Calcium channels in the surface membrane of heart cells play a central role in excitation-contraction coupling, in normal pacemaker activity and in arrhythmogenesis. As the major pathway for Ca influx into the cell, slow Ca channels help determine the free intracellular C 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 broad variety of other cell types. 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 following protein kinase C activation in Aplysia neurones. This project proposes to investigate the basic mechanism(s) of Ca channel regulation by (Ca2+)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 influence the number of functional channels. Single channel recordings will enable characterization of the changes in gating behavior underlying positive and negative regulation. For example, is there a change of open state probability as (Ca2+)i rises? Direct simultaneous measurements of (Ca2+)i (using fura-2) and ICa will establish quantitatively the relationship between these two variables, and will provide direct evidence regarding the (Ca2+)-dependence of positive regulation and inactivation. Finally, measurements of (Ca2+)i and Ca channel current during (Ca2+)i oscillations will help clarify the non-monotonic recovery from inactivation of ICa during Ca overload. Ca2+-regulation of Ca channels, while ubiquitous, remains poorly characterized at the most basic level. A clearer understanding of Ca channel regulation by (Ca2+)i, as sought by the proposed project, will lead to consideration of specific cellular mediators (e.g., protein kinase C). Such understanding also promises to help elucidate the mechanism of arrhythmias associated with Ca overload states.
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