The force and rate of the heart beat are regulated through release of acetylcholine (ACh) and norepinephrine (NE) by the autonomic nervous system, however, the molecular mechanisms of this regulation are poorly understood. While it is certain NE acts partly through the cAMP system the mechanisms of ACh action are unknown. An understanding of these mechanisms is important for understanding the normal functioning heart and for understanding cardiac disease. For example, T.N. James and others have implicated cardiac neuropathies with certain kinds of sudden death. ACh decreases heart rate by activating a hyperpolarizing background potassium current and decreases the force of contraction by decreasing intracellular calcium. The slow onset and long duration in response to a brief pulse of ACh suggest a multistep mechanism with one or more enzymatic reactions. The experiments in this proposal will test the hypothesis that ACh activation of potassium current and inhibition of calcium current in the heart occurs via coupling of GTP binding proteins to various effector proteins. The hypothesis will be tested within the framework of a collision-coupling model for receptor/GTP protein interaction. Whole cell and single channel currents will be measured in the frog atrium and sinus venosus by patch clamp methods. The general approach will be to perfuse cells intracellularly with a variety of molecular probes. Examples of such probes are GTP and GTP analogs, Bordetella pertussis, and inhibitors and activators of various protein kinases. The results from this work will contribute to the understanding of the molecular mechanisms which regulate the heart and facilitate an understanding of cholinergic mechanisms elsewhere in the body.
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