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Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-β-cyclodextrin (MβCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein-coupled phospholipase Cβ (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. Here, we studied the effects of MβCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MβCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MβCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MβCD suppressed a constitutively active TRP mutant-channel, trpP365, suggesting that TRP channel activity is a target of MβCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MβCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MβCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating., Competing Interests: Conflict of interest The authors declare that they have no conflict of interests with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Physiological activation by light of the Drosophila TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP 2 ) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. In this study, we re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells. Unlike previous studies, we added OAG into the cytosol via a patch-clamp pipette and observed robust activation of the expressed TRPL channels. However, TRPL channel activation was much slower than the physiologically activated TRPL by light. Therefore, we used a picosecond-fast optically activated DAG analogue, OptoDArG. Inactive OptoDArG was added into the intracellular solution with the patch-clamp pipette, and it slowly accumulated on the surface membrane of the recorded HEK cell in the dark. A fast application of intense UV light to the recorded cell resulted in a robust and relatively fast TRPL-dependent current that was greatly accelerated by the constitutively active TRPL F557I pore-region mutation. However, this current of the mutant channel was still considerably slower than the native light-induced TRPL current, suggesting that DAG alone is not sufficient for TRPL channel activation under physiological conditions., Competing Interests: The authors declare no conflict of interest.

The Drosophila light-activated Transient Receptor Potential (TRP) channel is the founding member of a large and diverse family of channel proteins. The Drosophila TRP (dTRP) channel, which generates the electrical response to light has been investigated in a great detail two decades before the first mammalian TRP channel was discovered. Thus, dTRP is unique among members of the TRP channel superfamily because its physiological role and the enzymatic cascade underlying its activation are established. In this article we outline the research leading to elucidation of dTRP as the light activated channel and focus on a major physiological property of the dTRP channel, which is indirect activation via a cascade of enzymatic reactions. These detailed pioneering studies, based on the genetic dissection approach, revealed that light activation of the Drosophila TRP channel is mediated by G-Protein-Coupled Receptor (GPCR)-dependent enzymatic cascade, in which phospholipase C β (PLC) is a crucial component. This physiological mechanism of Drosophila TRP channel activation was later found in mammalian TRPC channels. However, the initial studies on the mammalian TRPV1 channel indicated that it is activated directly by capsaicin, low pH and hot temperature (>42 °C). This mechanism of activation was apparently at odds with the activation mechanism of the TRPC channels in general and the Drosophila light activated TRP/TRPL channels in particular, which are target of a GPCR-activated PLC cascade. Subsequent studies have indicated that under physiological conditions TRPV1 is also target of a GPCR-activated PLC cascade in the generation of inflammatory pain. The Drosophila light-activated TRP channel is still a useful experimental paradigm because its physiological function as the light-activated channel is known, powerful genetic techniques can be applied to its further analysis, and signaling molecules involved in the activation of these channels are available.

Transient Receptor Potential (TRP) channels constitute a large superfamily of polymodal channel proteins with diverse roles in many physiological and sensory systems that function both as ionotropic and metabotropic receptors. From the early days of TRP channel discovery, membrane lipids were suggested to play a fundamental role in channel activation and regulation. A prominent example is the Drosophila TRP and TRP-like (TRPL) channels, which are predominantly expressed in the visual system of Drosophila . Light activation of the TRP and TRPL channels, the founding members of the TRP channel superfamily, requires activation of phospholipase Cβ (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) into Diacylglycerol (DAG) and Inositol 1, 4,5-trisphosphate (IP 3 ). However, the events required for channel gating downstream of PLC activation are still under debate and led to several hypotheses regarding the mechanisms by which lipids gate the channels. Despite many efforts, compelling evidence of the involvement of DAG accumulation, PIP 2 depletion or IP 3 -mediated Ca 2+ release in light activation of the TRP/TRPL channels are still lacking. Exogeneous application of poly unsaturated fatty acids (PUFAs), a product of DAG hydrolysis was demonstrated as an efficient way to activate the Drosophila TRP/TRPL channels. However, compelling evidence for the involvement of PUFAs in physiological light-activation of the TRP/TRPL channels is still lacking. Light-induced mechanical force generation was measured in photoreceptor cells prior to channel opening. This mechanical force depends on PLC activity, suggesting that the enzymatic activity of PLC converting PIP 2 into DAG generates membrane tension, leading to mechanical gating of the channels. In this review, we will present the roles of membrane lipids in light activation of Drosophila TRP channels and present the many advantages of this model system in the exploration of TRP channel activation under physiological conditions.

