CELLULAR BIOLOGY OF CARDIAC SODIUM-CALCIUM EXCHANGE

描述(申请人提供)：心肌细胞膜Na-Ca交换器(NCX)是从心肌细胞中排出钙的主要机制。因此，交换器是细胞内钙和心肌收缩能力的重要调节器。这一应用的长期目标是进一步了解钠-钙交换器在兴奋-收缩(EC)耦合中的意义。钠钙交换器水平的遗传变化对EC偶联产生了意想不到的影响。提出的两个项目如下：1.心脏内皮细胞偶联对Na-Ca交换器基因改变的适应。在PI的实验室里，已经建立了钠钙交换水平变化较大的小鼠。其中最有趣的是转基因纯合子NCX过表达(活性增加3倍)和心脏特异性NCX基因敲除(KO)小鼠。NCX KO小鼠出人意料地活到成年，并展示了令人惊讶的EC偶联通路的可塑性。心肌通过减少80%的钙内流来适应主要的钙外流机制的缺失。建议进行实验，以探索各种机制对这种适应的贡献。2.EC偶联增益变化的生化相关性。肌膜Na-Ca交换器的过表达导致二氢吡啶受体(DHPR或L型钙通道)膜环境的改变；在野生型心肌细胞中，DHPR位于脂筏中，而在NCX高表达的心肌细胞中，DHPR不再分布在脂筏中。与这种重新分布相关的是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弱和增益下降。了解NCX如何与EC偶联机制相互作用，对于了解心力衰竭和其他病理过程中改变的钙流是至关重要的。NCX对EC偶联有意想不到的调节作用，并具有药理学意义。

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.