arr2 translocation, whether of endogenous Arr2 or GFP-tagged Arr2, appears essentially intact in ninaC-null mutants. In fact, far from being impaired, the results indicate that translocation can be rescued by ninaC mutations under conditions where translocation is slowed down by reduced Ca²⁺ influx. Although translocation was significantly slower in ninaC mutants following prolonged dark adaptation, it was never prevented and full translocation was always achieved within ~2-3 min of appropriate illumination (Hardie, 2012a).

A novel phenotype of ninaC^P235-null mutants, and also ninaC^Δ174 mutants lacking only the rhabdomeric p174 isoform, was the complete absence of an early rapid increase in fluorescence routinely observed during the first ~500 ms of measurements of Arr2-GFP fluorescence from the DPP of wild-type photoreceptors or dissociated ommatidia. With a time constant of ~260 ms, this rapid phase was ~40× faster than the overall translocation (τ ~10 s) itself the fastest protein translocation reported in a photoreceptor to date, and probably diffusionally limited (Satoh, 2010). It therefore seems unlikely that the fast phase represents a 40x faster, ninaC-dependent movement of Arr2 from cell body into the rhabdomere. Instead we suggest that it represents a change in the fluorescence efficiency of Arr2-GFP as it is released (via Ca²⁺ influx) from the rhabdomeric p174 NINAC isoform. A lower fluorescence when bound to NINAC might reflect crowding of the GFP-fluorophore, or could be due to some other feature of the nano-environment of the fluorophore when Arr2 is bound to NINAC in the microvilli. This interpretation was supported by the ability to eliminate the rapid phase by pre-illumination with long wavelength light, which induces Ca²⁺ influx without net change in M. The rapid phase then re-emerged with a time constant of ∼3 s in the dark, presumably representing rapid rebinding of Arr2-GFP to NINAC (Hardie, 2012a).

After more than a few minutes in the dark, Arr2 translocation into the rhabdomere became progressively slower, with clear functional consequences in a parallel slowing of the decay of the ERG. This gradual slowing was considerably more pronounced in ninaC-null mutants, where it si proposed that the slowing represents binding or sequestration of Arr2 via one or more NINAC-independent target(s) or compartment(s). Release from such sites also requires activation of the phototransduction cascade, and translocation could be accelerated back to levels typical of short dark-adaptation times by pre-illumination with bright orange light, which itself does not generate a net increase in M. It seems likely that the rise in Ca²⁺ is also responsible for release from this site; however, the involvement of other products of the phototransduction cascade cannot be excluded. The identity of this second site or compartment remains a subject for future investigation. Given previous reports that Arr2 can bind to phosphoinositides, negatively charged phosphoinositide species on endomembranes, which could be screened by Ca²⁺, might represent promising candidates. Drosophila Arr2 is an unusually basic (positively charged) protein and may thus have a strong tendency to bind to such sites. The finding that the slowing of translocation with dark adaptation was more pronounced in ninaC mutants suggests that one of the functions of cytosolic NINAC may be to prevent immobilization of Arr2 by this alternative potential sink. Because the Ca²⁺-dependent release of Arr2 from NINAC occurs on a subsecond timescale, this then allows more rapid translocation (and hence recovery of the electrical response) after a period in the dark (Hardie, 2012a).

These results demonstrate that Arr2 translocation is accelerated by Ca²⁺ influx, and suggest that this is mediated by a disinhibitory mechanism, whereby NINAC p132 binds to Arr2 under low Ca²⁺ conditions in the dark, rapidly releasing it in response to Ca²⁺ influx associated with the photoresponse. Although inferred from essentially independent experiments, this mechanism is strikingly similar to one previously proposed for the rapid, Ca²⁺-dependent inactivation of M during the light response itself (Liu, 2008). In that study the time constant of M inactivation by Arr2 was found to be accelerated from ~200 ms under Ca²⁺-free conditions to ~20 ms following Ca²⁺ influx. This Ca²⁺ dependence was eliminated in both ninaC-null mutants, and in ninaC^Δ174 mutants lacking the rhabdomeric p174 (but not in ninaC^Δ132 mutants lacking cytosolic p132). The results also indicated a disinhibitory mechanism, leading to the proposal that Arr2 in the microvilli was bound to rhabdomeric NINAC p174 under low Ca²⁺ conditions in the dark, thus hindering its diffusional access to activated M. Ca²⁺ influx via the first activated TRP channels, then rapidly releases Arr2, allowing it to diffuse, bind to, and inactivate M (Hardie, 2012a).

NINAC p132 and p174 share a common CaM binding site (CBS) and although p174 has a second CBS not found in p132 (Porter, 1993; Porter, 1995), the pronounced slowing of translocation with dark adaptation in null ninaC^P235 mutants was recapitulated in mutants lacking the common CBS. It is therefore suggested that essentially the same mechanism underlies the Ca²⁺-dependent rapid translocation of Arr2, but now acting via NINAC p132 rather than p174 and working over much larger distances (several micrometers as opposed to the nanometer dimensions of single microvilli) and hence slower timescales (Hardie, 2012a).

The proposed interaction between NINAC and Arr2 finds some support from biochemical data reporting coimmunoprecipitation of Arr2 and NINAC in extracts from whole heads (Lee, 2004). However, that study also reported that both Arr2 and NINAC had significant in vitro affinity for phosphoinositides. It was proposed that the NINAC/Arr2 association was indirect and mediated by both Arr2 and NINAC binding to phosphoinositide-rich membrane. Although the current results clearly indicate that Ca²⁺-dependent modulation of Arr2 binding to M (Liu, 2008) and Ca²⁺-dependent translocation of Arr2 are both dependent upon NINAC, the possibility cannot be excluded that the interaction is mediated indirectly via a NINAC-dependent target. Ultimate verification will require direct demonstration of NINAC/Arr2 binding and its dependence upon Ca²⁺/CaM (Hardie, 2012a).

