Cardiac disease continues to be the leading cause of death in this country. Drugs that affect blood pressure are important therapeutic agents, and calcium channel blockers have been validated in a number of large clinical trials as effective in not only lowering blood pressure but also in reducing the risks of coronary heart disease and stroke. Although they are generally thought to preferentially block L-type calcium channels in the vascular system, some of them have also been reported to block T-type calcium channels as well. We propose to investigate the binding determinants and molecular basis of inhibition of three calcium channel blockers: mibefradil, as a prototype drug for which the T-type calcium channel is the primary target, verapamil, a first generation calcium channel blocker (phenylalkylamine), which continues to be effective for treatment of hypertension and also is a first line therapy for paroxysmal atrial tachycardia, and amlodipine, a highly prescribed fourth generation dihydropyridine (DHP). Our experimental focus will be on the T-type calcium channel as an important primary or secondary target of these drugs, although we also propose to collaborate with an L-channel laboratory for comparative analysis between drugs and T- and L-channels and to study voltage dependent Na channels as a secondary target of mibefradil. Our long term goal is to understand the control of the molecular substrate of drugs that target cardiac ion channels, i.e. the structural bases of their state dependent affinities. We have chosen these three agents because they are classes of therapeutic agents that are relevant to or attractive for clinical use, interact with T-channels in distinct ways, and provide us with the opportunity to discover important structural determinants of kinetic function. Our strategy combines mutagenesis, whole cell, single channel, and gating current electrophysiology, and molecular modeling.
