In the immune response the activation of lymphocytes by specific antigens or mitogens begins with membrane events and culminates in cell division. The intracellular signals from surface receptors to the nucleus remain unclear, but may involve ionic channels, integral membrane proteins that gate the flow of ions across the membrane. Using the gigaohm seal patch recording technique on individual T lymphocytes, in parallel with biochemical and immunological studies, the functional requirement for ionic channels in mitogenesis will be assessed. The patch recording technique allows ionic channels in small cells, such as lymphocytes, to be studied with resolution to the single channel level. Expression of ionic channels in resting and activated human T-cells and a clonal T-cell line will be determined through whole cell and isolated patch recording. Protocols will be used to test for the existence of potassium channels, calcium-activated channels, calcium channels, and sodium channels. We will study the modulation of channel properties by mitogenic and non-mitogenic lectins, and by a mitogenic clonospecific antibody against the T-cell receptor. The dependence of mitogen-stimulated cellular events on functioning potassium channels will be examined in detail. The effect of potassium channel blockers on mitogen-stimulated changes in membrane potential, intracellular calcium concentration, and on capping of cell surface receptors will be explored. The surface distribution and gating properties of potassium channels will be determined in capped lymphocytes. Potassium channel blockers will be tested for effects on phospholipid turnover, various aspects of protein synthesis, interleukin-2 production, and the expression of transferrin receptor and HLA-DR antigen on T-cells. Time windowing experiments will determine periods during which T cell activation is most sensitive to potassium channel block. Other known modulators of ionic channels will be tested for effects on thymidine incorporation. Interleukins, suppressor factors, and cyclosporin A will be tested for effects on ionic channel function. Single cell recording in parallel with biochemical experiments will probe the mechanism of clinically important modulators of the immune response, as well as providing clues regarding the role of ionic channels in the transformation of a resting cell to a proliferating state.
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