The long term objective of the proposed research is to study the molecular, physiological and biological mechanisms of several forms of retinal degeneration using the approach of genetic dissection of Drosophila phototransduction. The unifying theme of these studies is analysis of the role, mechanism and physiological consequences underlying translocation of signaling molecules, specifically the Gq-protein and the TRPL channel. The impaired localization of Gq will be studied in the constitutively active rhodopsin mutant ninaEpp100 that leads to retinal degeneration. The hitherto unknown gating mechanism of the light sensitive channels TRP and TRPL will be investigated using activation of these channels in the dark in response to metabolic stress with particular emphasis on phosphorylation and dephosphorylation reactions. Finally, the activity dependent (i.e. light dependent) translocation of Gq from the membranes to the cytosol, which is accompanied by marked changes of the cortical actin cytoskeleton in the photoreceptor cell, will be studied in a variety of visual mutants and transgenic flies. Two hypotheses on the functional role of translocation of Gq to the cytosol and the accompanied morphological changes will be tested: i) that it serves as a desensitization mechanism during continuous illumination; ii) that it serves as a link between the rhabdomere and the cortical actin through interaction of Gq and the Drac protein. Both direct effect of Gq alpha and Drac1 and indirect effect through activation of Drac1 exchange factor will be tested. To study the above specific aims powerful techniques will be employed including: electrophysiology, biochemistry, molecular genetics, immunoelectron microscopy, immunofluorescence and microfluorimetry. Constitutive activity and translocation of critical signaling molecules, including the photopigment, transducin and TRP homologue channels have been found in mammalian tissues. We have indications that malfunction of these regulatory processes lead to retinal degeneration in Drosophila and possibly in mammalian retina. This study is thus expected to shed light on mechanisms that associate several forms of retinal degeneration with malfunction of fundamental regulatory mechanisms.
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