In addition to regulating electrical activity in the nervous system, ion channels mediate very different cellular mechanisms within the immune system. The earliest events signaling the binding of antigen to surface receptors in T lymphocytes and mast cells include the activation of ion channels and a rise in cytosolic [Ca2+]. This """"""""[Ca2+]i signal"""""""" links membrane antigen receptors to gene expression, lymphokine secretion and cell proliferation essential to the immune response. It has become clear that ion channels mediate a variety of cellular behaviors in lymphocytes, including the [Ca2+]i signal, secretion of lymphokines, killing of target cells, progression through the cell cycle, and volume regulation in response to osmotic stress. Ion channels in cells of the immune system offer promising targets for therapeutic agents in the treatment of a variety of disorders, including immunodeficiency, autoimmunity, leukemias and lymphomas, graft rejection, and inflammation. Patch-clamp and video- imaging techniques are being used at the single-cell level to investigate intracellular and intercellular signaling mechanisms. The proposed experiments on T lymphocytes and mast cells are divided into three sections corresponding to the sequence of events following antigen stimulation. (1) Intracellular signaling leading from the engagement of membrane receptors to the [Ca2+]i signal: Here the main issues are the mode of T-cell receptor engagement, and the role of kinases, G-proteins, and ion channels in mediating the [Ca2+]i signal. Antigen-specific T cell lines will be used to investigate activation by contact with antigen-presenting B cells. The roles of kinases, G-proteins, and ion channels will be probed by intracellular injection of specific peptides that mimic effector regions of signaling molecules and by using specific channel blockers. Genetically altered cell lines and mice will be used in these experiments. (2) Activation of the interleukin-2 gene by the rise in [Ca2+]i: The goal here is to develop and exploit a single-cell assay of gene expression. A T-cell hybridoma engineered to express bacterial beta-galactosidase when the interleukin-2 gene is activated for transcription will be used to study gene expression at the single-cell level. (3) Secretion of molecules which have effects on neighboring cells: Signaling mechanisms mediated by ATP receptors will be investigated. Reporter cells with P2 receptors will be used to detect ATP released locally. The distribution of ATP receptors in cells of the immune system will be determined. Collectively, these experiments make use of a single-cell approach to clarify signal transduction mechanisms vital to immune system function.
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