Modulation of Cardiac E-C Coupling by Sorcin

描述（由申请人提供）：兴奋-收缩（E-C）偶联是心肌中的钙依赖性过程。 表面膜的去极化允许少量的Ca内流（内向Ca电流，伊卡）。 然后，伊卡引起Ca从肌浆网（SR）释放，这一过程称为Ca诱导的Ca释放（CICR）。 负责CICR的蛋白质是Ca释放通道/ryanodine受体（RyR）。 孤立的RyR是“渴望钙反应者”，当（钙）上升时迅速打开，只要（钙）保持高水平，就保持打开。 RyR充当Ca指示剂的这种意愿是令人惊讶的，因为在细胞环境中，尽管（Ca）高，RyR仍快速闭合，从而避免SR Ca耗尽和Ca溢出细胞溶质区室。 允许体内RyR闭合的机制尚不清楚。 该提案将确定sorcin，一种新的钙结合蛋白抑制剂RyRs，是否是一个重要组成部分的机制，淬灭CICR在完整的细胞。 具体目的是：1）建立sorcin调节心脏RyRs的结构决定因素。 我们将确定Sorcin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手中的一个或几个中结合Ca的sorcin突变体以及不能进行磷酸化的sorcin突变体;特别感兴趣的是F112 L-sorcin，一种与家族性肥厚性心肌病和高血压相关的sorcin突变体。 2)探讨Sorcin对心肌细胞钙释放和E-C偶联的调节作用。 我们将确定Sorcin在完整和透化心肌细胞中局部和整体钙释放的影响。 我们还将确定心室细胞中sorcin过表达和消融表达的影响。 最后，我们将描述sorcin无效转基因小鼠。

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.