Drosophila photoreceptors respond to oscillating light of high frequency (∼100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. Here, we report that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca 2+ -dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC). Electroretinogram measurements of the frequency response to oscillating lights in vivo revealed that dark-reared flies expressing wild-type TRP exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation. Strikingly, preventing phosphorylation of the S936-TRP site by alanine substitution in transgenic Drosophila ( trp S936A ) abolished the difference in frequency response between dark-adapted and light-adapted flies, resulting in high-frequency response also in dark-adapted flies. In contrast, inserting a phosphomimetic mutation by substituting the S936-TRP site to aspartic acid ( trp S936D ) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies. Light-adapted rdgC mutant flies showed relatively high S936-TRP phosphorylation levels and light-dark phosphorylation dynamics. These findings suggest that RDGC is one but not the only phosphatase involved in pS936-TRP dephosphorylation. Together, this study indicates that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions. SIGNIFICANCE STATEMENT Drosophila photoreceptors exhibit high temporal resolution as manifested in frequency response to oscillating light of high frequency (≤∼100 Hz). Light rearing conditions modulate the maximal frequency detected by photoreceptors, thus enabling them to maintain high sensitivity to light and high temporal resolution. However, the precise mechanisms for this process are not fully understood. Here, we show by combination of biochemistry and in vivo electrophysiology that transient receptor potential (TRP) channel dephosphorylation at a specific site is a fast, light-activated and Ca 2+ -dependent regulatory process. TRP dephosphorylation affects the detection limit of oscillating light according to the adaptation state of the photoreceptor cells by shifting the detection limit to higher frequencies upon light adaptation. This novel mechanism thus adjusts dynamic processing of visual information under varying light conditions., (Copyright © 2017 the authors 0270-6474/17/374213-12$15.00/0.)

Cholesterol is an essential compound of higher eukaryotic cell membranes and a known modulator of ion channel activity. Changes in phospholipids and cholesterol composition of cell membranes are known to alter the activity of ion channels. However, there is little knowledge on the effects of cholesterol on transient receptor potential (TRP) channels. In this study, we explore the effects of cholesterol depletion on the Drosophila photoreceptor channel TRP-like (TRPL), when expressed in tissue culture cells. Depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) induced fast (<100s) suppression of spontaneous TRPL channel activity, a typical state of expressed TRPL channels in Drosophila S2 cells. An equally fast suppression of receptor-induced TRPL channel activity in HEK293 cells, downstream of phospholipase C (PLC) activation, was also induced by MβCD. Biochemical experiments showed binding of TRPL to immobilized cholesterol, suggesting direct binding of cholesterol to TRPL. Exploring the effects of several mutations in a putative cholesterol-binding site of TRPL was inconclusive as some did not render the channel insensitive to cholesterol depletion while others rendered the channel inactive. We conclude that (i) cholesterol is essential for TRPL channel activity, (ii) TRPL channels interact with cholesterol, and (iii) the binding site of cholesterol in TRPL differs from the putative binding site of TRPV1. Thus, the fast and strong effects of cholesterol depletion on the TRPL channel activity suggest that cholesterol is an important component of fly photoreceptor signaling membrane., (© 2017 Elsevier Inc. All rights reserved.)

Family members of the cationic transient receptor potential (TRP) channels serve as sensors and transducers of environmental stimuli. The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements for channel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilies has been described, even within a subfamily (such as TRPC) not all members co-assemble with each other. In Drosophila photoreceptors two TRPC channels, TRP and TRP-like protein (TRPL) are expressed together in photoreceptors where they generate the light-induced current. The formation of functional TRP-TRPL heteromultimers in cell culture and in vitro has been reported. However, functional in vivo assays have shown that each channel functions independently of the other. Therefore, the issue of whether TRP and TRPL form heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers, and the structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP and TRPL channels, in vivo. Interaction studies of tagged and native channels as well as native and chimeric TRP-TRPL channels using co-immunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We found that TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that the transmembrane regions of TRP and TRPL do not determine assemble specificity of these channels. However, the C-terminal regions of both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels.

