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

本研究的长期目标是了解H+的机制 由胃壁细胞分泌。一个动机是世界范围内 消化性溃疡的患病率与溃疡的关系 病理生理学和酸分泌;同样重要的是澄清 一个完整的膜蛋白如何将ATP的标量能量 转化为质子梯度的矢量能量。假设驱动 研究是α和β亚基之间的特异性相互作用 的H，K-ATP酶是核心的形成百万倍的H+梯度 穿过壁细胞顶膜对这一假设的检验 需要α和β亚基二级的详细知识， 三级结构多年来， 分离的细胞和膜囊泡已经详尽地定义了 酶的动力学特性，而生物化学和分子生物学 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的 表达系统，其中α和β亚基的作用 可以研究对H，K-ATP酶功能的突变。到目前为止，没有H，K-ATP酶 已经报道表达系统产生足够的功能酶 用于动力学研究。对这一建议的初步研究构成了 基于两个具体目标：1）优化和验证昆虫细胞 用于H，K-ATP酶结构-功能研究的表达系统;和2） 在昆虫细胞中研究外源组胺H2 受体与内源性信号通路和外源性H，K-ATP酶。 昆虫细胞将感染重组杆状病毒携带 H，K-ATP酶α和β亚基cDNA和H2受体DNA。H，K-ATP酶 通过免疫显微镜和免疫印迹监测表达， 功能将通过BCECF测量为细胞质碱化 荧光，以及SCH 28080敏感性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、胞浆cAMP升高和胞浆[Ca 2 +]增加。 本研究将提供H，K-ATP酶α和β亚基的详细信息 相互作用，将阐明信号转导机制，最终 在酸分泌中，将告知H，K-ATP酶定点突变， 这将有助于理解H+的分子机制 运输

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.