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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Physiology Study Section (PHY)
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Shapiro, Bert I
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Ohio State University
Schools of Medicine
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Hassock, Sheila R; Zhu, Michael X; Trost, Claudia et al. (2002) Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel. Blood 100:2801-11
Zhang, Z; Tang, Y; Zhu, M X (2001) Increased inwardly rectifying potassium currents in HEK-293 cells expressing murine transient receptor potential 4. Biochem J 354:717-25
Zhang, Z; Tang, J; Tikunova, S et al. (2001) Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain. Proc Natl Acad Sci U S A 98:3168-73
Tang, J; Lin, Y; Zhang, Z et al. (2001) Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels. J Biol Chem 276:21303-10
Birnbaumer, L; Boulay, G; Brown, D et al. (2000) Mechanism of capacitative Ca2+ entry (CCE): interaction between IP3 receptor and TRP links the internal calcium storage compartment to plasma membrane CCE channels. Recent Prog Horm Res 55:127-61; discussion 161-2
Tang, Y; Tang, J; Chen, Z et al. (2000) Association of mammalian trp4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF. J Biol Chem 275:37559-64
Boulay, G; Brown, D M; Qin, N et al. (1999) Modulation of Ca(2+) entry by polypeptides of the inositol 1,4, 5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca(2+) entry. Proc Natl Acad Sci U S A 96:14955-60
Wang, W; O'Connell, B; Dykeman, R et al. (1999) Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein. Am J Physiol 276:C969-79
Vannier, B; Peyton, M; Boulay, G et al. (1999) Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel. Proc Natl Acad Sci U S A 96:2060-4
Zhu, X; Jiang, M; Birnbaumer, L (1998) Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry. J Biol Chem 273:133-42

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