The regulated multisink model proposed in this study differs fundamentally from an earlier model in which NINAC was proposed as a molecular motor transporting Arr2 in phosphoinositide-rich vesicles (Lee, 2004). By contrast it shows strong parallels with current models for arrestin translocation in vertebrate rods (Calvert, 2006; Slepak, 2008). Here, phosphorylated rhodopsin represents the light-activated sink in the outer segments, while microtubules have been proposed as the cytosolic sink in the inner segments. There is also evidence indicating light-regulated acceleration of translocation in vertebrate rods. The mechanism is unclear; however, intriguingly a recent study has implicated roles for PLC and protein kinase C possibly stimulating release of arrestin from its cytosolic sink (Hardie, 2012a and references therein).

Such regulated-sink models have the advantage of simplicity: directed translocation requires no more than diffusion coupled with regulated binding, can rapidly transport virtually unlimited quantities of protein, and per se consumes essentially no energy. While it can be conveniently studied in photoreceptors with their distinctive polarized morphologies and high concentrations of transduction machinery, translocation according to the same general principles may represent a general and elegant solution to the problem of directed movements of signaling proteins (Hardie, 2012a).

Common mechanisms regulating dark noise and quantum bump amplification in Drosophila photoreceptors

Absolute visual thresholds are limited by 'dark noise,' which in Drosophila photoreceptors is dominated by brief (~10 ms), small (~2 pA) inward current events, occurring at ~2/s, believed to reflect spontaneous G protein activations. These dark events were increased in rate and amplitude by a point mutation in myosin III (NINAC), which disrupts its interaction with the scaffolding protein, INAD. This phenotype mimics that previously described in null mutants of ninaC (no inactivation no afterpotential; encoding myosin III) and an associated protein, retinophilin (rtp). Dark noise was similarly increased in heterozygote mutants of diacylglycerol kinase (rdgA/+). Dark noise in ninaC, rtp, and rdgA/+ mutants was greatly suppressed by mutations of the G_q α-subunit (Gαq) and the major light-sensitive channel (trp) but not rhodopsin. ninaC, rtp, and rdgA/+ mutations also all facilitated residual light responses in Gαq and PLC hypomorphs. Raising cytosolic Ca²⁺ in the submicromolar range increased dark noise, facilitated activation of transient receptor potential (TRP) channels by exogenous agonist, and again facilitated light responses in Gαq hypomorphs. These results indicate that RTP, NINAC, INAD, and diacylglycerol kinase, together with a Ca²⁺-dependent threshold, share common roles in suppressing dark noise and regulating quantum bump generation; consequently, most spontaneous G protein activations fail to generate dark events under normal conditions. By contrast, quantum bump generation is reliable but delayed until sufficient G proteins and PLC are activated to overcome threshold, thereby ensuring generation of full-size bumps with high quantum efficiency (Chu, 2013).

EVOLUTIONARY HOMOLOGS

Drosophila TRP gamma

Cellular calcium homeostasis is regulated by hormones and neurotransmitters, resulting in the activation of a variety of proteins, in particular, channel proteins of the plasma membrane and of intracellular compartments. Such channels are, for example, TRP channels of the TRPC protein family that are activated by various mediators from receptor-stimulated signaling cascades. In Drosophila, two TRPC channels, TRP and TRPL, are involved in phototransduction. In addition, a third Drosophila TRPC channel, TRPgamma, has been identified and described as an auxiliary subunit of TRPL. The current data show that heterologously expressed TRPgamma forms a receptor-activated, outwardly rectifying cation channel independent from TRPL co-expression. Analysis of the activation mechanism revealed that TRPgamma is activated by various polyunsaturated fatty acids generated in a phospholipase C- and phospholipase A(2)-dependent manner. The most potent activator of TRPgamma, the stable analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid, induced currents in single channel recordings. Upon heterologous expression TRPgamma forms a homomeric channel complex that is activated by polyunsaturated fatty acids as mediators of receptor-dependent signaling pathways. Reverse transcription PCR analysis showed that TRPgamma is expressed in Drosophila heads and bodies. Its body-wide expression pattern and its activation mechanism suggest that TRPgamma forms a fly cation channel responsible for the regulation of intracellular calcium in a variety of hormonal signaling cascades (Jors, 2006).

TRP channels in other invertebrates

Transient receptor potential (TRP) channels mediate light-induced Ca(2+) entry and the electrical response in Drosophila photoreceptors. The role of TRP channels in other invertebrate photoreceptors is unknown, particularly those, exemplified by Limulus ventral eye photoreceptors, in which calcium release from intracellular stores is prominent. cDNA encoding three variants of a Limulus TRP channel were amplified. LptrpA and LptrpB encode proteins of 896 and 923 amino acids, that differ by a 27 amino acid insert within the C-terminus. LptrpC encodes an alternative 63 amino acid sequence in the pore domain compared with LptrpB. LptrpB and LptrpC are present in ventral eye mRNA, while LptrpA is only present in brain mRNA. In situ hybridization indicates the presence of Lptrp in photoreceptors of the Limulus ventral eye. Some canonical TRP channels can be activated by diacylglycerol analogs. Injection of a diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), into Limulus photoreceptors can activate an inward current with electrical characteristics similar to the light-activated current. However, simultaneous elevation of cytosolic calcium concentration appears to be necessary. Illumination attenuates the response to OAG injections and vice versa. These results provide molecular and pharmacological evidence for a TRP channel in Limulus ventral eye that may contribute to the light-sensitive conductance (Bandyopadhyay, 2004).

