Potassium channels of the cardiac cell membrane play a major role in initiating and controlling the heart beat. Thus, for example, cardiac pacemaker activity is slowed by the neurotransmitter acetylcholine by way of its activation of an inwardly-rectifying potassium channel in the cll membrane. The mechanism of this activation is complex. It is thought to proceed through the stimulation of membrane-bound muscarinic receptors which activate a signal-transducing guanine nucleotide binding protein. Interaction of this activated transducer molecule with the potassium channel, either directly or indirectly by way of intermediate steps, results in channel opening which produces the electrophysiological effect. The objective is to elucidate the molecular mechanisms by which the function of cardiac potassium channels is controlled by neuroendocrine, pharmacological and biochemical factors. Initially, the project will combine electrophysiological and biochemical techniques to establish the molecular mechanisms of the control of K+ channels in living heart cells produced by enzymatic disruption of the adult heart. Specifically, the ability of purified quanine nucleotide binding proteins to open K+ channels will be examined to establish which of the signal transducing G-proteins activate K+ channels and whether or not activation occurs by direct interaction of the G-protein with the channel protein. These studies will be followed by efforts to incorporate, from vesicles of heart sarcolemma into lipid bilayer membranes, functional K+ channels that respond to modulation by the appropriate G-protein. Reconstitution of the signal transducing complex in a simple, chemically well-defined system is necessary for a molecular understanding of the mechanism by which the system functions. It allows one to make molecular changes, for example by altering the G-protein or the membrane lipid composition, specifically designed to test hypotheses concerning the function of the K+ channel and its interaction with G-protein. Later stages of the project will also attempt to isolate and biochemically characterize the K+ channel protein by taking advantage of its specific interaction with G-protein. If successful, this procedure should permit the sequencing and eventual cloning of the K+ channel.
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