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

这项研究的长期目标是了解H+的机制 胃壁细胞分泌。一个动机是世界范围内的 消化性溃疡的患病率及其与溃疡的关系 病理生理学和胃酸分泌；同样重要的是澄清 一种完整的膜蛋白如何转换ATP的标量能量 转化为质子梯度的矢量能量。推动假说的因素 这项研究是α和β亚基之间的特定相互作用 H，K-ATPase是形成百万倍H+梯度的中心 穿过壁细胞顶膜。对这一假设的检验 需要详细的阿尔法和贝塔亚基二级和 三级结构。多年来，生理测量在 分离的细胞和膜泡已经详尽地定义了 酶的动力学性质，而生化和分子生物学 各种方法已经阐明了H，K-ATPase结构组织的各个方面。 然而，结构与功能的关联需要一种异源 表达系统中α和β亚基的作用 可以研究H，K-ATPase功能的突变。到目前为止，没有H，K-ATPase 据报道，表达系统可以产生足够的功能酶 用于动力学研究。对这项建议的初步研究形成了 基于两个具体目标：1)优化和验证昆虫细胞 用于H，K-ATPase结构和功能研究的表达系统； 在昆虫细胞中研究外源性组胺H2的相互作用 具有内源性信号通路和外源性H，K-ATPase的受体。 昆虫细胞将感染携带病毒的重组杆状病毒 H，K-ATPaseα和β亚基cDNA和H2受体DNA。H，K-ATPase 表达将通过免疫显微镜和免疫印迹进行监测，以及 功能将通过BCECF的细胞质碱化来测量 荧光和AS SCH28080敏感的K+依赖的ATPase活性和 α亚基磷酸化。受体功能将通过以下方式进行评估 激动剂结合、激活内源性G蛋白和腺苷 环化酶、胞浆cAMP升高和胞浆[Ca~(2+)]升高。 这项研究将提供H，K-ATPaseα和β亚基的详细信息 相互作用，将阐明最终的信号转导机制 在酸分泌中，将通知H，K-ATPase的定点突变， 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.