investigator's application): Intracellular Ca2+ release elicited by inositol triphosphate is often followed by Ca2+ entry from the extracellular space, which by refilling the stores, play an important role in many cellular processes, such as the proliferation of T lymphocytes. The Drosophila trp gene is known to encode a plasma membrane light-sensitive ion channels that serves critical functions in Ca2+ entry for phototransduction in insect. Expression of the Trp protein in insect Sf9 cells and Xenopus oocytes is accompanied by an increase in Ca2+ influx in response to internal store depletion. The mammalian trp homologs are thus likely to form the voltage-independent channels responsible for Ca2+ influx in non-excitable cells. At lease six non-allelic trp-related genes are present in a mammalian genome. Full-length cDNA for three Trps were cloned and two human Trps (Htrp1 and Htrp3) showed to facilitate Ca2+ entry when expressed in COS cells. Expression of antisense mouse trp sequences in murine L cells almost completely abolished the endogenous Ca2+ influx. These results provide evidence that mammalian Trp proteins are involved in Ca2+ entry. This application is aimed to fully investigate the role of the mammalian Trp proteins in Ca2+ influx. This will be achieved by studying function and regulatory mechanisms of cloned Trps expressed in cultured mammalian cells in transient and stable manners. First, upon activation of cell surface receptors or store-depletion by thapsigargin, intracellular Ca2+ concentrations will be monitored by fura-2 fluorescence measurement and whole cell currents will be recorded by patch clamping. Ion selectivity for each Trp and effects of Ca2+ channels blockers will be investigated. Second, the molecular diversity of the trp gene family and its expression in T lymphocytes will be studied. Third, proteins that associate with Trp will be identified and cloned via immuno-coprecipitation and using the yeast two-hybrid system. Fourth, the membrane topology of a Trp will be studies through analysis of its glycosylation and phosphorylation sites and epitope cleavage patterns. The overall expectation of these projects is to gain a deep understanding of the function, regulation, structural organization, and subtype distribution of Trp proteins and their influence on human health. To accomplish this, the investigators will use approaches drawn from the field of cell biology, biophysics, molecular biology, pharmacology, and biochemistry.
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