Structure and Function of the Cardiac Na+-Ca2+ Exchanger

描述（由申请人提供）：心肌细胞的质膜（肌膜）具有高水平的Na+-Ca2+交换活性。Na+- Ca2+交换是细胞内Ca2+的重要调节因子，因此是心肌收缩性的主要决定因素。Na+-Ca2+交换上调，可能在肥厚和心力衰竭期间具有额外的意义。研究心脏Na+-Ca2+交换分子的结构和分子特性对了解交换分子在生理和病理生理中的作用具有重要意义。为实现这一目标，本建议的具体目的如下：结构和规则。Na+-Ca2+交换器由非运输的细胞内Ca2+调节。Ca2+调控位点的结构最近被确定。目的有两个：a.确定Na+-Ca2+交换器胞内环的其他片段的结构。b.应用结构信息在分子水平上理解Ca2+调控。2. 螺旋包装，二聚化和运输途径。目的是进一步研究Na+-Ca2+交换剂内和交换剂二聚体之间跨膜段的包装排列。突变方法将用于了解离子转运的机制和途径。3. 荧光共振能量转移（FRET）在Na+-Ca2+交换剂研究中的应用。目的是应用FRET来研究交换剂在体外、细胞和活的有机体中的构象变化。FRET也将应用于研究二聚化的交换。潜在的假设是，增加对Na+- Ca2+交换分子的结构，功能和调节的知识将直接导致更好地理解交换分子在生理和病理生理环境中的作用。增加的知识可能会有药理学和临床应用。

DESCRIPTION (provided by applicant): The plasma membrane (sarcolemma) of cardiac myocytes has high levels of Na+-Ca2+ exchange activity. Na+- Ca2+ exchange is an important regulator of intracellular Ca2+ and thus a major determinant of myocardial contractility. Na+-Ca2+ exchange is upregulated and may take on added significance during hypertrophy and heart failure. It is important to investigate the structure and molecular properties of the cardiac Na+-Ca2+ exchange molecule to gain understanding of the role of the exchanger in physiology and pathophysiology. Towards this goal, the specific aims of the proposal are as follows: 1. Structure and Regulation. The Na+-Ca2+ exchanger is regulated by nontransported intracellular Ca2+. The structure of the Ca2+ regulatory site has recently been determined. The goals are twofold: a. Determine the structures of other segments of the intracellular loop of the Na+-Ca2+ exchanger. b. Apply structural information to understand Ca2+ regulation at the molecular level. 2. Helix Packing, Dimerization, and Transport Pathway. The aim is to further investigate the packing arrangement of transmembrane segments within the Na+-Ca2+ exchanger and between exchanger dimers. Mutational approaches will be used to understand the ion translocation mechanism and pathway. 3. Application of Fluorescent Resonance Energy Transfer (FRET) to Study the Na+-Ca2+ Exchanger. The aim is to apply FRET to investigate conformational changes of the exchanger in vitro, in cells, and in a living organism. FRET will also be applied to investigate dimerization of the exchanger. The underlying hypothesis is that increased knowledge of the structure, function, and regulation of the Na+- Ca2+ exchanger molecule will directly lead to a better understanding of the role of the exchanger in physiological and pathophysiological settings. The increased knowledge will perhaps have pharmacological and clinical applications.