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),

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).

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