This project focuses on molecular properties and regulation of ion channels in T lymphocytes, taking advantage of parallel advances in electrophysiology, molecular biology and video imaging techniques. Our goal is to understand the role of ion channels in the immune response and other cell behavior. Using patch-clamp techniques, we have characterized a diverse set of functionally significant K+, Ca2+, and Cl- channels in human and mouse T cells and cell lines. These channels are differentially expressed depending on the developmental and activation state and have been shown to contribute to T-cell receptor signaling leading to gene expression, secretion of lymphokines, and cell proliferation. In addition, the channels serve to regulate cell volume, and may play a role in stabilizing the contact between a T cell and an antigen-presenting cell. Channels are now being targeted for the development of novel immunosuppressive drugs. Through the proposed experiments, we plan to continue our studies of five main channels. Using site-directed mutants and a novel mammalian microinjection system for expression, we will characterize the inactivation mechanism and drug block of the voltage- activated K+ channel. The action of progesterone and classical """"""""calcium antagonists"""""""" on the K+ channel will be investigated in detail. We will screen several scorpion toxin mutants for differential effects in binding to the outer vestibule of voltage-activated and calcium-activated K+ channels. Calcium signaling and gene expression depend crucially on a low-conductance Ca2+-influx channel that opens when intracellular Ca2+ stores are released. We will investigate the activation mechanism of this channel, using solutions that magnify the size of single channels. Chloride channels in lymphocytes are activated by cell swelling. We will investigate the nucleotide dependence of Cl-channel activation. Video- imaging experiments will be performed to investigate the roles of Cl channels in Ca2+ signaling, gene expression, motility and volume regulation. Through the proposed experiments we hope to define mechanisms that regulate ion channels and corresponding cell functions that underlie the immune response.
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