MODULATORY MECHANISMS OF RYANODINE RECEPTOR ADAPTATION

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In cardiac and in skeletal muscle, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is central to excitation-contraction coupling, the process that links membrane depolarization to mechanical contraction. However, 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, a self-limiting mechanism stabilizes CICR and prevents depletion of Ca2+ from the SR. The identity of this mechanism is unknown. A fundamental property of Ca2+ release channels/ryanodine receptors (RYR) termed adaptation may be the mechanism that counters the inherent positive feedback of CICR. Adaptation allows RYRs to close (adapt) even though an increased [Ca2+] around the channel is maintained. However, the time constant of adaptation measured in vitro (seconds) is much slower than that of the negative feedback mechanism that controls CICR in vivo (milliseconds). Thus, it is essential to establish the physiological relevance of RYR adaptation. This proposal seeks to determine mechanisms of cardiac and skeletal RYR adaptation to establish its physiological significance. Single RYRs will be reconstituted in lipid bilayers and the (Ca2+) in the microenvironment of the channel will be changed by laser flash photolysis of caged Ca2+. The (Ca2+) change produced by this system may be controlled by varying the concentration of caged Ca2+, the power output of the laser lamp, and the rate of Ca2+ diffusion. Moreover, the use of NP-EGTA, a novel caged Ca2+ with low affinity for Mg2+, will allow us to establish the RYR response to Ca2+ in the presence of physiological concentrations of Mg and ATP, two critical modulators of RYR function. We propose: (1) to define the elementary properties of adaptation in cardiac and skeletal RYRs; (2) to measure RYR adaptation in the presence of all relevant modulators of Ca2+ release; (3) to evaluate the functional consequences of RYR phosphorylation on the kinetics of adaptation, and (4) to identify a role for FKBP12 in the mechanism of RYR adaptation using RYRs expressed without FKBP12 and RYRs coexpressed with FKBP12. These studies are 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, and recover from the adapted state. The work will consequently provide fundamental new information on the regulation of contraction in cardiac and skeletal muscle.