The voltage-dependent calcium channel plays a significant role in a number of cellular and physiological processes, including contraction of heart and smooth muscle and in the functioning of neurons and secretory systems. The long-term objectives of this direction of research, of which this proposal is only the first step, is to understand the molecular mechanism of the functioning of this important protein and its physiological and pharmacological modulation by various processes and agents. Initially, as outlined in this proposal, the mechanism of action of a structurally diverse group of agents known as calcium antagonists will be investigated. The calcium antagonists block voltage-dependent calcium channels and include three structurally distinct families of agents: the dihydropyridines (e.g. nifedipine), the phenylalkylamines (e.g. verapamil), and the benzothiazepines (e.g. diltiazem). The dihydropyridines, in fact, now include two """"""""calcium agonists"""""""" which activate the voltage-dependent calcium channel. These calcium antagonists are used extensively in the treatment of a number of cardiovascular disorders including cardiac arrhythmias, angina pectoris, and systemic hypertension. To study the molecular basis of these agents' actions on the voltage-dependent calcium channel, an integrated multilevel approach will be used. The model system to be utilized for these studies is the GH3 cell line, a cloned pituitary tumor which has already proved extremely useful in studies of the electrophysiology of calcium channel function and is an ideal tissue source for both biochemical and electrophysiological studies. Biochemical studies of the mechanism of action of these agents will utilize radioligand binding techniques to study the interactions of these agents with the calcium channel. The effects of these agents on Ca++ currents will be studied using electrophysiological patch clamp techniques, characterizing whole cell and single channel currents. To study biochemically the functional consequences of these interactions changes in the Ca++ permeability of the channel using radioactive Ca++ fluxes and fluorescence of a Ca++ sensitive optical indicator, Indol, will be characterized. By studying the action of these agents in parallel on a number of levels in the same tissue preparation, a complete picture of the molecular mechanism of their effects on the voltage-dependent calcium channel will emerge. In addition the normal physiological functioning of the calcium channel will be better understood as a consequence of these investigations.
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