Modulation of Cardiac E-C Coupling by Sorcin

描述(申请人提供)：兴奋-收缩(E-C)偶联是心肌中一个钙依赖的过程。表面膜的去极化允许少量的钙内流(内向钙电流，ICa)。然后，ICA引起肌浆网(SR)的钙释放，这个过程被称为钙诱导钙释放(CICR)。负责CICR的蛋白质是钙释放通道/Ryanodine受体(RyR)。 孤立的RyR是“急切的钙反应者”，当(Ca)上升时，它会迅速开放，只要(Ca)保持在高水平，它就会保持开放。RyRs作为钙指示剂的这种意愿是令人惊讶的，因为在细胞环境中，RyRs尽管(Ca)很高，但很快就关闭了，从而避免了SR Ca的耗竭和胞质中Ca的溢出。允许在体内关闭RyR的机制(S)尚不清楚。 这一提议将确定RyRs的一种新的钙结合蛋白抑制剂Sorcin是否是在完整细胞中猝灭CICR的机制的重要组成部分。其具体目的是：1)建立山梨素调节心脏RyRs的结构决定因素。我们将确定山梨素与RyRs相互作用的动力学以及磷酸化所起的作用。此外，我们将获得不能在其五个E-F手中的一个或几个上结合钙的山梨素突变体，以及无法进行磷酸化的山梨素突变体；特别感兴趣的是F112L-山梨素，这是一种与家族性肥厚型心肌病和高血压相关的山梨素突变体。2)探讨山梨素对心肌细胞钙释放和E-C偶联的调节作用。我们将确定山梨素对完整和通透性心肌细胞局部和整体钙释放的影响。我们还将确定在心室细胞中过表达和消融表达Sorin的效果。最后，我们将鉴定一只山梨素缺失的转基因小鼠。

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.