Ion channels in the surface membrane of heart cells underlie excitability, act as receptors for anti-arrhythmic drugs, and initiate excitation contraction coupling. This proposal focuses on the factors that control the opening and closing of cardiac Na and Ca channels. These two channel types open and later inactivate in response to membrane depolarization. The inactivation process of Na channels is voltage-dependent, but that of cardiac L-type Ca channels is considerably more complex. Both voltage and Ca influence the inactivation of Ca channels, but the way that the two factors interact is not clear. Clarification of this process is the major goal of the present proposal. We will utilize the patch clamp technique to record currents through single Na or Ca channels in guinea pig ventricular myocytes. For Na channels, we have formulated a model that describes quantitatively the decay of current during depolarization. We will test this model and extend our observations on Na channels while applying the insights thereby gained to the more complex gating of Ca channels. We will obtain recordings from drug-free Ca channels with the physiological charge carrier, Ca. The data will be interpreted in terms of a Markov gating model that will make it possible to determine explicitly the rate constants for inactivation and for channel closure by deactivation. The interpretations will be checked by convolution analysis. The roles of Ca and voltage in the inactivation of Ca channals will be probed by comparing the single-channel behavior 2 with various charge carriers, by using proteolytic enzymes, by buffering Ca2+, and by phosphorylating the channels irreversibly during activation of cyclic-AMP dependent protein kinase. We hope thereby to distinguish between the effects of Ca within the permeation pathway and cytoplasmic Ca2+(acting directly on the channel or via a cytoplasmic effector). The results of each intervention will be interpreted in terms of the quantitative models of Ca channel gating, with the goal of pinpointing the molecular correlates of each modification. Several lines of evidence now support the idea that current flow through Ca channels is facilitated by a modest increase in the intracellular free Ca concentration. We will characterize the effects of channel phosphorylation by Ca2+-calmodulin dependent protein kinase to determine whether it mediates this process. The proposed work promises to provide important new insights into the molecular mechanisms of ion channel regulation in heart cells
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