Apoptotic cells undergo a series of morphological changes. These changes are dependent on caspase cleavage of downstream targets, but which targets are significant and how they facilitate the death process are not well understood. In Caenorhabditis elegans an increase in the refractility of the dying cell is a hallmark morphological change that is caspase dependent. This study identified a presumptive transient receptor potential (TRP) cation channel, CED-11, that acts in the dying cell to promote the increase in apoptotic cell refractility. CED-11 is required for multiple other morphological changes during apoptosis, including an increase in electron density as visualized by electron microscopy and a decrease in cell volume. In ced-11 mutants, the degradation of apoptotic cells is delayed. Mutation of ced-11 does not cause an increase in cell survival but can enhance cell survival in other cell-death mutants, indicating that ced-11 facilitates the death process. In short, ced-11 acts downstream of caspase activation to promote the shrinkage, death, and degradation of apoptotic cells (Driscoll, 2017).

Subcellular localization of TRP channels

Ca(2+) influx via plasma membrane Trp3 channels is proposed to be regulated by a reversible interaction with inositol trisphosphate receptor [IP(3)R] in the endoplasmic reticulum. Condensation of the cortical actin layer has been suggested to physically disrupt this interaction and inhibit Trp3-mediated Ca(2+) influx. This study examines the effect of cytoskeletal reorganization on the localization and function of Trp3 and key Ca(2+) signaling proteins. Calyculin-A treatment resulted in formation of condensed actin layer at the plasma membrane; internalization of Trp3, Galpha(q/11), phospholipase Cbeta, and caveolin-1, and attenuation of 1-oleoyl-2-acetyl-sn-glycerol- and ATP-stimulated Sr(2+) influx. Importantly, Trp3 and IP(3)R-3 remained co-localized inside the cell and were co-immunoprecipitated. Jasplakinolide also induced internalization of Trp3 and caveolin-1. Pretreatment of cells with cytochalasin D or staurosporine did not affect Trp3 but prevented calyculin-A-induced effects. Based on these data, it is suggest that Trp3 is assembled in a caveolar Ca(2+) signaling complex with IP(3)R, SERCA, Galpha(q/11), phospholipase Cbeta, caveolin-1, and ezrin. Furthermore, the data demonstrate that conditions that stabilize cortical actin induce loss of Trp3 activity due to internalization of the Trp3-signaling complex, not disruption of IP(3)R-Trp3 interaction. This suggests that localization of the Trp3-associated signaling complex, rather than Trp3-IP(3)R coupling, depends on the status of the actin cytoskeleton (Lockwich, 2001).

Receptor-coupled [Ca2+]i increase is initiated in the apical region of epithelial cells and has been associated with apically localized Ca2+-signaling proteins. However, localization of Ca2+ channels that are regulated by such Ca2+-signaling events has not yet been established. This study examines the localization of TRPC channels in polarized epithelial cells and demonstrates a role for TRPC3 in apical Ca2+ uptake. Endogenously and exogenously expressed TRPC3 localizes apically in polarized Madin-Darby canine kidney cells (MDCK) and salivary gland epithelial cells. In contrast, TRPC1 localizes basolaterally, whereas TRPC6 is detected in both locations. Localization of Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and phospholipase Cbeta1 and -beta2 is also predominantly apical. TRPC3 co-immunoprecipitates with endogenous TRPC6, phospholipase Cbetas, Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and syntaxin 3 but not with TRPC1. Furthermore, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-stimulated apical 45Ca2+ uptake is higher in TRPC3-MDCK cells compared with control (MDCK) cells. Bradykinin-stimulated apical 45Ca2+ uptake and transepithelial 45Ca2+ flux are also higher in TRPC3-expressing cells. Consistent with this, OAG induces [Ca2+]i increase in the apical, but not basal, region of TRPC3-MDCK cells that is blocked by EGTA addition to the apical medium. Most importantly, (1) TRPC3 is detected in the apical region of rat submandibular gland ducts, whereas TRPC6 is present in apical as well as basolateral regions of ducts and acini; and (2) OAG stimulates Ca2+ influx into dispersed ductal cells. These data demonstrate functional localization of TRPC3/TRPC6 channels in the apical region of polarized epithelial cells. In salivary gland ducts this could contribute to the regulation of salivary [Ca2+] and secretion (Bandyopadhyay, 2005).

Protein interactions of TRP family members

Mammalian homologues of Drosophila Trp have been implicated to form channels that are activated following the depletion of Ca(2+) from internal stores. Recent studies indicate that actin redistribution is required for the activation of these channels. Murine Trp4 and Trp5, as well as phospholipase C beta1 and beta2 interact with the first PDZ domain of NHERF, regulatory factor of the Na+/H+ exchanger. The association of Trp4 and phospholipase C-beta1 with NHERF in vivo in an HEK293 cell line expressing Trp4 and in adult mouse brain has been demonstrated by immuno-coprecipitation. NHERF is a two PDZ domain-containing protein that associates with the actin cytoskeleton via interactions with members of ezrin/radixin/moesin family. Thus, store-operated channels involving Trp4 and Trp5 can form signaling complexes with phospholipase C isozymes via interactions with NHERF, thereby linking the lipase and the channels to the actin cytoskeleton. The interaction with the PDZ protein may constitute an important mechanism for distribution and regulation of store-operated channels (Tang, 2000).

