Baruch Minke

The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors

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

Novel Ca2+ channels underlying transduction in Drosophila photoreceptors: implications for phosphoinositide-mediated Ca2+ mobilization

Drosophila photoreceptors are excellent models for studies of the ubiquitous phosphoinositide signalling cascade. Recent studies suggest that light-induced phosphoinositide hydrolysis in Drosophila leads to the activation of two classes of channels. One is selective for Ca2+ and absent in the transient receptor potential mutant trp. The trp gene product, which shows some structural similarity to vertebrate voltage-gated Ca2+ channels, may thus define a novel family of second-messenger-operated Ca2+ channels generally responsible for the widespread but poorly understood phenomenon of phosphoinositide-mediated Ca2+ entry. The other channel is a non-selective cation channel that requires Ca2+ for activation. As well as being a major charge carrier for the light-induced current, Ca2+ influx via the trp-dependent channels appears to be required for refilling Ca2+ stores sensitive to inositol 1,4,5-trisphosphate and for feedback regulation (light adaptation) of the transduction cascade.

The Light Response of Drosophila Photoreceptors Is Accompanied by an increase in Cellular Calcium: Effects of Specific Mutations

Photoreceptors of dissociated Drosophila retinae were loaded with the fluorescent Ca2+ indicators, fluo-3 and Calcium Green-5N. In fluo-3-loaded, wild-type photoreceptors, a rapid increase in fluorescence (Ca2+ signal) accompanied the light-evoked inward current. Removal of extracellular Ca2+ greatly reduced the Ca2+ signal, indicating Ca2+ influx as its major cause. In Calcium Green-5N-loaded trp mutants, which lack a large fraction of the Ca2+ permeability underlying the light-evoked inward current, the Ca2+ signal was smaller relative to wild-type photoreceptors. Fluo-3-loaded norpA mutant photoreceptors, which lack a light-activated phospholipase C, generated no light-evoked inward current and no Ca2+ signal. The phosphoinositide pathway therefore appears necessary for both excitation and changes in cytosolic free Ca2+ concentration.

Light-regulated subcellular translocation of Drosophila TRPL channels induces long-term adaptation and modifies the light-induced current

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.

Drosophila photoreceptors and signaling mechanisms

Fly eyes have been a useful biological system in which fundamental principles of sensory signaling have been elucidated. The physiological optics of the fly compound eye, which was discovered in the Musca, Calliphora and Drosophila flies, has been widely exploited in pioneering genetic and developmental studies. The detailed photochemical cycle of bistable photopigments has been elucidated in Drosophila using the genetic approach. Studies of Drosophila phototransduction using the genetic approach have led to the discovery of novel proteins crucial to many biological processes. A notable example is the discovery of the inactivation no afterpotential D scaffold protein, which binds the light-activated channel, its activator the phospholipase C and it regulator protein kinase C. An additional protein discovered in the Drosophila eye is the light-activated channel transient receptor potential (TRP), the founding member of the diverse and widely spread TRP channel superfamily. The fly eye has thus played a major role in the molecular identification of processes and proteins with prime importance.

Carvacrol is a novel inhibitor of Drosophila TRPL and mammalian TRPM7 channels.

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.

Drosophila mutant with a transducer defect.

The trp is a conditional phototransduction mutant of Drosophila. Direct electrical measurements and shot noise analysis suggest that a prolonged intense light causes in the mutant a reduction in the quantum efficiency for quantum bump production that does not arise from bleaching of the visual pigment. This effect depends on the duration of the light and only weakly on its intensity. In the normal fly, an intense blue light that shifts the visual pigment from rhodopsin to metarhodopsin, induces an excitatory process manifested by a prolonged depolarizing after potential (PDA). In the mutant, the PDA has a small amplitude and bump noise is superimposed on the response. It can thus be shown that the excitatory process underlying the PDA is also present in those trp mutants where the PDA voltage response is small or absent. It is suggested that the absence of the PDA voltage response in the mutant is probably due to a defect in an intermediate process, which links the excitatory process to the membrane conductance change.

Phospholipase C and termination of G-protein-mediated signalling in vivo

In Drosophila photoreceptors, phospholipase C (PLC) and other signalling components form multiprotein structures through the PDZ scaffold protein INAD. Association between PLC and INAD is important for termination of responses to light; the underlying mechanism is, however, unclear. Here we report that the maintenance of large amounts of PLC in the signalling membranes by association with INAD facilitates response termination, and show that PLC functions as a GTPase-activating protein (GAP). The inactivation of the G protein by its target, the PLC, is crucial for reliable production of single-photon responses and for the high temporal and intensity resolution of the response to light.

Calmodulin binding to Drosophila NinaC required for termination of phototransduction.

The ninaC locus encodes two unconventional myosins, p132 and p174, consisting of fused protein kinase and myosin head domains expressed in Drosophila photoreceptor cells. NinaC are the major calmodulin‐binding proteins in the retina and the NinaC‐calmodulin interaction is required for the normal subcellular localization of calmodulin as well as for normal photo‐transduction. In the current report, we present evidence for two calmodulin‐binding sites in NinaC, C1 and C2, which have different in vitro binding properties. C1 was found to be common to both p132 and p174 while C2 was unique to p174. To address the requirements for calmodulin binding at each site in vivo, we generated transgenic flies expressing ninaC genes deleted for either C1 or C2. We found that the spatial localization of calmodulin depended on binding to both C1 and C2. Furthermore, mutation of either site resulted in a defective photoresponse. A prolonged depolarization afterpotential (PDA) was elicited at lower light intensities than necessary to produce a PDA in wild‐type flies. These results suggest that calmodulin binding to both C1 and C2 is required in vivo for termination of phototransduction.

Regulation of light‐dependent Gqα translocation and morphological changes in fly photoreceptors

Heterotrimeric G‐proteins relay signals between membrane‐bound receptors and downstream effectors. Little is known, however, about the regulation of Gα subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gqα to the cytosol, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqα affect these processes. Epistatic analysis showed that Gqα is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqβ is essential for targeting of Gqα to the membrane and suggested that Gqβ is also needed for efficient activation of Gqα by rhodopsin. Our results support the ‘two‐signal model’ hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity‐dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors.