An increase in the level of intracellular free calcium ([Ca2+]i) occupies a central role in the receptor-driven activation of many cell types, including T lymphocytes. In T cells, antigen recognition by the T cell receptor triggers Ca2+ release from intracellular stores and stimulates Ca2+ influx through channels in the plasma membrane. New physiological approaches directed at the level of single cells have revealed complexities in this response, such as [Ca2+]i oscillations, and have provided direct evidence for distinct classes of Ca2+ and K+ channels that contribute to the Ca2+ signaling process. The long-term objectives of this proposal are to understand the molecular mechanisms that regulate Ca2+ signaling during T-cell activation. The proposed experiments specifically seek to define the properties, regulation, and distribution of Ca2+ channels in T cells and to understand the role of K+ channels in modulating Ca2+ signaling. Fluorescence video- imaging and patch-clamp recording methods will be combined to characterize receptor-regulated Ca2+ channels in terms of their pharmacological profile, ionic selectivity, and single-channel properties. The subcellular mechanisms that link the antigen receptor to activation of Ca2+ channels are largely unknown. Pharmacological probes will be applied to distinguish the role of inositol phosphates and intracellular Ca2+ stores in mediating Ca2+-channel activation. Advances in video microscopy have made it possible to image intracellular Ca2+ with high temporal and spatial resolution in single cells. We will use these techniques to explore the physical association between Ca2+ channels and the antigen receptor, by visualizing sites of Ca2+ influx after capping the antigen receptor on the cell surface. Experiments with recently identified, highly specific K+- channel blockers reveal that voltage-gated and Ca2+-activated K+ channels contribute to oscillatory Ca2+ signaling, possibly through an influence on the membrane potential. We seek to establish the link between K+ channels, membrane potential, and [Ca2+]i in imaging experiments using voltage- and Ca2+-dependent dyes and photolysis of caged Ca2+. Through their influence on Ca2+ signaling in T cells, Ca2+ and K+ channels represent sensitive control points for modulation of the immune response. By extending our knowledge of ion channel properties and regulatory mechanisms, these studies may lead to the identification and rational design of drugs to control T-cell responsitivity. In this way, the proposed research may find future therapeutic application to a wide variety of human diseases, including immunodeficiencies such as AIDS, autoimmune disorders, allergy, graft rejection, and disorders associated with acute and chronic inflammation.
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