Mammalian homologs of the Drosophila TRP protein have been shown to form cation-permeable channels in the plasma membrane but very little is known about the mechanisms that control their cell surface localization. Recently it has been demonstrated that the last three C-terminal amino acids (TRL) of TRPC4 comprise a PDZ-interacting domain that binds to the scaffold protein EBP50 [ezrin/moesin/radixin-binding phosphoprotein 50]. In this report, the influence of the TRL motif on the subcellular distribution of TRPC4 was examined in human embryonic kidney (HEK) 293 cells. The consequences of the interaction between EBP50 and the membrane-cytoskeletal adaptors of the ezrin/radixin/moesin (ERM) family was examined for the cell surface expression of TRPC4. Using immunofluorescence microscopy, it was found that the mutant lacking the TRL motif accumulated into cell outgrowths and exhibited a punctate distribution pattern whereas the wild-type channel was evenly distributed on the cell surface. Deletion of the PDZ-interacting domain also decreased the expression of TRPC4 in the plasma membrane by 2.4-fold, as assessed by cell surface biotinylation experiments. Finally, in a large percentage of cells co-expressing TRPC4 and an EBP50 mutant lacking the ERM-binding site, TRPC4 was not present in the plasma membrane but co-localized with the truncated scaffold in a perinuclear compartment (most probably representing the Golgi apparatus) and in vesicles associated with actin filaments. The data demonstrate that the PDZ-interacting domain of TRPC4 controls its localization and surface expression in transfected HEK293 cells. They also point to a yet unexplored role of the EBP50-ERM complex in the regulation of protein insertion into the plasma membrane (Merv, 2002).

TRPC1-7 proteins are members of a family of mammalian non-specific cation channels that mediate receptor-operated, phospholipase Cbeta/Cgamma dependent Ca(2+) influx in various cell types. TRPC4 and TRPC5 form a subfamily within TRPCs. Uniquely in the TRPC family, these channels possess a C-terminal 'VTTRL' motif that binds to PDZ-domains of the scaffolding protein, EBP50 (NHERF1). The functional effects of EBP50 on TRPC4/5 activity have not been investigated. Rat TRPC5 (rTRPC5) was cloned, functionally expressed in HEK293 cell, and channel regulation was studied with patch-clamp techniques. Both rTRPC5 and its VTTRL deletion mutant (r5dV) localize to the plasma membrane. rTRPC5 does not display any significant basal activity in unstimulated HEK293 cells. In cells co-expressing rTRPC5 and H1 histamine receptor, rTRPC5 current evoked by GTPgammaS or histamine develops in two phases: a slowly developing, small inward current followed by a rapidly developing, transient, large inward current. Each phase has a characteristic non-linear current-voltage (I-V) relationship. Deletion of the VTTRL motif has no detectable effect on the biophysical properties of the channel. Co-expression of EBP50 with rTRPC5 causes a significant delay in the time-to-peak of the histamine-evoked, transient large inward current. EBP50 does not modify the activation kinetics of the VTTRL-deletion mutant. It is concluded that the VTTRL motif is not necessary for activation of TRPC5, but may mediate the modulatory effect of EBP50 on TRPC5 activation kinetics (Obukhov, 2004).

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca(2+) store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. Two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation is mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induces the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and is accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na+/H+ exchanger regulatory factor. Furthermore, this translocation and association is defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs is concomitant with a reduction in Ca(2+) influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function (Odell, 2005).

Mammalian TRPC channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs reveals the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, it was found that the second ankyrin-like repeat domain of TRPC6 interacts with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, it was shown that MxA interacts with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced either agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacts specifically with the second ankyrin-like repeat domain of TRPCs and suggests that monomeric MxA regulates the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulates the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling (Lussier, 2005).

The acrosome reaction, the first step of the fertilization, is induced by calcium influx through TRPC channels. The molecular nature of TRPC involved is still a debated question. In mouse, TRPC2 plays the most important role and is responsible for the calcium plateau. However, TRPC1 and TRPC5 are also localized in the acrosomal crescent of the sperm head and may participate in calcium signaling, especially in TRPC2-deficient mice. Activation of TRPC channels is an unresolved question in germ and somatic cells as well. In particular, in sperm, little is known concerning the molecular events leading to TRPC2 activation. From the discovery of IP3R binding domains on TRPC2, it has been suggested that TRPC channel activation may be due to a conformational coupling between IP3R and TRPC channels. Moreover, recent data demonstrate that junctate, an IP3R associated protein, participates also in the gating of some TRPC. This study shows that junctate is expressed in sperm and co-localizes with the IP3R in the acrosomal crescent of the anterior head of rodent sperm. Consistent with its specific localization, pull-down experiments show that junctate interacts with TRPC2 and TRPC5 but not with TRPC1. Focus was placed on the interaction between TRPC2 and junctate; the N-terminus of junctate interacts with the C-terminus of TRPC2, both in vitro and in a heterologous expression system. Junctate binds to TRPC2 independently of the calcium concentration and the junctate binding site does not overlap with the common IP3R/calmodulin binding sites. TRPC2 gating is downstream phospholipase C activation, which is a key and necessary step during the acrosome reaction. TRPC2 may then be activated directly by diacylglycerol (DAG), as in neurons of the vomeronasal organ. The present study investigated whether DAG could promote the acrosome reaction. It was found that 100 microM OAG, a permeant DAG analogue, was unable to trigger the acrosome reaction. Altogether, these results provide a new hypothesis concerning sperm TRPC2 gating: TRPC2 activation may be due to modifications of its interaction with both junctate and IP3R, induced by depletion of calcium from the acrosomal vesicle (Stamboulian, 2005).

