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，是心脏中兴奋-收缩耦合的新型调节剂。我们之前已经描述了 sorcin 与肌浆网的心脏 Ca 释放通道/ryanodine 受体 (RyR2) 的关联。使用体外和体内功能测定，我们发现 sorcin 1) 直接与 RyR2s 结合并具有快速动力学，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 yR2s 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蛋白靶标转位到膜结合蛋白靶标，5) 减弱 Ca 火花和 Ca 瞬变在完整细胞中，6) 被 PKA 磷酸化，从而减弱其对 RyR2s 的抑制作用。最近，我们培育了一个具有 SRI 基因消融的小鼠品系，SRI 是人类和多种动物物种中 sorcin 的编码基因。在基础条件下，这些小鼠的心室肌细胞表现出明显正常的 Ca 瞬变和收缩。然而，sorcin-ko 小鼠的表观平衡是不稳定的，因为 a) 运动测试很快使这些小鼠陷入异常心电活动（快速心律失常） 和心脏骤停），b）sorcin-ko 心脏的 β 肾上腺素刺激导致心律失常和颤动，c）用异丙肾上腺素刺激的分离心肌细胞显示 Ca 振荡、后收缩和延迟后除极。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。该提案的目标是确定 sorcin 在心脏电电耦合中的功能作用，在很大程度上确定其缺失影响的分子和细胞功能，并剖析导致异常电行为的机制。我们假设在正常心室肌细胞中，sorcin 至少与 e-c 耦合的四个关键参与者结合，其总体作用是细胞质 Ca2 清除剂。我们建议：1）确定sorcin与e-c偶联关键蛋白相互作用的分子决定因素； 2）确定sorcin在正常电-电耦合中的综合作用，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.