The molecular mechanism of phototransduction and perturbations in this signalling pathway leading to retinal degeneration will be studied. Substantial advances that we have made in understanding the gating mechanism of the light-activated ion channels, the molecular mechanism of light adaptation and in termination of the phototransduction process will be further examined and gaps of knowledge analyzed in detail. Changes in [Ca2+]1, and of Ca in the intracellular stores play a key role in controlling light excitation, adaptation, termination of the light response and retinal degeneration. The target molecules for our studies are: a) The trp protein that we have recently identified as a Ca2+ channel needed for a sustained receptor potential and to replenish the inositol trisphosphate-sensitive Ca2+ stores. b) The trp like protein (trp1) a calmodulin binding membrane protein with a striking homology to trp. c) Protein kinase C (PKC) a kinase activated by diacylglycerol and tumor promoters with largely unknown physiological functions. d) The arrestin protein that is phosphorylated by a Ca2+ dependent kinase and terminates metarhodopsin activity. e) The rhodopsin (R) phosphatase which dephosphorylates the inactive R and thereby makes it ready for excitation. Transgenic Drosophila flies transformed by epitope-tagged and site- directed modified trp gene in a null trp background will be used to identify the signal for its specific cellular localization and the regulatory gating mechanism of its light induced current (LIC). The null mutant of the Drosophila eye-PKC (inaC[P209]) will be employed to test the hypothesis that PKC mediates light adaptation by terminating Ca2+ release from the InsP3-sensitive Ca2+ stores. Recombinant pure arrestin will be used to study the role of arrestin phosphorylation and its -relevance to arrestin-mediated quenching of G-protein and R-phosphatase activities. Simultaneous whole cell voltage clamp recording and microfluorimetric measurements of free Ca2+ will be applied to the novel preparation of isolated ommatidia of -Drosophila mutants. The hypothesis that photoreceptor degeneration results from a toxic increase in cellular Ca2+ due to excessive phosphorylation of key-proteins will be tested using retinal degeneration Drosophila mutants and in vivo phosphorylation. Toxic increase in total Ca of cellular stores will be estimated by energy dispersive X-ray analysis and Laser Micro. Mass Analysis (LAMMA) of shock- freezed preparations. Chemicals which antagonize the toxic increase in [Ca2+]i will be tested for their efficacy as therapeutic means to prevent light induced retinal degeneration. GRANT=R01EY04939 The goal is to understand the control of the chromophore of rhodopsin over the processes of visual excitation and bleaching adaptation. The hypothesis is that the rhodopsin has a distinCtly different conformation from its product of bleaching, opsin, and these differences in conformation affect the interaction with other retinal proteins which in turn control the sensitivity of the photoreceptor. The alteration of the chromophore structure allows the manipulation of the physiology and the biochemistry of the photoreceptor. Derivatives of the chromophore, chosen to test some specific aspect of the interaction of the chromophore with the protein, will be synthesized. Physiological measurements in isolated rods and cones will be related to in vitro measurements of the biochemical processes that are involved. The native and analogue pigments will be studied by a variety of mass spectral and other spectroscopic techniques and related to the biochemical findings. Both the transduction process and the mechanisms of pigment regeneration and termination of bleaching adaptation by retinoids will be studied. The role of retinoid transport and the retinoid binding proteins will be examined through the use of retinoid analogues. For certain conditions of clinical retinal degenerations, data from several laboratories reveal the presence of point mutations or polymorphisms in various retinal proteins, which would lead to abnormal interactions of these proteins in the visual transduction and adaption processes. Derivatives of the chromophore of rhodopsin can induce abnormal conformations of rhodopsin which in turn affect the interaction with proteins in the transduction process. The experiments proposed here offer a means for studying these clinical disorders at the basic biochemical and physiological levels.
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