Calcium channels in the surface membrane of heart cells play a central role in excitation-contraction coupling, in normal pacemaker acitivity and in arrhythmogenesis. As the major pathway for Ca influx into the cell, Ca channels help determine the free intracellular Ca concentration, (Ca2+)i. Several lines of evidence indicate that (Ca2+)i in turn exerts feedback control on the availability of Ca channels. An increase in (Ca2+)i has been implicated in the decay of Ca current during depolarization (""""""""inactivation""""""""), as in a number of other preparations. An increase in (Ca2+)i is also believed to exert opposing positive feedback effects on ICa under certain conditions, such as during digitalis inotropy in the heart and during C kinase activation in Aplysia neurones. This project proposes to investigate the basic mechanism(s) of Ca channel regulation by (Ca+)i, under normal conditions and during """"""""Ca overload"""""""" states associated with triggered arrhythmias. Membrane currents will be measured using several variants of the gigaseal (""""""""patch"""""""") voltage clamp technique in single mammalian ventricular cells. Whole-cell Ca current (ICa) recordings will be used to test simple kinetic schemes for Ca-dependent inactivation. Ensemble fluctuation analysis of whole-cell currents will determine whether or not changes in (Ca2+)i affect the number of functional channels. Single channel recordings will enable characterization of the changes in gating behavior underlying positive and negative regulation. Direct simultaneous measurements of (Ca2+)i and ICa will establish quantitatively the relationship between these two variables, and will provide direct evidence regarding the relative roles of positive regulation and Ca-dependent inactivation. Finally, measurements of (Ca2+)i during the recovery from inactivation of ICa will help establish the basis of the oscillatory restitution that occurs during Ca overload. Ca-regulation of Ca channels, while ubiquitous, remains poorly characterized at the most basic level. A clearer understanding of the regulation of Ca channels, as sought by the proposed project, will be of fundamental importance. Such understanding also promises to help elucidate the mechanism of arrhythmias associated with Ca overload states.
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