The molecular mechanism of inositide-mediated phototransduction and defects in this signaling pathway leading to retinal degeneration will be studied. The proposed project will focus on two crucial molecules: TRP and RDGB. TRP is a novel calcium-permeable channel, whose mechanism of activation by light is completely vague. RDGB is a phosphatidylinositol (PI) transfer protein (PITP), but its role in phototransduction and the mechanism underlying retinal degeneration in the absence of RDGB is completely unknown. The overall objective of this project is to study the mechanism of retinal degeneration caused by mutations in the trp and rdgB genes. The hypothesis that deregulation of the TRP channel activity leads to the observed rapid retinal degeneration will be examined in the novel semi-dominant trpP365 gain-of-function mutant, in transgenic flies, in Xenopus oocytes and in Caenorhabditis elegans in which this mutant TRP protein will be heterologously expressed. The studies of RDGB will probe the mechanism underlying retinal degeneration in rdgB mutants through investigation of RDGB's role in phototransduction and in the inositol-lipid pathway, especially its function as PITP in renewal of phosphoinositides and in vesicular transport and secretion. All the criteria that the investigators have developed to study phototransduction will be applied using biochemical and electrophysiological techniques and microfluorimetry. The evolutionary conservation of TRP and RDGB suggests that they represent prototypic members of novel families of important proteins that are generally responsible for the widespread phenomena of inositol lipid signaling and calcium homeostasis. This study is thus expected to reveal how TRP is activated, the role of RDGB in phototransduction and to shed light on the mechanism that associates retinal degeneration with deregulation of channel activity in trp and abnormal calcium homeostasis and defects in phosphoinositide pathways in rdgB.
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