The long-term objective of the proposed research is to understand the molecular mechanisms underlying vision in the fruitfly, Drosophila melanogaster. The goal of the current proposal is to understand the mechanisms regulating TRP, a novel plasma membrane Ca2+ channel specifically expressed in Drosophila photoreceptor cells. Changes in the concentration of intracellular Ca2+ are crucial for signal transduction in virtually every cell. During the last couple of years, it has been found that one of the most effective and prevalent Ca2+ influx pathways, referred to as capacitative Ca2+ entry, occurs via Ca2+ selective ion channels in the plasma membrane that are activated following depletion of intracellular Ca2+ stores. The mechanisms regulating these ion channels are poorly understood and the molecular identification of the proteins responsible for these plasma membrane Ca2+ conductances have been elusive. Based on a variety of electrophysiological, pharmacological and molecular analyses, it appears that TRP is the archetypical member of the family of capacitative Ca2+ entry channels. The approach to characterizing TRP takes advantage of a combination of molecular, biochemical, electrophysiological, pharmacological, genetic and germline transformation techniques. To study the mechanisms regulating TRP we plan to: 1) test the hypothesis that TRP function is regulated by interaction with calmodulin, 2) test the hypothesis that TRP localization to the base of the rhabdomeres is mediated by interaction with alpha-actinin, through the TRP ankyrin repeat, 3) test the hypothesis that TRP is regulated by interaction with GTP, 4) test whether the PEST signal is important for TRP function, 5) characterize the effects of systematic deletions in TRP on subunit assembly, activation and inactivation, and 6) characterize TRP expressed in the neural cell line, NG115-401L. The results of the proposed experiments should contribute not only to understanding visual physiology but to the widespread phenomenon of capacitative Ca2+ entry which has been proposed to be important in such diverse cellular processes as fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signaling. Several studies indicate that Ca2+ influx is important in memory involving NMDA receptors and other studies suggest that blockage of Ca2+ influx is important in controlling certain types of epileptic seizures.
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