verbatim): In cardiac and skeletal muscle, the dihydropyridine receptor (DHPR) of the external membrane and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum are key components of excitation-contraction (EC) coupling, the series of events that link an electrical stimulus (depolarization) to a mechanical contraction. Ca2+ induced Ca2+ release (CICR) is a Ca2+-amplification process with an increasingly evident participation in skeletal muscle and a well-established role in cardiac muscle. Nevertheless, a fundamental problem hinders our understanding of CICR. Because Ca2+ is the triggering signal and the output signal of CICR, the process is expected to be self-perpetuating and all-or-none. In intact cells however, CICR is finely graded and may be arrested by suppressing the inward Ca2+ current, ICa. Thus, self-limiting mechanisms of Ca2+ release operate in vivo to stabilize CICR and to prevent depletion of Ca2+ from the SR. The identity of these mechanisms is unknown. In this proposal, we will investigate if adaptation and Ca2+-dependent inactivation, two mechanisms intrinsic to RyRs, modulate the RyR response to Ca2+ and counter the inherently positive feedback of CICR. These different and probably complementary mechanisms may endow the RyRs with the functional flexibility to both transiently increase their level of response to incoming Ca2+ stimuli (adaptation) and to turn offtheir activity once a critical Ca2+ level has been reached at their cytosolic face (Ca2+-dependent inactivation). Using single-channel recording systems that allow fast and calibrated changes of [Ca2+] in the medium around RyRs, we propose to study the dynamic interaction of RyRs with cellular ligands that accelerate adaptation and Ca2+-dependent inactivation, specifically, Ca2+ ions, Mg2+ ions, and cADPr.
The specific aims are: 1) To determine the RyR response to Ca2+ using a whole range of fast and calibrated Ca2+ stimuli. 2) To probe for the structural determinants of adaptation and Ca2+ dependent inactivation. 3) To elucidate the mechanism of action of cADPr on cardiac RyRs. The information derived from these studies will be important to understand how Ca2+ and other cytosolic factors control the number of open channels at any given time, and the rate at which RyRs open, adapt, inactivate, and recover from the inactivated state.
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