Modulation of Cardiac E-C Coupling by Sorcin

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DESCRIPTION (provided by applicant): Excitation-contraction (E-C) coupling is a Ca-dependent process in cardiac muscle. Depolarization of the surface membrane permits a small influx of Ca (the inward Ca current, ICa). ICa then evokes Ca release from the sarcoplasmic reticulum (SR), a process termed Ca-induced Ca release (CICR). The protein responsible for CICR is the Ca release channel/ryanodine receptor (RyR). Isolated RyRs are "eager Ca responders" that quickly open when (Ca) rises and remain open as long as (Ca) remains high. This willingness of RyRs to act as Ca indicators is surprising given that in cellular settings RyRs quickly close despite (Ca) being high, thereby avoiding SR Ca depletion and overflowing of cytosolic compartments with Ca. The mechanism(s) that allows RyR closure in vivo is unknown. This proposal will determine whether sorcin, a novel Ca-binding protein inhibitor of RyRs, is an important component of the mechanism that quenches CICR in intact cells. The specific aims are: 1) To establish the structural determinants of sorcin modulation of cardiac RyRs. We will determine the kinetics of sorcin interaction with RyRs and the role played by phosphorylation. Also, we will obtain sorcin mutants that are unable to bind Ca in one or several of its five E-F hands as well as sorcin mutants unable to undergo phosphorylation; of special interest is F112L-sorcin, a sorcin mutant associated with Familial Hypertrophic Cardiomyopathy and hypertension. 2) To determine the role of sorcin in regulation of Ca release and E-C coupling of cardiac cells. We will determine effects of sorcin in localized and global Ca release in intact and permeabilized cardiac myocytes. We will also determine the effects of overexpression and ablated expression of sorcin in ventricular cells. Finally, we will characterize a sorcin-null transgenic mouse.