BIOGENESIS AND FUNCTION OF NA/K ATPASE SUBUNITS

NA/K ATP酶亚基的生物发生和功能

• 批准号: 2182495
• 负责人: KUNIO TAKEYASU
• 金额: $ 8.76万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182495
• 关键词: Xenopus oocyte; atomic force microscopy; calcium transporting ATPase; cations; chimeric proteins; chymotrypsin; conformation; electron microscopy; enzyme mechanism; enzyme structure; ion transport; molecular cloning; ouabain; potassium; protein sequence; sodium; sodium potassium exchanging ATPase; structural biology; tissue /cell culture; toxin; transfection; trypsin; vanadium

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.

Na/K-ATP酶（钠泵;强心苷的受体，如 哇巴因）是一种多亚基整合膜蛋白，其功能 （Na+和K+离子的ATP依赖性转运）在维持 和调节心脏功能。 在此前的支持下，我们 确定了哇巴因结合区、Na和K敏感区， 亚基组装域，并提出了一个“功能域模型”， 钠泵α亚单位 阐明空间组织 这些功能结构域的研究对于理解 钠泵功能的机制。 我们最近开始使用 原子力显微镜（AFM）是一种先进的物理技术， 几埃的分辨率，并确定了通道状结构的 泵分子 这是AFM技术引入的第一个例子 生物医学科学的新信息。 根据我们的新 信息和技术技能，我们建议确定的作用， α亚基的Na+和K+敏感结构域（离子传感器）， 阐明这些功能域的空间组织。 这 将通过实现以下具体目标来实现这一目标： 1：确定离子敏感结构域在Na/K- ATP酶α-亚基（氨基末端69个氨基酸（Met 1-Leu 69）， Na+和羧基末端161个氨基酸（Ser 830-COOH）（K+），和 以确定离子传输的关键区域。 目标2：将 从初级结构到高级结构。 目标1将是 通过使用重组DNA和基因转移技术来解决。 使用 在先前的研究中构建的所有钠泵/钙泵嵌合体 以及一组在组织中表达的新嵌合体- 培养细胞和非洲爪蟾卵母细胞，我们将研究离子的作用- 敏感域和搜索关键域所需的离子 运输 目标2将通过使用结构中的技术来解决 生物学 在过去的四年里，我们已经开发出了 准备技术的使用原子力显微镜，现在提出相关 “通道样”结构与钠的独特构象， 通过比较已知离子的充分表征的生物化学效应， 和毒素的影响 我们计划使用电子 显微镜（EM）作为比较标准，并用于一般分析 蛋白质制剂在整个建议，虽然EM提供较低 决议。 这些目标的实现将回答以下问题 理解分子结构的基本问题 离子泵的功能关系一般：“如何做Na+-和 α亚单位的K+感受器调节钠泵活性？", “Na+和/或K+对α亚基的哪些结构域是必需的 易位？离子和抑制剂是如何修饰“通道- 比如钠泵蛋白的构象". 所提出的方法 也有可能检测到P型的构象变化， ATP酶，并将是第一次有机会可视化的实际变化， 离子泵的分子结构