EPITHELIAL H+ TRANSPORT--STRUCTURE OF H,K-ATPASE

上皮H运输--H,K-ATP酶的结构

• 批准号: 2458773
• 负责人: ADAM J SMOLKA
• 金额: $ 21.1万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2458773
• 关键词: apical membrane; conformation; enzyme structure; gastric acid; gastric mucosa; gastrointestinal absorption /transport; gel electrophoresis; histamine receptor; hydrogen potassium exchanging ATPase; hydrogen transport; ion transport; laboratory mouse; membrane transport proteins; microsomes; monoclonal antibody; potassium; protein structure function; site directed mutagenesis; transfection

The long term goal of this study is to understand the mechanism of H+ secretion by gastric parietal cells. One motivation is the world-wide prevalence of peptic ulcer disease and the relationship between ulcer pathophysiology and acid secretion; equally important is clarification of how an integral membrane protein converts the scalar energy of ATP into the vectorial energy of a proton gradient. The hypothesis driving the study is that specific interactions between alpha and Beta subunits of the H,K-ATPase are central to formation of a million-fold H+ gradient across the parietal cell apical membrane. Tests of this hypothesis require detailed knowledge of alpha and Beta subunit secondary and tertiary structure. Over the years, physiological measurements in isolated cells and membrane vesicles have defined exhaustively the enzyme's kinetic properties, while biochemical and molecular biological approaches have clarified aspects of H,K-ATPase structural organization. However, correlation of structure to function needs a heterologous expression system in which the effects of alpha and Beta subunit mutations on H,K-ATPase function can be studied. To date, no H,K-ATPase expression system has been reported to produce enough functional enzyme for kinetic studies. The preliminary studies for this proposal form the basis of two specific aims: 1) To optimize and validate the insect cell expression system for structure-function studies of H,K-ATPase; and 2) To investigate in insect cells the interaction of exogenous histamine H2 receptor with endogenous signalling pathways and exogenous H,K-ATPase. Insect cells will be infected with recombinant baculoviruses carrying H,K-ATPase alpha and Beta subunit cDNAs and H2 receptor DNA. H,K-ATPase expression will be monitored by immunomicroscopy and immunoblotting, and function will be measured as cytoplasmic alkalinization by BCECF fluorescence, and as SCH28080-sensitive K+-dependent ATPase activity and alpha subunit phosphorylation. Receptor function will be assessed by agonist binding, activation of endogenous G protein and adenylate cyclase, cytoplasmic cAMP elevation, and increases in cytoplasmic [Ca2+]. This study will provide details of H,K-ATPase alpha and Beta subunit interaction, will elucidate signal transduction mechanisms culminating in acid secretion, will inform site-directed mutagenesis of H,K-ATPase, and will contribute to understanding of the molecular mechanism of H+ transport.

本研究的长期目标是了解 H+ 的机制 由胃壁细胞分泌。动力之一是全世界 消化性溃疡病患病率与溃疡病的关系 病理生理学和酸分泌；同样重要的是澄清 完整膜蛋白如何转换 ATP 标量能量 转化为质子梯度的矢量能。假设驱动 该研究表明 α 和 Beta 亚基之间的特定相互作用 H,K-ATPase 的数量对于形成百万倍 H+ 梯度至关重要 穿过壁细胞顶膜。对该假设的检验 需要详细了解 α 和 Beta 亚基的次级和 三级结构。多年来，生理测量 分离的细胞和膜囊泡已经详尽地定义了 酶的动力学特性，而生化和分子生物学 这些方法已经阐明了 H,K-ATP 酶结构组织的各个方面。 然而，结构与功能的相关性需要异源 表达系统中α和β亚基的作用 可以研究 H,K-ATP 酶功能的突变。迄今为止，还没有 H,K-ATP 酶 据报道表达系统可以产生足够的功能酶 用于动力学研究。本提案的初步研究形成 两个具体目标的基础：1) 优化和验证昆虫细胞 H,K-ATP酶结构功能研究的表达系统；和 2) 研究昆虫细胞中外源组胺 H2 的相互作用 具有内源性信号通路和外源性 H,K-ATP 酶的受体。 昆虫细胞将被携带的重组杆状病毒感染 H,K-ATPase α 和 Beta 亚基 cDNA 以及 H2 受体 DNA。 H,K-ATP酶 将通过免疫显微镜和免疫印迹监测表达，并且 功能将通过 BCECF 测量为细胞质碱化 荧光，以及 SCH28080 敏感的 K+ 依赖性 ATP 酶活性和 α亚基磷酸化。受体功能将通过以下方式评估 激动剂结合、内源性 G 蛋白和腺苷酸的激活 环化酶、细胞质 cAMP 升高和细胞质 [Ca2+] 增加。 本研究将提供 H,K-ATPase α 和 Beta 亚基的详细信息 相互作用，将阐明信号转导机制 在酸分泌中，将告知 H,K-ATP 酶的定点诱变， 并将有助于理解H+的分子机制 运输。