Mammalian TRPC genes encode a family of nonselective cation channels that are activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. In Drosophila photoreceptor cells, TRP channels are found in large, multimolecular signaling complexes in association with the PDZ-containing scaffolding protein, INAD. A similar mammalian TRPC 'signalplex' has been proposed, but has yet to be defined. In the present study, affinity-purified polyclonal antibodies against TRPC5 and TRPC6 were used to immunoprecipitate signalplex components from rat brain lysates. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, digested with trypsin, and sequenced by mass spectrometry. Proteins identified in the immunoprecipitates included cytoskeletal proteins spectrin, myosin, actin, drebrin, tubulin, and neurabin; endocytic vesicle-associated proteins clathrin, dynamin and AP-2, and the plasmalemmal Na(+)/K(+)-ATPase (NKA) pump. Several of these interactions were confirmed by reciprocal immunoprecipitation followed by Western blot analysis. In lysates from rat kidney, TRPC6, but not TRPC3, was found to coimmunoprecipitate with the NKA pump. Likewise, TRPC6, stably expressed in human embryonic kidney (HEK) cells, coimmunoprecipitated with endogenous NKA and colocalized with the pump to the plasmalemma when examined by immunofluorescence microscopy. Cell surface biotinylation experiments, in intact HEK cells, confirms that both the Na(+) pump and TRPC6 are present in the surface membrane and appear to interact. Lastly, TRPC6 coimmunoprecipitated with the NKA pump when the proteins were coexpressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. These observations suggest that TRPC6 and the Na(+) pump are part of a functional complex that may be involved in ion transport and homeostasis in both the brain and kidney (Goel, 2005).

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. The activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade was studied along with the role of calmodulin (CaM) on the activation. Exogenous application of 10 microM CaM through patch pipette accelerates the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 to 1.56 min. A novel CaM-binding site was identified located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuates the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 renders the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, high intracellular free Ca2+ inhibits the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrate that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation (Ordaz, 2005).

Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) are known to be inhibited by tyrosine kinase inhibitors. Activation of C-type transient receptor potential channel (TRPC) isoform 3 (TRPC3), a cation channel thought to be involved in SOCE and/or ROCE, was recently shown to depend on src tyrosine kinase activity. What is not known is the step at which src acts on TRPC3 and whether the role for tyrosine kinases in ROCE or SOCE is a general phenomenon. Using in vitro and in cell protein-protein interaction assays it is now reported that src phosphorylates TRPC3 at Y226 and that formation of phospho-Y226 is essential for TRPC3 activation. This requirement is unique for TRPC3 because (1) mutation of the cognate tyrosines of the closely related TRPC6 and TRPC7 have no effect; (2) TRPC6 and TRPC7 are activated in src-, yes-, and fyn-deficient cells; and (3) src, but not yes or fyn, rescue TRPC3 activation in src-, yes-, and fyn-deficient cells. The Src homology 2 domain of src interacts with either the N or the C termini of all TRPCs, suggesting that other tyrosine kinases may play a role in ion fluxes mediated by TRPCs other than TRPC3. A side-by-side comparison of the effects of genistein (a general tyrosine kinase inhibitor) on endogenous ROCE and SOCE in mouse fibroblasts, HEK and COS-7 cells, and on ROCE in HEK cells mediated by TRPC3, TRPC6, TRPC7, and TRPC5 showed differences that argue for ROCE and SOCE channels to be heterogeneous (Kawasaki, 2006).

Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca(2+) influx in vertebrate cells. Intracellular Ca(2+) is involved in the receptor-mediated activation of mammalian TRPC5 channels. This study investigated the role of calmodulin, an important sensor of changes in intracellular Ca(2+), and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca(2+) entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, is abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impairs the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique is inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch is unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions is reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca(2+)-calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC (Shimizu, 2006).

Formation of TRP channel heterodimers

Endogenously expressed TRPC homologs were investigated for their role in forming store-operated, 1-oleoyl-2-acetyl-sn-glycerol-stimulated, or carbachol (CCh)-stimulated calcium entry pathways in HEK-293 cells. Measurement of thapsigargin-stimulated Ba(2+) entry indicate that the individual suppression of TRPC1, TRPC3, or TRPC7 protein levels, by siRNA techniques, dramatically inhibits store-operated calcium entry (SOCE), whereas suppression of TRPC4 or TRPC6 has no effect. Combined suppression of TRPC1-TRPC3, TRPC1-TRPC7, TRPC3-TRPC7, or TRPC1-TRPC3-TRPC7 gives only slightly more inhibition of SOCE than seen with suppression of TRPC1 alone (68%), suggesting that these three TRPC homologs work in tandem to mediate a large component of SOCE. Evidence from co-immunoprecipitation experiments indicates that a TRPC1-TRPC3-TRPC7 complex, predicted from siRNA results, does exist. The suppression of either TRPC3 or TRPC7, but not TRPC1, induces a high Ba(2+) leak flux that is inhibited by 2-APB and SKF96365, suggesting that the influx is via leaky store-operated channels. The high Ba(2+) leak flux is eliminated by co-suppression of TRPC1-TRPC3 or TRPC1-TRPC7. For 1-oleoyl-2-acetyl-sn-glycerol-stimulated cells, siRNA data indicate that TRPC1 plays no role in mediating Ba(2+) entry, which appears to be mediated by the participation of TRPC3, TRPC4, TRPC6, and TRPC7. CCh-stimulated Ba(2+) entry, in contrast, can be inhibited by suppression of any of the five endogenously expressed TRPC homologs, with the degree of inhibition being consistent with CCh stimulation of both store-operated and receptor-operated channels. In summary, endogenous TRPC1, TRPC3, and TRPC7 participate in forming heteromeric store-operated channels, whereas TRPC3 and TRPC7 can also participate in forming heteromeric receptor-operated channels (Zagranichnaya, 2005).

Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because TRPC proteins have been proposed as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), the TRPC subunit composition of endogenous endothelial TRPC channels was studied. A redox-sensitive TRPC3-TRPC4 channel complex is described. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex is supported by four lines of evidence as follows: (1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. (2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. (3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. (4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. TRPC3 and TRPC4 are proposed as subunits of native endothelial cation channels that are governed by the cellular redox state (Poteser, 2006).

Activation of TRP channels by lipids

Many ion channels are regulated by lipids, but prominent motifs for lipid binding have not been identified in most ion channels. Phospholipase Cgamma1 (PLC-gamma1) binds to and regulates TRPC3 channels, components of agonist-induced Ca2+ entry into cells. This interaction requires a domain in PLC-gamma1 that includes a partial pleckstrin homology (PH) domain that functions as consensus lipid-binding and protein-binding sequence. A gestalt algorithm was developed to detect hitherto 'invisible' PH and PH-like domains, and the partial PH domain of PLC-gamma1 interacts with a complementary partial PH-like domain in TRPC3 to elicit lipid binding and cell-surface expression of TRPC3. These findings imply a far greater abundance of PH domains than previously appreciated, and suggest that intermolecular PH-like domains represent a widespread signalling mode (van Rossum, 2005).

TRPC3 has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. This study investigates the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments have demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibits a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contains a haemagglutinin epitope tag in the second extracellular loop, confers antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation increased surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. It is suggested the cholesterol content of the plasma membrane is a determinant of cellular TRPC3 activity and evidence is provided for cholesterol dependence of TRPC3 surface expression (Grazini, 2006).

TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. One of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurs downstream and independently of G-protein signaling. The effect is not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activates TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation shows dependence on side-chain length and the chemical head-group. The data reveal a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor (Fleming, 2006).

Ionic regulation of TRP channels

Members of the TRP cation channel family control a wide variety of cellular functions by regulating calcium influx. In neurons, TRP channels may also modulate cell excitability. TRPC5 is a neuronal TRP channel that plays a role in controlling neurite extension in the hippocampus. Transiently expressed TRPC5 exhibits a doubly rectifying current-voltage relationship characterized by relatively large inward currents and a unique outwardly rectifying current with a 'flat' segment between +10 and +40 mV that may be attributable to Mg2+ block. Intracellular Mg2+ blocks the outward current through TRPC5 with an IC50 of 457 microM. The block is mediated by a cytosolic aspartate residue, D633, situated between the termination of the sixth transmembrane domain and the 'TRP box.' The substitution of noncharged or positively charged residues for the negatively charged D633 resulted in a channel with markedly reduced inward currents, in addition to decreased Mg2+ block. This suggests that electrostatic attraction of cations by D633 may contribute to inward current amplitude in TRPC5. It is proposed that cytosolic negatively charged residues can modulate the conductivity of TRP cation channels (Obukhov, 2005).

TRP channel variants

A full-length RIKEN mouse cDNA has been identified that encodes a putative variant of the C3-type TRPC that differs from the previously cloned murine TRPC3 cDNA in that it has a 5' extension stemming from inclusion of an additional exon (exon 0). The extended cDNA adds 62 aa to the sequence of the murine TRPC3. A cDNA encoding the human homologue of this extended TRPC3 has a highly homologous 73-aa N-terminal extension, referred to as hTRPC3a. A query of the GenBank genomic database predicts the existence of a similar gene product also found in rats. Transient expression of the longer TRPC3a in human embryonic kidney (HEK) cells showed that it mediates Ca2+ entry in response to stimulation of the Gq-phospholipase C beta pathway, which is similar to that mediated by the shorter hTRPC3. However, after isolation of HEK cells expressing hTRPC3 in stable form, TRPC3a gave rise to Ca2+-entry channels that are not only activated by the Gq-phospholipase C beta pathway (receptor-activated Ca entry) but also by thapsigargin triggered store depletion. In conjunction with findings that TRPC1, TRPC2, TRPC4, TRPC5, and TRPC7, can each mediate store-depletion-activated Ca2+ entry in mammalian cells, these findings with hTRC3a support the proposal that TRPCs form capacitative Ca-entry channels (Yildirim, 2005).

An exquisitely specific PDZ/target recognition revealed by the structure of INAD PDZ3 in complex with TRP channel tail

The vast majority of PDZ domains are known to bind to a few C-terminal tail residues of target proteins with modest binding affinities and specificities. Such promiscuous PDZ/target interactions are not compatible with highly specific physiological functions of PDZ domain proteins and their targets. This study reports an unexpected PDZ/target binding occurring between the scaffold protein Inactivation no afterpotential D (INAD) and Transient receptor potential (TRP) channel in Drosophila photoreceptors. The C-terminal 15 residues of TRP are required for the specific interaction with INAD PDZ3. The INAD PDZ3/TRP peptide complex structure reveals that only the extreme C-terminal Leu of TRP binds to the canonical αB/βB groove of INAD PDZ3. The rest of the TRP peptide, by forming a β hairpin structure, binds to a surface away from the αB/βB groove of PDZ3 and contributes to the majority of the binding energy. Thus, the INAD PDZ3/TRP channel interaction is exquisitely specific and represents a new mode of PDZ/target recognitions (Ye, 2016).

