CELLULAR BIOLOGY OF CARDIAC SODIUM-CALCIUM EXCHANGE

描述（由申请人提供）：心脏肌层钠钙交换器（NCX）是从肌细胞中挤出钙的主要机制。因此，交换剂是细胞内钙离子和心脏收缩性的重要调节剂。这项应用的长期目标是进一步了解Na-Ca交换器在激发-收缩（EC）耦合中的意义。Na-Ca交换器水平的遗传改变对EC偶联产生了意想不到的影响。建议的两个项目如下：心脏EC偶联对基因改变的Na-Ca交换器水平的适应性。在PI的实验室中，已经创造出Na-Ca交换剂水平发生较大变化的小鼠。这些小鼠中最有趣的是转基因纯合子NCX过表达者（活性增加3倍）和心脏特异性NCX敲除（KO）小鼠。NCX KO小鼠意外地活到了成年，并表现出令人惊讶的EC偶联途径的可塑性。心肌通过减少钙流入心肌细胞的80%来适应初级钙外排机制的缺失。提出了实验来探讨各种机制对这种适应的贡献。2. EC耦合增益改变的生化相关性。肌层Na-Ca交换器的过表达导致二氢吡啶受体（DHPR或l型Ca通道）的膜环境发生变化；在野生型肌细胞中，DHPR位于脂筏中，而在过表达ncx的肌细胞中，DHPR不再在脂筏中分离。与这种再分布相关的是EC耦合增益的降低。我们假设NCX的过表达改变了dhpr和ryanodine受体之间的物理关系，从而导致这些蛋白质之间的偶联减少和增益降低。了解NCX如何与EC耦合机制相互作用对于理解心力衰竭和其他病理中钙通量的改变至关重要。NCX对EC偶联具有意想不到的调节作用和药理意义。

DESCRIPTION (provided by applicant): The cardiac sarcolemmal Na-Ca exchanger (NCX) is the primary mechanism for the extrusion of Ca from myocytes. As such, the exchanger is an important regulator of intracellular Ca and cardiac contractility. The long-term objective of this application is to further understand the significance of the Na-Ca exchanger in excitation-contraction (EC) coupling. Genetic alterations in the level of the Na-Ca exchanger have had unexpected effects on EC coupling. The two proposed projects are as follows: 1. Adaptations of cardiac EC coupling to genetically altered levels of Na-Ca exchanger. Mice with large changes in the level of the Na-Ca exchanger have been created in the laboratory of the PI. The most interesting of these mice are transgenic homozygous NCX overexpressors (>3-fold increase in activity) and cardiac-specific NCX knockout (KO) mice. The NCX KO mice unexpectedly live to adulthood and demonstrate a surprising plasticity of EC coupling pathways. The myocardium adapts to the absence of the primary Ca efflux mechanism by decreasing Ca influx into myocytes by 80%. Experiments are proposed to explore the contributions of various mechanisms to this adaptation. 2. A biochemical correlate of altered gain of EC coupling. Overexpression of the sarcolemmal Na-Ca exchanger induces a change in the membrane environment of the dihydropyridine receptor (DHPR or L-type Ca channel); the DHPR is located in lipid rafts in wild type myocytes, but the DHPR no longer fractionates in lipid rafts in NCX-overexpressing myocytes. Correlating with this re-distribution is a decrease in the gain of EC coupling. We hypothesize that overexpression of NCX changes the physical relationship between DHPRs and ryanodine receptors which leads to diminished coupling between these proteins and a decrease in gain. Understanding how NCX interacts with the EC coupling machinery is essential to understanding the altered Ca fluxes in heart failure and other pathologies. NCX has unexpected regulatory effects on EC coupling with pharmacological implications.