Cytosolic Calcium Sweeper in Cardiac Myocytes

描述（由申请人提供）：Sorcin是一种在哺乳动物组织中广泛表达的~22-kDa Ca结合蛋白，是心脏兴奋-收缩偶联的新型调节剂。我们以前的特点与心脏钙释放通道/Ryanodine受体（RyR 2）的肌浆网sorcin协会。使用体外和体内功能测定，我们发现sorcin 1）以快速动力学直接结合RyR 2s，快速抑制单通道活性，2）经历Ca依赖性构象变化，从而调节其对yR 2s的亲和力，3）定位于心室心肌细胞中的z线，4）以Ca依赖性方式从可溶性易位至膜结合蛋白靶，5）减弱完整细胞中的Ca火花和Ca瞬变，6）被PKA磷酸化，这反过来减弱其对RyR 2的抑制作用。最近，我们产生了一个基因消除SRI的小鼠品系，SRI是人类和多种动物物种中编码sorcin的基因。在基础条件下，这些小鼠的心室肌细胞显示出明显正常的Ca瞬变和收缩。然而，sorcin-ko小鼠中的表观平衡是不稳定的，因为a）运动测试很快使这些小鼠进入异常的心脏电活动（快速性心律失常 和心脏骤停），B）sorcin-ko心脏的β肾上腺素能刺激导致心律失常和纤维性颤动，和c）用异丙肾上腺素刺激的分离的心肌细胞显示Ca振荡、后收缩和延迟的后去极化。因此，sorcin的消融使基础心脏活动几乎完好无损，但导致自律性增加。这项计划的目标是确定sorcin在心脏电-电偶联中的功能作用，主要是确定其缺失所影响的分子和细胞功能，并剖析导致异常电行为的机制。我们假设，在正常的心室肌细胞，sorcin结合到至少四个关键球员的电子-C耦合，其整体效果是一个胞质Ca 2+清扫。我们建议：1）鉴定sorcin与E-C偶联的关键蛋白相互作用的分子决定因素;和2）确定sorcin在正常E-C偶联中的综合作用，以及在其缺失时导致异常电行为的病理生理机制。

DESCRIPTION (provided by applicant): Sorcin, a ~22-kDa Ca-binding protein widely expressed in mammalian tissues, is a novel regulator of excitation-contraction coupling in the heart. We have previously characterized the association of sorcin with the cardiac Ca release channel/ryanodine receptor (RyR2) of the sarcoplasmic reticulum. Using in vitro and in vivo functional assays, we found that sorcin 1) binds to RyR2s directly and with fast kinetics, rapidly inhibiting single channel activity, 2) undergoes Ca-dependent conformational changes, thereby modulating its affinity for yR2s, 3) localizes to z-lines in ventricular cardiomyocytes, 4) translocates from soluble to membrane bound protein targets in a Ca-dependent manner, 5) attenuates Ca sparks and Ca transients in intact cells, 6) is Phosphorylated by PKA, which in turn attenuates its inhibitory effect on RyR2s. Recently, we generated a Mouse line with genetic ablation of SRI, the gene encoding for sorcin in humans and multiple animal species. Under basal conditions, ventricular myocytes from these mice show apparently normal Ca transients and contractions. However, the apparent equilibrium in sorcin-ko mice is precarious, because a) exercise tests quickly throw these mice into aberrant cardiac electrical activity (tachyarrhythmias and sudden cardiac arrest), b) beta adrenergic stimulation of sorcin-ko hearts leads to arrhythmias and fibrillation, and c) isolated cardiomyocytes stimulated with Isoproterenol display Ca oscillations, aftercontractions, and delayed afterdepolarizations. Thus, ablation of sorcin leaves basal cardiac activity almost intact, but leads to increased automaticity. The goal of this proposal is to determine the functional role of sorcin in e-c coupling of the heart, in great part y identifying the molecular and cellular functions affected by its absence, and dissecting the mechanisms that lead to aberrant electrical behavior. We hypothesize that in normal ventricular myocytes, sorcin binds to at least four key players of e-c coupling, with its overall effect being that of a cytosolic Ca2+ sweeper. We propose: 1) to identify the molecular determinants of sorcin interaction with key proteins of e-c coupling; and 2) to determine the integrated role of sorcin in normal e-c coupling, and the pathophysiological mechanisms that lead to aberrant electrical behavior in its absence.