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

描述（由申请方提供）：心肌细胞的质膜（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+的重要调节剂，因此是心肌收缩力的主要决定因素。Na+-Ca 2+交换在肥大和心力衰竭期间上调，并且可能具有额外的意义。研究心脏Na+-Ca 2+交换分子的结构和分子特性对于了解该交换分子在生理和病理生理中的作用具有重要意义。为了实现这一目标，该提案的具体目标如下：1.结构和规则。Na ~+-Ca ~（2+）交换受非转运的胞内Ca ~（2+）调节。Ca 2+调节位点的结构最近已被确定。目标是双重的：A。确定Na+-Ca 2+交换器细胞内环其他片段的结构。B.应用结构信息在分子水平上理解Ca 2+调节。2.阻碍包装、二聚化和运输途径。目的是进一步研究跨膜段内的Na+-Ca 2+交换器和交换器二聚体之间的包装安排。突变方法将用于理解离子易位机制和途径。3.应用荧光共振能量转移技术研究Na+-Ca 2+交换体。其目的是应用FRET研究在体外，在细胞中，并在一个活的有机体中的交换器的构象变化。FRET也将被应用于研究交换剂的二聚化。基本的假设是，增加知识的结构，功能和调节的Na+-Ca 2+交换分子将直接导致更好地了解交换器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.