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.
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