The Drosophila light activated TRP and TRPL channels have been a model for TRPC channel gating. Several gating mechanisms have been proposed following experiments conducted on photoreceptor and tissue cultured cells. However, conclusive evidence for any mechanism is still lacking. Here, we show that the Drosophila TRPL channel expressed in tissue cultured cells is constitutively active in S2 cells but is silent in HEK cells. Modulations of TRPL channel activity in different expression system by pharmacology or specific enzymes, which change the lipid content of the plasma membrane, resulted in conflicting effects. These findings demonstrate the difficulty in elucidating TRPC gating, as channel behavior is expression system dependent. However, clues on the gating mechanism may arise from understanding how different expression systems affect TRPC channel activation.

In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P(2) in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P(2) was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P(2) is not an inhibitor of TRPL channel activation. PI(4,5)P(2) hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P(2) levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P(2) is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.

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

Transient receptor potential (TRP) channels are polymodal cellular sensors involved in a wide variety of cellular processes, mainly by changing membrane voltage and increasing cellular Ca(2+). This review outlines in detail the history of the founding member of the TRP family, the Drosophila TRP channel. The field began with a spontaneous mutation in the trp gene that led to a blind mutant during prolonged intense light. It was this mutant that allowed for the discovery of the first TRP channels. A combination of electrophysiological, biochemical, Ca(2+) measurements, and genetic studies in flies and in other invertebrates pointed to TRP as a novel phosphoinositide-regulated and Ca(2+)-permeable channel. The cloning and sequencing of the trp gene provided its molecular identity. These seminal findings led to the isolation of the first mammalian homologues of the Drosophila TRP channels. We now know that TRP channel proteins are conserved through evolution and are found in most organisms, tissues, and cell-types. The TRP channel superfamily is classified into seven related subfamilies: TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN. A great deal is known today about participation of TRP channels in many biological processes, including initiation of pain, thermoregulation, salivary fluid secretion, inflammation, cardiovascular regulation, smooth muscle tone, pressure regulation, Ca(2+) and Mg(2+) homeostasis, and lysosomal function. The native Drosophila photoreceptor cells, where the founding member of the TRP channels superfamily was found, is still a useful preparation to study basic features of this remarkable channel.

Transient receptor potential (TRP) channels are essential components of biological sensors that detect changes in the environment in response to a myriad of stimuli. A major difficulty in the study of TRP channels is the lack of pharmacological agents that modulate most members of the TRP superfamily. Notable exceptions are the thermoTRPs, which respond to either cold or hot temperatures and are modulated by a relatively large number of chemical agents. In the present study we demonstrate by patch clamp whole cell recordings from Schneider 2 and Drosophila photoreceptor cells that carvacrol, a known activator of the thermoTRPs, TRPV3 and TRPA1 is an inhibitor of the Drosophila TRPL channels, which belongs to the TRPC subfamily. We also show that additional activators of TRPV3, thymol, eugenol, cinnamaldehyde and menthol are all inhibitors of the TRPL channel. Furthermore, carvacrol also inhibits the mammalian TRPM7 heterologously expressed in HEK cells and ectopically expressed in a primary culture of CA3-CA1 hippocampal brain neurons. This study, thus, identifies a novel inhibitor of TRPC and TRPM channels. Our finding that the activity of the non-thermoTRPs, TRPL and TRPM7 channels is modulated by the same compound as thermoTRPs, suggests that common mechanisms of channel modulation characterize TRP channels.

Light adaptation is a process that enables photoreceptor cells to operate over a wide range of light intensities without saturation. In invertebrate photoreceptors, fast adaptation is mediated by a Ca2+-dependent negative-feedback mechanism, which mainly affects the terminal steps of the cascade. Therefore, the response to each photon is smaller as light intensity increases, accommodating both high sensitivity and a vast dynamic range. Here, we describe a novel type of adaptation, which is mediated by one of the first steps in the phototransduction cascade affecting the sensitivity to absorbed photons. Long exposure to light resulted in dramatic reduction in the probability of each absorbed photon to elicit a response, whereas the size and shape of each single photon response did not change. To dissect the molecular mechanism underlying this form of adaptation we used a series of Drosophila mutants. Genetic dissection showed a pivotal role for light-induced translocation of Gq alpha between the signaling membrane and the cytosol. Biochemical studies revealed that the sensitivity to light depends on membrane Gq alpha concentration, which was modulated either by light or by mutations that impaired its targeting to the membrane. We conclude that long-term adaptation is mediated by the movement of Gq alpha from the signaling membrane to the cytosol, thereby reducing the probability of each photon to elicit a response. The slow time scale of this adaptation fits well with day/night light intensity changes, because there is no need to maintain single photon sensitivity during daytime.

