Cytosolic Calcium Sweeper in Cardiac Myocytes

描述(申请人提供)：Sorcin是一种在哺乳动物组织中广泛表达的~22 kDa钙结合蛋白，是一种新的心脏兴奋-收缩偶联调节因子。我们先前已经确定了山梨素与肌浆网的心肌钙释放通道/兰诺定受体(RyR2)之间的联系。在体外和体内的功能分析中，我们发现，索菌素1)以快速的动力学方式直接与RyR2结合，迅速抑制单通道活动，2)经历钙依赖的构象变化，从而调节其对yR2的亲和力，3)定位于心肌细胞的z线，4)以钙依赖的方式从可溶性蛋白转位到膜结合的蛋白靶标，5)减弱完整细胞中的钙火花和钙瞬变，6)被PKA磷酸化，从而减弱其对RyR2的抑制作用。最近，我们建立了一个基因消融SRI的小鼠系，SRI是人类和多种动物中编码山梨素的基因。在基础状态下，这些小鼠的心室肌细胞表现出明显的正常钙瞬变和收缩。然而，索尔辛-KO小鼠的表观平衡是不稳定的，因为a)运动测试迅速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)β肾上腺素能刺激心脏导致心律失常和纤颤，以及c)用异丙肾上腺素刺激分离的心肌细胞显示钙振荡、后收缩和延迟后除极。因此，消融山梨素使基础心脏活动几乎保持不变，但会增加自律性。这项建议的目的是确定索尔菌素在心脏e-c偶联中的功能作用，主要是确定它的缺失对分子和细胞功能的影响，并剖析导致异常电行为的机制。我们推测，在正常的心室肌细胞中，山梨素与至少四个e-c偶联的关键分子结合，其总体作用是胞内钙离子清扫器。我们建议：1)确定索尔菌素与e-c偶联关键蛋白相互作用的分子决定因素；2)确定索尔菌素在正常的e-c偶联中的整合作用，以及在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.