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

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The long term goal of this work is to understand the mechanism of H+ secretion by parietal cells of mammalian gastric mucosa. The pathophysiology of gastric ulcers is intimately related to H,K-ATPase activity, which is inhibited directly by omeprazole and indirectly by H2 antagonists. A poorly understood aspect of H+ transport in epithelia is the mechanisms by which the scalar energy of ATP is converted into vectorial energy manifested in a H+ gradient. The H,K-ATPase is a unique experimental model for this mechanism, being readily isolated from the gastric mucosa, where it forms a million fold gradient of H+ across the gastric epithelium. Cloning and sequencing of H,K-ATPase cDNA has revealed the primary structure of the pump. Hydropathy analysis predicts multiple transmembrane alpha-helices which conceivably form H+ and K+ pores. Experimental confirmation or refutation important in modelling these mechanisms is knowledge of H,K-ATPase conformational changes occurring during transport. In this study, the orientation of the H,K-ATPase in the parietal cell apical membrane will be probed with monoclonal antibodies (Mab). The sidedness of Mab and by immunoelectron microscopy. Epitopic domains of the H,K-ATPase. Ligand-induced (E1-E2) and stimulation-induced conformational transitions will be documented using Mab, gastric vesicles, and isolated parietal cells. The study will provide direct evidence for H+ pump topography, will clarify conformational transitions essential to transport, will aid in the interpretation of H,K-ATPase electron diffraction data, will inform site-directed mutagenesis, and will contribute to understanding of the molecular mechanism of H+ transport.