BIOGENESIS AND FUNCTION OF NA/K ATPASE SUBUNITS

Na/K-ATPase(钠泵；心脏糖苷的受体，如 哇巴因(哇巴因)是一种多亚基完整膜蛋白，其功能 (依赖于三磷酸腺苷的钠和钾离子的运输)在维持中是必不可少的 和心脏功能的调节。在之前的支持下，我们 鉴定了哇巴因结合区、钠和钾敏感片段，以及 亚基组装域，并提出了一个功能域模型 钠泵α亚基。对空间组织的阐释 这些功能结构域对于理解分子是必不可少的 钠泵功能的机制。我们最近开始使用一种 发展起来的物理技术，原子力显微镜(AFM)，作为一种 较小的埃分辨率，并确定了通道状的结构 泵浦分子。这是原子力显微镜技术引入的第一个例子 生物医学科学的新信息。在我们新的基础上 信息和技术技能，我们建议确定以下角色 α亚基的Na和K敏感结构域(离子传感器)和 阐明这些功能域的空间组织。这 通过实现以下具体目标来实现目标：目标 1：确定离子敏感结构域在Na/K-中的调节作用 ATPaseα亚基(氨基末端69个氨基酸(MET1-Leu69)) Na和羧端161个氨基酸(K)的Ser830-COOH)，以及 以确定离子传输的关键区域。目标2：将 从初级结构到高阶结构。目标1将是 通过使用重组DNA和基因转移技术来解决。vbl.使用 上一次构建的所有钠泵/钙泵嵌合体 支持年份以及在组织中表达的一组新的嵌合体- 培养细胞和非洲爪哇卵母细胞，我们将研究离子- 敏感域并搜索离子所需的关键域 运输。目标2将通过使用结构中的技术来实现 生物学。在过去的四年里，我们研制出了 原子力显微镜的制备技术，现在建议相关 形成了钠离子的独特构象-- 通过比较已知离子的充分表征的生化效应来泵浦 钠泵结构上的毒素。我们计划使用电子 显微镜(EM)作为比较标准和用于一般分析 蛋白质制剂贯穿整个提案，尽管EM提供了较低的 决心。这些目标的实现将回答以下问题 了解分子结构的基本问题- 离子泵的一般函数关系：Na~-和 阿尔法亚单位的钾传感器调节钠泵活动？“， α亚基的哪些结构域对钠和/或钾是必需的 易位？“，以及”离子和抑制剂如何改变‘通道-- 比如“钠泵蛋白的构象？”。建议的方法 也有可能检测到P-型的构象变化- ATPase，这将是第一个将实际变化可视化的机会 离子泵的分子结构。

The Na/K-ATPase (sodium-pump; a receptor for cardiac glycosides, such as ouabain) is a multi-subunit integral membrane protein whose function (ATP-dependent transport of Na+ and K+ ions) is essential in maintenance and regulation of cardiac functions. Under the previous support, we identified the ouabain-binding region, Na- and K-sensitive segments, and the subunit assembly domain, and proposed a 'functional domain model' of the sodium-pump alpha subunit. Elucidation of the spatial organization of these functional domains is essential for understanding the molecular mechanism of the sodium-pump function. We have started to use a recently developed physical technique, atomic force microscopy (AFM) which as a few angstrom resolution, and identified a channel-like structure of the pump molecule. This is the first example that AFM technique brought in new information to biomedical sciences. On the basis of our new information and technical skills, we propose to identify the roles of Na+- and K+-sensitive domains (ion sensors) of the alpha subunit and elucidate the spatial organization of these functional domains. This goal will be attained by accomplishing the following specific aims: Aim 1: to define the regulatory roles of ion-sensitive domains in the Na/K- ATPase alpha-subunit (the amino-terminal 69 amino acids (Met1-Leu69) for Na+ and the carboxy-terminal 161 amino acids (Ser830-COOH) for K+), and to identify critical domains for ion-transport. Aim 2: to correlate the primary structure to the higher-order structures. Aim 1 will be addressed by using recombinant DNA and gene transfer techniques. Using all the sodium-pump/calcium-pump chimeras constructed in the previous support years as well as a new set of chimeras expressed in tissue- cultured cells and Xenopus oocytes, we will examine the roles of the ion- sensitive domains and search for the critical domains required for ion transport. Aim 2 will be addressed by using the techniques in structural biology. Over the past four years, we have developed specimen preparation techniques for the use of AFM, and now propose to correlate the 'channel-like' structure to a distinct conformation of the sodium- pump by comparing well-characterized biochemical effects of known ions and toxins on the sodium-pump structure. We plan to use electron microscopy (EM) as a comparative standard and for general analyses of the protein preparations throughout this proposal, although EM provides lower resolutions. Accomplishments of these aims will answer the following fundamental questions towards understanding the molecular structure- function relationship of ion pumps in general: "How do the Na+- and the K+-sensors of the alpha subunit regulate the sodium-pump activity?", "Which domains of the alpha subunit are essential for Na+ and/or K+ translocation?", and "How do ions and inhibitors modify the 'channel- like' conformation of the sodium-pump protein?". The proposed approaches also have a potential to detect conformational changes of the P-type- ATPase, and will be the first opportunity to visualize actual changes in the molecular structure of ion pumps.