The light-activated channels of Drosophila photoreceptors transient receptor potential (TRP) and TRP-like (TRPL) show voltage-dependent conductance during illumination. Recent studies implied that mammalian members of the TRP family, which belong to the TRPV and TRPM subfamilies, are intrinsically voltage-gated channels. However, it is unclear whether the Drosophila TRPs, which belong to the TRPC subfamily, share the same voltage-dependent gating mechanism. Exploring the voltage dependence of Drosophila TRPL expressed in S2 cells, we found that the voltage dependence of this channel is not an intrinsic property since it became linear upon removal of divalent cations. We further found that Ca(2+) blocked TRPL in a voltage-dependent manner by an open channel block mechanism, which determines the frequency of channel openings and constitutes the sole parameter that underlies its voltage dependence. Whole cell recordings from a Drosophila mutant expressing only TRPL indicated that Ca(2+) block also accounts for the voltage dependence of the native TRPL channels. The open channel block by Ca(2+) that we characterized is a useful mechanism to improve the signal to noise ratio of the response to intense light when virtually all the large conductance TRPL channels are blocked and only the low conductance TRP channels with lower Ca(2+) affinity are active.

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

Recent studies in Drosophila melanogaster 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. As ezrin-radixin-moesin (ERM) proteins are known to regulate actin-membrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that 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, we show that 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, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.

A variety of rod opsin mutations result in autosomal dominant retinitis pigmentosa and congenital night blindness in humans. One subset of these mutations encodes constitutively active forms of the rod opsin protein. Some of these dominant rod opsin mutant proteins, which desensitize transgenic Xenopus rods, provide an animal model for congenital night blindness. In a genetic screen to identify retinal degeneration mutants in Drosophila, we identified a dominant mutation in the ninaE gene (NinaE(pp100)) that encodes the rhodopsin that is expressed in photoreceptors R1-R6. Deep pseudopupil analysis and histology showed that the degeneration was attributable to a light-independent apoptosis. Whole-cell recordings revealed that the NinaE(pp100) mutant photoreceptor cells were strongly desensitized, which partially masked their constitutive activity. This desensitization primarily resulted from both the persistent binding of arrestin (ARR2) to the NINAE(pp100) mutant opsin and the constitutive activity of the phototransduction cascade. Whereas mutations in several Drosophila genes other than ninaE were shown to induce photoreceptor cell apoptosis by stabilizing a rhodopsin-arrestin complex, NinaE(pp100) represented the first rhodopsin mutation that stabilized this protein complex. Additionally, the NinaE(pp100) mutation led to elevated levels of G(q)alpha in the cytosol, which mediated a novel retinal degeneration pathway. Eliminating both G(q)alpha and arrestin completely rescued the NinaE(pp100)-dependent photoreceptor cell death, which indicated that the degeneration is entirely dependent on both G(q)alpha and arrestin. Such a combination of multiple pathological pathways resulting from a single mutation may underlie several dominant retinal diseases in humans.

The Transient Receptor Potential (TRP) proteins constitute a large and diverse family of channel proteins, which is conserved through evolution. TRP channel proteins have critical functions in many tissues and cell types, but their gating mechanism is an enigma. In the present study patch-clamp whole-cell recordings was applied to measure the TRP- and TRP-like (TRPL)-dependent currents in isolated Drosophila ommatidia. Also, voltage responses to light and to metabolic stress were recorded from the eye in vivo. We report new insight into the gating of the Drosophila light-sensitive TRP and TRPL channels, by which both Ca2+ and protein dephosphorylation are required for channel activation. ATP depletion or inhibition of protein kinase C activated the TRP channels, while photo-release of caged ATP or application of phorbol ester antagonized channels openings in the dark. Furthermore, Mg(2+)-dependent stable phosphorylation event by ATPgammaS or protein phosphatase inhibition by calyculin A abolished activation of the TRP and TRPL channels. While a high reduction of cellular Ca2+ abolished channel activation, subsequent application of Ca2+ combined with ATP depletion induced a robust dark current that was reminiscent of light responses. The results suggest that the combined action of Ca2+ and protein dephosphorylation activate the TRP and TRPL channels, while protein phosphorylation by PKC antagonized channels openings.

