STRUCTURE/FUNCTION OF CARDIAC SODIUM/CALCIUM EXCHANGE

心脏钠/钙交换的结构/功能

• 批准号: 6530661
• 负责人: KENNETH PHILIPSON
• 金额: $ 42.11万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6530661
• 关键词: Xenopus oocyte; ankyrins; calcium channel; calcium flux; calcium ion; conformation; dogs; heart contraction; ion transport; membrane transport proteins; protein binding; protein isoforms; protein sequence; protein structure function; restriction fragment length polymorphism; sarcolemma; site directed mutagenesis; sodium channel; sodium ion; tissue /cell culture

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.

肌膜 Na+-Ca2+ 交换器是主要的 Ca2+ 流出机制 在心肌细胞中。 因此，交换器在其中起着至关重要的作用。 心肌收缩力的调节。这方面的分子知识 必需转运蛋白将有助于了解正常和患病的情况 心。 这项研究的长期目标是阐明 Na+-Ca2+交换器的结构和功能，以更好地理解 运输机制和监管。 为实现这一目标，具体目标如下： 1.离子易位。 Na+ 和 Ca2+ 通过交换移动的途径 蛋白质将被测定。 半胱氨酸扫描诱变将 开始分析氨基酸残基的重要性 假设跨膜片段在离子中很重要 易位。 巯基影响的系统分析 半胱氨酸突变体试剂将有助于确定残基的可及性 并将提供易位结构的信息 途径（例如，孔宽度和深度；α-螺旋度）。 更远 芳香族残基、含羟基残基的诱变，以及 还计划在交换器之间保留残留物。 突变体将是 在非洲爪蟾卵母细胞中表达并通过 45Ca2+ 测量进行评估 通量和交换电流。 2. 监管。 Na+-Ca2+交换器是 受到多样化和复杂的监管。 例如，交换器 通过非转运 Ca2+ 与高亲和力的结合来调节 细胞内位点。 此外，交换器还具有自动调节功能 区域，具有锚蛋白结合位点，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、分子生物学来确定 生物学和电生理学。 3.结构。二级方面 Na+-Ca2+交换蛋白的三级结构为 决定。 拓扑将通过多种技术进行评估。 一个 使用半胱氨酸诱变和蛋白质组合的项目 生物化学也正在启动以确定α-的包装 膜内的螺旋。