The receptors for a large number of important antiarrhythmic and anti- ischemic drugs reside within cardiac ion channels. Sodium channels conduct the electrical impulse, while calcium channels underlie the action potential plateau and initiate excitation-contraction coupling. Thus, it is no wonder that drugs which block sodium or calcium channels exert powerful influences on cardiac function. The importance of such drugs transcends the cardiovascular system: sodium channel blockers are perhaps even more useful clinically as local anesthetics than as antiarrhythmics, and calcium channel blockers are now known to modulate processes as diverse as secretion and memory. Despite their therapeutic importance, our understanding of the mechanisms of action of these agents remains sketchy. Most of what we do know comes from studies of drug action on native channels in intact tissue or in primary isolated cells. While such studies have led to important concepts regarding drug action, including the modulated receptor and guarded receptor hypotheses, native channels offer a limited potential for resolving the salient questions. The present proposal explores, at the molecular level, the mechanism of action of drugs that block sodium and calcium channels. For sodium channels, we focus on lidocaine and related compounds; for L-type calcium channels, dihydropyridines and phenylalkylamines will be investigated. The complementary DNA for sodium or calcium channels is utilized to induce the expression of channels in a cell type where such channels are normally not present. The unique advantages of this approach include the ability to determine the primary structure of the expressed channels; wild-type channels can be investigated, or the structure can be altered selectively by site-directed mutagenesis. The function of the expressed channels is assayed electrophysiologically at the whole-cell and single-channel levels. The laboratory has extensive experience with these methods. Preliminary data are presented defining the precise location within the electrical field of several residues that line the sodium channel pore a,nd that determine tetrodotoxin sensitivity. Blockers act by occluding, directly or indirectly, the pore of the channels. The specific drugs of interest here, local anesthetics arid calcium channel blockers, exhibit the additional complexity of state- dependent binding: many of these agents stabilize or bind preferentially to the inactivated state of the channels. Thus, the first step will be to define the structures that underlie the permeation and inactivation mechanisms of sodium and calcium channels. We will then investigate in detail the interactions of drugs with the various structurally defined regions, by comparing their effects on wild-type and mutant channels. This program of investigation promises to elucidate the fundamental principles of action of clinically important pharmacologic agents that modify cardiac excitability, induce local anesthesia, and serve as antihypertensives.
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