investigator's application): The elevation of intracellular free calcium concentration by antigen plays a pivotal role in determining the fate and functions of T lymphocytes. The long term goals of this proposal are to elucidate the mechanisms that generate and regulate the calcium signal, and to understand how it activates specific T-cell activation genes. Work over the past five years indicates that Ca2+ signaling in T cells is sustained by capacitative Ca2+ entry, a widespread signaling mechanism by which the depletion of intracellular Ca2+ stores activated Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels. Relatively little is known about how these channels are regulated and how their activity determines responses such as T-cell activation or tolerance. Four specifics aims are proposed to elucidate the regulation and functions of CRAC channels in T cells using a combination of microscopic imaging and patch-clamp techniques: 1) Determine the dependence of CRAC channel activation on store content and intracellular Ca2+. 2) Investigate the mechanism by which intracellular Ca2+ feeds back to slowly inactivate CRAC channels. 3) Establish the role of mitochondria in modulating the Ca2+ signal and test the hypothesis that they influence CRAC channel function by interfering with slow inactivation. 4) Examine the role of [Ca2+]i oscillations in increasing the efficiency and/or the specificity of transcriptional responses, using reporter genes driven by two Ca2+-sensitive transcriptions factors, NFAT and AP-1. The significance of these studies is that they will describe for the first time several cellular processes that control the dynamic behavior of depletion-activated Ca2+ channels. Moreover, in many cells the specific consequences of dynamic Ca2+ signals such as oscillations has been largely unexplored, and these studies will assess their impact on a definable and physiologically important end point, gene activation. Given the requirement for CRAC channel function in T-cell activation, these studies may ultimately expose a number of control points for the immune response, leading to new strategies for immunomodulation with important therapeutic benefit in the prevention or treatment of disorders such as AIDS and autoimmune disease.
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