REGULATORY MECHANISMS OF K+ CHANNEL FUNCTION IN HEART

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Receptor-modulated ion channels play a primary role in the neuroendocrine regulation of cardiac function. Thus, for example, cholinergic activation of cardiac muscarinic receptors may affect the initiation and propagation of the heart beat by opening potassium channels in the cardiac sarcolemma. The long-range objective of this project is to understand quantitatively and at the level of molecular detail, how these regulatory processes operate, with particular emphasis on the recently discovered role of guanine nucleotide binding proteins (G-proteins) in the receptor effector coupling process. Specifically, muscarinic activation of the inwardly rectifying potassium channel K+(m) will be studied in isolated cardiac atrial myocytes. Use of this well defined system should facilitate the attainment of the following three primary objectives. 1. Identification of the critical steps involved in the coupling process in vivo and its modeling in terms of clearly defined chemical kinetic processes. Whole-cell gigaseal recording will be used to follow the evolution of K+(m) 1. in response to manipulations activating receptor or G-protein in a manner appropriate for revealing the specific kinetic steps involved in receptor-channel coupling. The results will be used to construct a mathematical model to be tested and refined by comparing its predictions with experiment. 2. Identification of the type and moiety of G-protein(s) that activate K+(m) in vivo by characterizing the effects of intracellularly injected, highly purifiid G-protein components on the ionic currents of cardiac myocytes. The results will be carefully related to those seen at the single-channel level with excised patches in order to insure that these two different techniques yield consistent and physiologically relevant conclusions. 3. Efforts will be made to reconstitute the control of K(m) channels in lipid bilayer membranes by G-protein and receptor, with the ultimate goal of elucidating the structural requirements for a functional regulatory system. This project is likely to further our understanding of the neurohormonal regulation of normal and abnormal cardiac function. Moreover, since G-proteins have been found to couple receptors to effectors in virtually all cell types, the results are bound to be of more general significance with respect to the control of cell function by extracellular signals.