STRUCTURE/FUNCTION OF CARDIAC SODIUM/CALCIUM EXCHANGE

肌膜Na~+-Ca~(2+)交换是钙离子外流的主要机制 在心肌细胞中。因此，交易所在 心肌收缩能力的调节。关于这一点的分子知识 必要的运输者将帮助了解正常和疾病 红心。这项研究的长期目标是阐明 进一步了解Na~+-Ca~(2+)交换器的结构和功能 运输的机制和调节。 为实现这一目标，具体目标如下：1.离子转运。 Na~+和Ca~(2+)通过交换的运动途径 蛋白质将被测定。半胱氨酸扫描突变将是 开始分析氨基酸残基在人体内的重要性 跨膜片段被认为在离子交换中是重要的 易位。巯基作用的系统分析 半胱氨酸突变体上的试剂将有助于确定残基的可及性 并将提供有关易位结构的信息 路径(例如，孔的宽度和深度；阿尔法螺旋度)。进一步 芳香族残基、含羟基残基和 在交易所之间保存的残留物也在计划中。变种人将会是 非洲爪哇卵母细胞表达及~(45)Ca~(2+)测定 通量和交换电流。2.监管。钠-钙离子交换器是 受到不同和复杂的监管。例如，交换器 受未转运的钙离子与高亲和力结合的调节 胞内位置。此外，该交换机还具有自动调节功能 区域，具有一个锚定蛋白结合部位，并受阴离子刺激 磷脂，如PIP2。这些现象的分子基础将 结合蛋白质生物化学、分子生物学 生物学和电生理学。3.结构。次要的各方面 和Na+-Ca2+交换蛋白的三级结构 下定决心。拓扑将通过各种技术进行评估。一个 结合半胱氨酸诱变和蛋白质的项目 生物化学也在启动以确定α-的包装 膜内的螺旋。

The sarcolemmal Na+-Ca2+ exchanger is the dominant Ca2+ efflux mechanism in myocardial cells. Thus, the exchanger has a critical role in the regulation of myocardial contractility. Molecular knowledge of this essential transporter will help understand both normal and diseased hearts. The long-term objective of this research is to elucidate the structure and function of the Na+-Ca2+ exchanger to better understand the mechanism and regulation of transport. Toward this goal, specific aims are as follows: 1. Ion translocation. The pathway for the movement of Na+ and Ca2+ through the exchange protein will be determined. Cysteine-scanning mutagenesis will be initiated to analyze the importance of amino acids residues in transmembrane segments hypothesized to be important in ion translocation. Systematic analysis of the effects of sulfhydryl reagents on cysteine mutants will help determine residue accessibility and will provide information on the structure of the translocation pathway (e.g., pore width and depth; alpha-helicity). Further mutagenesis of aromatic residues, hydroxyl-containing residues, and residues conserved among exchangers is also planned. Mutants will be expressed in Xenopus oocytes and assessed by measurements of 45Ca2+ fluxes and exchange currents. 2. Regulation. The Na+-Ca2+ exchanger is subject to diverse and complex regulation. For example, the exchanger is regulated by the binding of nontransported Ca2+ to a high affinity intracellular site. In addition, the exchanger has an autoregulatory region, has an ankyrin-binding site, and is stimulated by anionic phospholipids such as PIP2. The molecular bases of these phenomena will be determined using a combination of protein biochemistry, molecular biology, and electrophysiology. 3. Structure. Aspects of the secondary and tertiary structure of the Na+-Ca2+ exchange protein will be determined. Topology will be assessed by a variety of techniques. A project using a combination of cysteine mutagenesis and protein biochemistry is also being initiated to determine the packing of alpha- helices within the membrane.