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

描述(申请人提供)：心肌细胞质膜(肌膜)具有高水平的Na+-Ca~(2+)交换活性。Na+-Ca~(2+)交换是细胞内Ca~(2+)的重要调节因子，因此是心肌收缩能力的主要决定因素。Na+-Ca~(2+)交换上调，在肥厚和心力衰竭时可能具有额外的意义。研究心肌Na+-Ca~(2+)交换分子的结构和分子性质对于了解该交换分子在生理学和病理生理学中的作用具有重要意义。为实现这一目标，《建议》的具体目标如下：1.结构和监管。Na+-Ca~(2+)交换受细胞内非转运的Ca~(2+)调节。最近已经确定了钙离子调控位点的结构。目标有两个：a.确定Na+-Ca~(2+)交换器细胞内环的其他部分的结构。B.应用结构信息在分子水平上了解钙离子的调节。2.螺旋堆积、二聚化和运输途径。目的是进一步研究Na+-Ca~(2+)交换器内和交换器二聚体之间跨膜片段的堆积排列。突变方法将被用来理解离子转移的机制和途径。3.应用荧光共振能量转移(FRET)技术研究Na~+-Ca~(2+)交换剂。其目的是应用FRET来研究交换器在体外、细胞内和活体中的构象变化。FRET还将用于研究交换器的二聚化。潜在的假设是，对Na+-Ca~(2+)交换分子的结构、功能和调节的了解的增加将直接导致对交换分子在生理和病理生理环境中的作用的更好的理解。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.