verbatim): In mammalian cardiac muscle the process of excitation contraction (E-C) coupling is mediated by Ca entry through voltage dependent dihydropyridine receptor channels (DHPRs) in the surface membrane, which activated Ca release through ryanodine receptor channels (RyRs) in the membrane of the sarcoplasmic reticulum (SR). The overall goal of this proposal is to understand how SR Ca release is controlled by Ca and the mechanisms underlying two pathological alterations of E-C coupling: failure of release and spontaneous release. To accomplish this goal, we will use a unique combination of three approaches. (1) The first, which has been developed in the P.I.'s laboratory, integrates a pulsed UV laser into a RyR reconstitution (i.e., bilayer) system, such that caged Ca (i.e., DM-nitrophen or NP-EGTA) is used to generate fast (less than 1 ms) Ca spikes that mimic the Ca trigger associated with single DHPR channel openings to activate single RyR channels. Preliminary experiments have assessed the kinetic limits of the effective trigger signal and have quantified the RyR response with submillisecond time resolution. This type of information, unobtainable until now, will provide a conceptual framework for understanding activation, modulation and failure of Ca release in the heart. (2) The second approach uses confocal Ca imaging, in both intact and permeabilized cardiac myocytes, to study the activity and interactions or RyRs in their native environment. Preliminary studies, using the combination of RyR reconstitution and imaging techniques have revealed a novel form of regulation of Ca release that involves Sr luminal Ca-sensing sites. This type of regulation might be responsible for the initiation and prolongation of spontaneous Ca release under conditions of excessive cellular Ca load. (3) The third approach employs mathematical modeling of single-channel gating and intracellular Ca dynamics. It will provide a framework for evaluating the experimental results and for investigating phenomena that are not accessible to measurement. The goals of this project are to utilize these three approaches to define the key Ca signaling mechanism at three levels of the E-C coupling process: (1) interaction between the activities of single DHPRs and RyRs; (2) interaction among adjacent RyRs forming a functional release unit; and (3) interaction between adjacent release units. These studies will provide new knowledge regarding the operation of E-C coupling in normal and diseased hearts and suggest rational strategies for the treatment of pathological conditions such as certain forms of heart failure and arrhythmia.
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