The Drosophila light-activated channel TRP is the founding member of a large and diverse family of channel proteins that is conserved throughout evolution. In spite of much progress, the gating mechanism of TRP channels is still unknown. However, recent studies have shown multi-faceted functions of the Drosophila light-sensitive TRP channel that may shed light on TRP gating. Accordingly, metabolic stress, which leads to depletion of cellular ATP, reversibly activates the Drosophila TRP and TRPL channels in the dark in a constitutive manner. In several Drosophila mutants, constitutive activity of TRP channels lead to a rapid retinal degeneration in the dark, while genetic elimination of TRP protects the cells from degeneration. Additional studies have shown that TRPL translocates in a light-dependent manner between the signaling membranes and the cell body. This light-activated translocation is accompanied by reversible morphological changes leading to partial and reversible collapse of the microvillar signaling membranes into the cytosol, which allows turnover of signaling molecules. These morphological changes are also blocked by genetic elimination of TRP channels. The link of TRP gating to the metabolic state and maintenance of cells makes cells expressing TRP extremely vulnerable to metabolic stress via a mechanism that may underlie retinal degeneration and neuronal cell death upon malfunction.

TRP channel proteins constitute a large and diverse family of proteins that are expressed in many tissues and cell types. This family was designated TRP because of a spontaneously occurring Drosophila mutant lacking TRP that responded to a continuous light with a transient receptor potential (hence TRP). In addition to responses to light, TRPs mediate responses to nerve growth factor, pheromones, olfaction, mechanical, chemical, temperature, pH, osmolarity, vasorelaxation of blood vessels, and metabolic stress. Furthermore, mutations in several members of TRP-related channel proteins are responsible for several diseases, such as several tumors and neurodegenerative disorders. TRP-related channel proteins are found in a variety of organisms, tissues, and cell types, including nonexcitable, smooth muscle, and neuronal cells. The large functional diversity of TRPs is also reflected in their diverse permeability to ions, although, in general, they are classified as nonselective cationic channels. The molecular domains that are conserved in all members of the TRP family constitute parts of the transmembrane domains and in most members also the ankyrin-like repeats at the NH2 terminal of the protein and a "TRP domain" at the COOH terminal, which is a highly conserved 25-amino acid stretch with still unknown function. All of the above features suggest that members of the TRP family are "special assignment" channels, which are recruited to diverse signaling pathways. The channels' roles and characteristics such as gating mechanism, regulation, and permeability are determined by evolution according to the specific functional requirements.

Drosophila phototransduction results in the opening of two classes of cation channels, composed of the channel subunits transient receptor potential (TRP), TRP-like (TRPL), and TRPgamma. Here, we report that one of these subunits, TRPL, is translocated back and forth between the signaling membrane and an intracellular compartment by a light-regulated mechanism. A high level of rhabdomeral TRPL, characteristic of dark-raised flies, is functionally manifested in the properties of the light-induced current. These flies are more sensitive than flies with no or reduced TRPL level to dim background lights, and they respond to a wider range of light intensities, which fit them to function better in darkness or dim background illumination. Thus, TRPL translocation represents a novel mechanism to fine tune visual responses.

The Drosophila light activated channel TRP is the founding member of a large and diverse family of channel proteins that is conserved throughout evolution. These channels are Ca2+ permeable and have been implicated as important component of cellular Ca2+ homeostasis in neuronal and non-neuronal cells. The power of the molecular genetics of Drosophila has yielded several mutants in which constitutive activity of TRP leads to a rapid retinal degeneration in the dark. Metabolic stress activates rapidly and reversibly the TRP channels in the dark in a constitutive manner by a still unknown mechanism. The link of TRP gating to the metabolic state of the cell is shared also by mammalian homologues of TRP and makes cells expressing TRP extremely vulnerable to metabolic stress, a mechanism that may underlie retinal degeneration and neuronal cell death.