MECHANISM AND REGULATION OF NA/K-ATPASE

这项研究的长期目标是提高我们对 NaK-ATP酶主动转运Na ~+和K ~+分子机制 (the钠泵）;一种质膜酶， 心肌的完整性和兴奋性，是受体的 洋地黄类药物的正性肌力作用，并调节心脏基因 参与心肌细胞的肥大生长。拟议 研究的重点是该实验室的最新进展， 离子转运功能的酶的结构， 跨膜结构域。在具体目标1的实验中，我们将使用 蛋白水解消化的和/或化学修饰的制剂 纯化的酶，以便（a）确定两个不同的位置， 阳离子掩蔽袋（结合位点及其进入通道） 在不同的跨膜螺旋内;和（B）表征 每个闭塞的结合部位和进入通道的特性 口袋，以及两个口袋之间的相互作用，通过实验， ~（86）RB+和~（22）Na+的闭塞-解除闭塞动力学。既然我们有 最近确定，催化ATP位点和变构ATP 网站是两个不同的实体，在研究具体目标2，我们将 首先使用仅含有 变构位点，以确定参与此 结合位点的化学修饰实验。然后，我们将改变 通过定点诱变鉴定残基，并进行功能性 研究昆虫细胞中表达的突变体，以阐明 假设的作用变构ATP网站的调节，这两个 阳离子闭塞口袋，并在磷中间物的周转。 在具体目标3的研究中，我们将继续我们的化学交联 消化制剂试验 纯化的NaK-ATP酶，以绘制三维包装的 跨膜螺旋，并将这些螺旋-螺旋相互作用与 多个阳离子吸附位点的功能及其进入 渠道这些研究将阐明结构与功能的关系， 一种对调节心脏收缩力至关重要的酶， 在正常和衰竭的心脏中生长。

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.