The long-term goal of this project is to provide a better understanding of the physiological basis of the regulation of normal and abnormal spontaneous activity in cardiac cells. With the recent demonstration that depolarization induced automaticity can occur in cardiac tissue, advances in our study of normal and abnormal automatic activity will be provided by a detailed study of cardiac outward currents. Furthermore, multiple overlapping variables have made study of these currents in syncytial preparations difficult. Thus, we propose to study outward current carrying channels from the ventricular myocardium of both man and the dog at the level of unitary currents. We have chosen the single channel reconstitution technique over the patch clamp technique for these studies, because both sides of a reconstituted channel as well as the membrane environment itself are accessible to well-defined independent experimental manipulations, and this is ideally suited for pharmacologic studies. We specifically propose to prepare sarcolemmal vesicles and reconstitute single ionic channels into artificial lipid bilayer membranes and characterize each channel type in terms of its conduction process, and in terms of its gating process. The conduction process will be evaluated in terms of selectivity, concentration-dependence, temperature dependence, lipid charge effects, blocker ion effects and Eyring rate theory. The gating process will be evaluated in terms of thermodynamics and kinetics. In each of the above, we propose to examine the mechanism of ionic channel blockade for lidocaine, quinidine and flecainide. These studies provide a unique opportunity for studying the fundamental electrophysiological properties of single cardiac channels from the human heart. In addition, they will advance our understanding of the electrophysiology of canine and human cardiac tissue as well as place on firmer ground the basis of antiarrhythmic drug action in cardiac tissue.
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