Effects of TRP channel mutation in mammals

Progressive kidney failure is a genetically and clinically heterogeneous group of disorders. Podocyte foot processes and the interposed glomerular slit diaphragm are essential components of the permeability barrier in the kidney. Mutations in genes encoding structural proteins of the podocyte lead to the development of proteinuria, resulting in progressive kidney failure and focal segmental glomerulosclerosis. The TRPC6 ion channel is expressed in podocytes and is a component of the glomerular slit diaphragm. Five families were identified with autosomal dominant focal segmental glomerulosclerosis in which disease segregated with mutations in the gene TRPC6 on chromosome 11q. Two of the TRPC6 mutants had increased current amplitudes. These data show that TRPC6 channel activity at the slit diaphragm is essential for proper regulation of podocyte structure and function (Reiser, 2005).

Among the TRPC subfamily of TRP channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, a TRPC6-deficient mouse model was developed. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, is completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6^-/- smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells results in increased basal activity, while TRPC6 expression in TRPC6^-/- smooth muscle cells reduces basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone (Dietrich, 2005).

Focal and segmental glomerulosclerosis (FSGS) is a kidney disorder of unknown etiology, and up to 20% of patients on dialysis have been diagnosed with it. A large family with hereditary FSGS carries a missense mutation in the TRPC6 gene on chromosome 11q, encoding the ion-channel protein TRPC6. The proline-to-glutamine substitution at position 112, which occurs in a highly conserved region of the protein, enhances TRPC6-mediated calcium signals in response to agonists such as angiotensin II and appears to alter the intracellular distribution of TRPC6 protein. Previous work has emphasized the importance of cytoskeletal and structural proteins in proteinuric kidney diseases. These findings suggest an alternative mechanism for the pathogenesis of glomerular disease (Winn, 2005).

TRPC4 knockdown suppresses epidermal growth factor-induced store-operated channel activation and growth in human corneal epithelial cells

Epidermal growth factor (EGF) in corneal epithelial cells stimulates proliferation by inducing capacitative calcium entry (CCE). However, neither the identity nor the mechanism of activation of the plasma membrane influx pathway that mediates CCE is known. Accordingly, it was determined, in human corneal epithelial cells, whether or not (1) CCE is dependent upon stimulation of storeoperated channel (SOC) activity; (2) whether the canonical TRP protein isoform TRPC4 is a component of such channels, and (3) whether suppression of TRPC4 protein expression decreases EGF-induced stimulation of SOC activity and proliferation. The whole cell patch-clamp technique was used to monitor TRPC4-mediated stimulation of SOC activity following intracellular calcium store depletion and induction of CCE. TRPC4 small interfering RNA transfection suppresses TRPC4 protein expression. Reverse transcription-PCR and Western blot analysis were used to assess knockdown efficiency of mRNA and protein expression. [(3)H]Thymidine incorporation was used to evaluate EGF-induced mitogenesis. Ca(2+) transients were measured by single-cell fluorescence imaging. TRPC4 knockdown decreased mRNA and protein expression by 89% and 87%, respectively. In these cells, EGF-induced SOC activation elicited by intracellular calcium store depletion was obviated; EGF-induced CCE fell by 76%; EGF-induced stimulation of SOC activity was eliminated, and EGF-induced increases in proliferation fell by 54%. Thus, TRPC4 is a component of SOC in human corneal epithelial cells whose activation by EGF is requisite for an optimum mitogenic response to this growth factor (Yang, 2005).

TRP and intracellular release of Ca²⁺

TRPC3 is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was 'knocked down' in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, it was found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated in TRPC3 KD. Based on these data, it is concluded that expression of TRPC3 is tightly regulated during muscle cell differentiation and it is proposed that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load (Lee, 2006).

Coupling between TRPC6 and L-type channels is important in mediating smooth muscle cell membrane potential and muscle contraction

The ubiquitously expressed TRPC ion channels are considered important in Ca2+ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, molecular, biochemical, and electrophysiological approaches were used to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca2+ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca2+ entry is unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca2+ entry was suppressed by more than 50%. In contrast, OAG-induced Ca2+ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca2+ entry bears the hallmarks of TRPC6 function; it is inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Importantly, OAG-induced Ca2+ entry is blocked by the potent L-type Ca2+ channel inhibitor, *nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca2+ entry. Calculations reveal that even 90% reduction of TRPC6 channels allows depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction (Soboloff, 2005).

Hydrogen peroxide regulation of TRP channels

LTRPC2 is a cation channel recently reported to be activated by adenosine diphosphate-ribose (ADP-ribose) and NAD. Since ADP-ribose can be formed from NAD and NAD is elevated during oxidative stress, whole cell currents and increases in the intercellular free calcium concentration [Ca(2+)](i) were studed in LTRPC2-transfected HEK 293 cells after stimulation with hydrogen peroxide (H₂O₂). Cation currents carried by monovalent cations and Ca(2+) are induced by H₂O₂ as well as by intracellular ADP-ribose but not by NAD. H₂O₂-induced currents develop slowly after a characteristic delay of 3-6 min and recedes after wash-out of H₂O₂. [Ca(2+)](i) is rapidly increased by H₂O₂ in LTRPC2-transfected cells as well as in control cells; however, in LTRPC2-transfected cells, H₂O₂ evokes a second delayed rise in [Ca(2+)](i). A splice variant of LTRPC2 with a deletion in the C terminus (amino acids 1292-1325) was identified in neutrophil granulocytes. This variant is stimulated by H₂O₂ as the wild type. However, it does not respond to ADP-ribose. It is concluded that activation of LTRPC2 by H₂O₂ is independent of ADP-ribose and that LTRPC2 may mediate the influx of Na(+) and Ca(2+) during oxidative stress, such as the respiratory burst in granulocytes (Wehage, 2002).

