Transmembrane Ca channels have an important dual function: they generate electrical signals which are used in many cells for conduction of impulses and for the control of rhythmical electrical activity, but Ca channels also deliver a specific chemical messenger to the cell in the form of Ca ions, whose message is decoded by intracellular Ca binding effector proteins. Ca channels therefore play a fundamental role in the regulation of key cellular processes in most excitable cells. In heart cells, Ca channels initiate contraction, help determine heart rate by contributing to the pacemaker depolarization, promote slow conduction in nodal regions, and support the action potential plateau, thus determining the duration of electrical excitability. In partially depolarized regions of the heart, Ca channels can initiate slowly conducted action potentials and promote arrhythmic activity. The L-type Ca channel is the pharmacological receptor for some of the most widely used drugs (Ca channel blockers) in the management of coronary heart disease, hypertension and cardiac arrhythmias. This grant application proposes to investigate the elementary functional properties of Ca channels. Patch clamp recordings will be used to study the openings and closings of single Ca channel molecules in intact cardiac cells and sympathetic neurons. The kinetics of individual openings and closings, and of transitions between longer lasting gating modes will be analyzed quantitatively in order to better understand the mechanisms and stimuli which govern the activity of Ca channels in cells. The amplitude of the unitary current flowing through single open Ca channels under various experimental conditions will be measured to learn more about the process of ion permeation and selectivity, a process to which the channels owe their capability of passing Ca ions at a high rate and yet to remain selectively permeant to Ca ions even in the presence of high concentrations of competing ions. Separate experiments on a K-channel for which we have the cloned cDNA will attempt to correlate functional domains with structurally and functionally conserved domains of voltage gated ion channels relevant for both K- and Ca channels. An antigenic epitope will be introduced into various domains of the channel sequence by in-vitro mutagenesis in an effort to locate key segments of the primary structure with respect to the plasma membrane. Mutations of positive and negative charges in the presumed membrane spanning regions of the channel will be used to establish the relative location and functional interactions between residues conserved in all voltage dependent ion channels. From the proposed combination of functional studies at the single channel level and manipulation of the channel structure by site-directed mutagenesis we hope to gain new insight into the molecular mechanisms which determine the function of these important regulators of cellular excitability.
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