MECHANISM AND REGULATION OF NA/K-ATPASE

NA/K-ATP酶的作用机制及调控

• 批准号: 6564883
• 负责人: AMIR ASKARI
• 金额: $ 32.14万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564883
• 关键词: active sites; allosteric site; animal tissue; binding sites; cardiac glycosides; cardiac myocytes; conformation; enzyme mechanism; enzyme structure; gene expression; heart contraction; heart pharmacology; membrane transport proteins; molecular cloning; potassium channel; protein structure function; sodium channel; sodium potassium exchanging ATPase

The long term objectives of this research are to advance our knowledge of the molecular mechanism of active transport of Na+ and K+ by NaK-ATPase (the sodium pump); an enzyme of the plasma membrane that maintains the integrity and the excitability of the myocardium, is the receptor for the positive inotropic actions of digitalis drugs, and regulates cardiac genes involved in the hypertrophic growth of the cardiac myocyte. The proposed studies are focused on the recent progress of this laboratory relating the ion transport function of the enzyme to the structures of its transmembrane domains. In experiments of Specific Aim 1, we shall use proteolytically digested and/or chemically modified preparations of the purified enzyme in order (a) to identify the locations of the two distinct cations occulation pockets (the binding sites and their access channels) within different transmembrane helices; and (b) to characterize the properties of the bindings sites and the access channels of each occlusion pocket, and the interactions among the two pockets, by experiments on occlusion-deocclusion kinetics of 86RB+ and 22Na+. Since we have established recently that the catalytic ATP site and the allosteric ATP site are two distinct entities, in studies of Specific Aim 2 we shall first use digested preparations of the enzyme that contain only the allosteric site to identify the amino acid residues involved in this binding site by chemical modification experiments. We shall then alter the identified residues by site-directed mutagenesis, and conduct functional studies on the mutants expressed in insect cells, in order to clarify the postulated roles of the allosteric ATP site in the regulation of the two cation occlusion pockets, and in the turnover of the phosphointermediate. In studies of Specific Aim 3 we shall continue our chemical cross-linking experiment on the digested preparations on the digested preparations of the purified NaK-ATPase to map the three-dimensional packing of the transmembrane helices, and to relate these helix-helix interactions to the functions of the multiple cation occlusion sites and their access channels. These studies will clarify structure-function relationships of an enzyme that is central to the regulation of cardiac contractility and growth in the normal and failing hearts.

这项研究的长期目标是增进我们对 NAK-ATPase主动转运Na+、K+的分子机制 (钠泵)；质膜上的一种酶，维持 心肌的完整性和兴奋性，是 洋地黄类药物的正性变力作用及其对心脏基因的调控 参与心肌细胞的肥大生长。建议数 研究集中在这个实验室的最新进展，涉及 酶对ITS结构的离子转运作用 跨膜结构域。在特定目标1的实验中，我们将使用 蛋白水解物和/或化学修饰的制剂 纯化后的酶(A)分别鉴定两个不同的位置 阳离子掩蔽袋(结合部位及其通道) 在不同的跨膜螺旋中；和(B)表征 每个咬合的结合部位和访问通道的属性 口袋，以及两个口袋之间的相互作用，通过对 ~(86)Rb~+和~(22)Na~+的遮挡-去遮挡动力学因为我们有 新近建立的催化三磷酸腺苷和变构三磷酸 场地是两个截然不同的实体，在具体目标的研究中，我们将 首先使用仅含有 变构位点来鉴定参与这一过程的氨基酸残基 结合部位的化学修饰实验。然后，我们将更改 通过定点突变鉴定残基，并进行功能性 在昆虫细胞中表达的突变体的研究，以澄清 变构ATP位点在两者调控中的假设作用 阳离子堵塞袋，并在磷化中间体的周转。 在具体目标3的研究中，我们将继续进行化学交联。 消解制剂对白藜芦醇消解制剂的影响 纯化的NAK-ATPase用于定位血管的三维堆积 跨膜螺旋，并将这些螺旋-螺旋相互作用与 多个阳离子封闭部位的功能及其通路 频道。这些研究将阐明结构与功能的关系 一种对心脏收缩能力的调节至关重要的酶 在正常和衰竭的心脏中成长。