TRP channels, neurite outgrowth and axon guidance

Ion channels formed by the TRP superfamily of proteins act as sensors for temperature, osmolarity, mechanical stress and taste. The growth cones of developing axons are responsible for sensing extracellular guidance factors, many of which trigger Ca2+ influx at the growth cone; however, the identity of the ion channels involved remains to be clarified. TRP-like channel activity exists in the growth cones of cultured Xenopus neurons and can be modulated by exposure to netrin-1 and brain-derived neurotrophic factor, two chemoattractants for axon guidance. Whole-cell recording from growth cones showed that netrin-1 induces a membrane depolarization, part of which remains after all major voltage-dependent channels are blocked. Furthermore, the membrane depolarization is sensitive to blockers of TRP channels. Pharmacological blockade of putative TRP currents or downregulation of Xenopus TRP-1 (xTRPC1) expression with a specific morpholino oligonucleotide abolishes the growth-cone turning and Ca2+ elevation induced by a netrin-1 gradient. Thus, TRPC currents reflect early events in the growth cone's detection of some extracellular guidance signals, resulting in membrane depolarization and cytoplasmic Ca2+ elevation that mediates the turning of growth cones (G. X. Wang, 2005).

Brain-derived neurotrophic factor (BDNF) is known to promote neuronal survival and differentiation and to guide axon extension both in vitro and in vivo. The BDNF-induced chemo-attraction of axonal growth cones requires Ca2+ signalling, but how Ca2+ is regulated by BDNF at the growth cone remains largely unclear. Extracellular application of BDNF triggers membrane currents resembling those through TRPC channels in rat pontine neurons and in Xenopus spinal neurons. In cultured cerebellar granule cells, TRPC channels contribute to the BDNF-induced elevation of Ca2+ at the growth cone and are required for BDNF-induced chemo-attractive turning. Several members of the TRPC family are highly expressed in these neurons, and both Ca2+ elevation and growth-cone turning induced by BDNF are abolished by pharmacological inhibition of TRPC channels, overexpression of a dominant-negative form of TRPC3 or TRPC6, or downregulation of TRPC3 expression via short interfering RNA. Thus, TRPC channel activity is essential for nerve-growth-cone guidance by BDNF (Li, 2005).

The calcium- and sodium-permeable transient receptor potential channel TRPC5 has an inhibitory role in neuronal outgrowth but the mechanisms governing its activity are poorly understood. A mechanism is proposed involving the neuronal calcium sensor-1 (NCS-1) protein. Inhibitory mutants of TRPC5 and NCS-1 enhance neurite outgrowth similarly. Mutant NCS-1 does not inhibit surface-expression of TRPC5 but generally suppresses channel activity, irrespective of whether it is evoked by carbachol, store depletion, lanthanides or elevated intracellular calcium. NCS-1 and TRPC5 are in the same protein complex in rat brain and NCS-1 directly binds to the TRPC5 C-terminus. The data suggest protein-protein interaction between NCS-1 and TRPC5, and the involvement of this protein complex in retardation of neurite outgrowth (Hui, 2006).

TRP channels and excitotoxicity

Excitotoxicity in brain ischemia triggers neuronal death and neurological disability, and yet these are not prevented by antiexcitotoxic therapy (AET) in humans. In neurons subjected to prolonged oxygen glucose deprivation (OGD), AET unmasks a dominant death mechanism perpetuated by a Ca2+-permeable nonselective cation conductance (IOGD). IOGD is activated by reactive oxygen/nitrogen species (ROS), and permits neuronal Ca2+ overload and further ROS production despite AET. IOGD currents corresponded to those evoked in HEK-293 cells expressing the nonselective cation conductance TRPM7. In cortical neurons, blocking IOGD or suppressing TRPM7 expression blocked TRPM7 currents, anoxic 45Ca2+ uptake, ROS production, and anoxic death. TRPM7 suppression eliminates the need for AET to rescue anoxic neurons and permits the survival of neurons previously destined to die from prolonged anoxia. Thus, excitotoxicity is a subset of a greater overall anoxic cell death mechanism, in which TRPM7 channels play a key role (Aarts, 2003).

In vivo tracking of phosphoinositides in Drosophila photoreceptors

In order to monitor phosphoinositide turnover during phospholipase C (PLC) mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, TbR332H (mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for PtdIns(4,5)P2, and the P4M domain from Legionella SidM for PtdIns4P. Using accurately calibrated illumination, these indicated that only approximately 50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by approximately 100-fold dimmer intensities in mutants lacking Ca2+ permeable TRP channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t(1/2) approximately 12 s) was faster than PtdIns(4,5)P2 (t(1/2) approximately 40s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca2+ and PKC-dependent inhibition of PLC is critical for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle (Hardie, 2015).

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Search PubMed for articles about Drosophila transient receptor potential

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date revised: 22 May 2022

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