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。为实现这一目标，该提案的具体目标如下： 1.结构：阻聚填料。Na+-Ca 2+交换器具有9个跨膜区段（TMS）。目的是确定这些TMS在质膜内的包装安排。该方法将主要使用半胱氨酸诱变与交联技术的组合。 2.机制与规范。这个目标有五个部分：A。充电运动。确定与交换剂离子移位相关的电荷移动中涉及的残基。最初的实验将使用嵌合体方法。B.失活机制。验证TMS 2在Na+依赖性失活中起关键作用的假设。Na+依赖性失活是Na+-Ca 2+交换活性的关键调节因子。C.第一重入回路在交换器功能中的作用。确定重入式回路结构在换热器调节和输送中的重要性。D. Ca 2+结合和Ca 2+诱导的构象变化的动力学。确定调节性Ca 2+结合的动力学和随后的构象变化。交换器转运Ca 2+，但也受Ca 2+在高亲和力调节位点的调节。 e. FRET在Na+-Ca ~（2+）交换剂研究中的应用应用荧光共振能量转移（FRET）技术监测活细胞内交换剂的构象变化。交换器将用绿色荧光蛋白（GFP）的变体标记。

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: Helix Packing. The Na+-Ca2+ exchanger has nine transmembrane segments (TMSs). The objective is to determine the packing arrangement of these TMSs within the plasma membrane. The approach will primarily use a combination of cysteine mutagenesis in conjunction with crosslinking techniques. 2. Mechanism and Regulation. There are five parts to this aim: a. Charge movements. To determine residues involved in charge movements associated with ion translocation by the exchanger. Initial experiments will use a chimera approach. b. Mechanism of inactivation. To test the hypothesis that TMS 2 has a key role in Na+-dependent inactivation. Na+-dependent inactivation is a key regulator of Na+-Ca2+ exchange activity. c. Role of the first reentrant loop in exchanger function. To determine the importance of the structure of a reentrant loop in regulation and transport by the exchanger. d. Kinetics of Ca2+ binding and Ca2+-induced conformational changes. To determine the kinetics of the binding of regulatory Ca2+ and the subsequent conformational change. The exchanger transports Ca2+ but is also regulated by Ca2+ at a high affinity regulatory site. e. Application of FRET to study the Na+-Ca2+ exchanger. To apply fluorescence resonance energy transfer (FRET) for monitoring conformational changes of the exchanger within a living cell. Exchangers will be labeled with variants of green fluorescent protein (GFP).