REGULATION OF THE CARDIAC NA+/CA++ EXCHANGER

该项目的总体目标是确定分子 心脏Na/Ca交换系统的调节机制。 的 需要检验的主要假设是， 涉及细胞膜的中心亲水结构域内的特定区域， 交换载体 Na/Ca交换系统是一种质膜 载体介导的转运过程， 离子在一个方向上的运动到一个单一的钙离子在相反的方向上的运动 方向 它是心肌细胞中主要的Ca流出过程， 在调节心脏收缩力中起主要作用。 克隆的 牛心脏Na/Ca交换器在CHO中永久表达 细胞和这些细胞中交换活动的调节行为 其特征在于（a）其对细胞的依赖性 ATP水平和细胞内Ca，（B）蛋白激酶和 磷酸酶活性和（c）可能参与 氨基磷脂转位酶活性介导ATP依赖性 调控 为了限定亲水结构域的区域， 参与运输活动的监管，一系列删除 将制备突变体，并研究它们的调节和功能特性 将通过在COS细胞中的瞬时表达来检测。 最后，具体 亲水结构域内可能涉及的区域 交易所活动的监管将被改变， 这些突变体的诱变和调节行为将被检查 在转染的COS和/或CHO细胞中。 了解机制 参与调节Na/Ca交换活性将提供重要的 对心脏收缩力调节的认识， 心力衰竭的病理生理学

The overall objective of this project is to define the molecular mechanism of regulation of the cardiac Na/Ca exchange system. The primary hypothesis to be tested is that regulation of exchange activity involves specific regions within the central hydrophilic domain of the exchange carrier. The Na/Ca exchange system is a plasma membrane carrier-mediated transport process which couples the movement of 3 Na ions in one direction to the movement of a single Ca ion in the opposite direction. It is the principal Ca efflux process in cardiac myocytes and plays a major role in regulating cardiac contractility. The cloned bovine cardiac Na/Ca exchanger has been permanently expressed in CHO cells and the regulatory behavior of exchange activity in these cells will be characterized with respect to (a) its dependence upon cellular ATP levels and intracellular Ca, (b) the influence of protein kinases and phosphatases on activity and (c) the possible involvement of aminophospholipid translocase activity in mediating ATP-dependent regulation. To define regions of the hydrophilic domain that are involved in the regulation of transport activity, a series of deletion mutants will be prepared and their regulatory and functional properties will be examined by transient expression in COS cells. Finally, specific regions within the hydrophilic domain that might be involved in the regulation of exchange activity will be altered by site-directed mutagenesis and the regulatory behavior of these mutants will be examine in transfected COS and/or CHO cells. Understanding the mechanisms involved in regulating Na/Ca exchange activity will provide important insights into the regulation of cardiac contractility and the pathophysiology of cardiac failure.