The long term goal of the research is to study the fundamental molecular, cell biology and physiological mechanisms underlying retinal degeneration and long-term adaptation through studies aiming to elucidate the mechanisms of activation, inactivation and translocation of signaling molecules. These molecules include the Gq, Dmoesin and the light activated channels TRP and TRPL, which cause cell death upon malfunction.
Our specific aims are: i) To explore the molecular mechanism underlying the activation of the TRP and TRPL channels by metabolic stress in the dark, by studying the synergistic effects of cytosolic Ca2+ and protein phosphorylation and dephosphorylation reactions on TRP and TRPL activation and gating mechanism. Transgenic Drosophila lacking putative phosphorylation sites will be generated and studied, as well as transgenic Drosophila lacking or over expressing protein kinases and protein phosphatases. ii) To apply structure-function analysis of the TRP and TRPL channels. Site-directed mutations in TRP and TRPL channels will be generated. The mutant channels will be heterologously expressed in a stable form in Schneider, S2, cells together with the main proteins of the INAD signaling complex (INAD scaffold protein, PLC and PKC). The heterologous expression will be followed by a step by step comparison of the effects of critical mutations in the TRP and TRPL channels expressed in S2 cells to the same mutations in the native system using transgenic flies, in vivo, iii) To study the mechanisms underlying light-induced translocation of Gqa and TRPL and their functional consequences. We will investigate the effect of Gqa translocation on long-term light adaptation. Furthermore, we will investigate the molecular mechanism underlying retinal degeneration induced by abnormal localization of active Gqa in the cytosol of the rhodopsin mutant ninaEPPlOO. We will, furthermore, test the hypothesis that light and phosphorylation-dependent translocation of the cytoskeletal protein, Dmoesin out of the rhabdomere triggers TRPL translocation, which induces long term adaptation. This study is thus expected to shed new light on major unsolved questions of TRP gating, which is involved in several diseases and on light induced translocation of key signaling molecules.
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