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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043138-05
Application #
2142792
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1990-08-01
Project End
1996-07-31
Budget Start
1994-08-01
Budget End
1996-07-31
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
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Gooz, M; Gooz, P; Smolka, A J (2001) Epithelial and bacterial metalloproteinases and their inhibitors in H. pylori infection of human gastric cells. Am J Physiol Gastrointest Liver Physiol 281:G823-32
Smolka, A J; Larsen, K A; Hammond, C E (2000) Location of a cytoplasmic epitope for monoclonal antibody HK 12.18 on H,K-ATPase alpha subunit. Biochem Biophys Res Commun 273:942-7
Gooz, M; Hammond, C E; Larsen, K et al. (2000) Inhibition of human gastric H(+)-K(+)-ATPase alpha-subunit gene expression by Helicobacter pylori. Am J Physiol Gastrointest Liver Physiol 278:G981-91
Smolka, A J; Larsen, K A; Schweinfest, C W et al. (1999) H,K-ATPase alpha subunit C-terminal membrane topology: epitope tags in the insect cell expression system. Biochem J 340 ( Pt 3):601-11
Kraut, J A; Hiura, J; Shin, J M et al. (1998) The Na(+)-K(+)-ATPase beta 1 subunit is associated with the HK alpha 2 protein in the rat kidney. Kidney Int 53:958-62
Kraut, J A; Hiura, J; Besancon, M et al. (1997) Effect of hypokalemia on the abundance of HK alpha 1 and HK alpha 2 protein in the rat kidney. Am J Physiol 272:F744-50
Harris, A W; Walker, M M; Smolka, A et al. (1996) Parietal cells in the duodenal bulb and their relation to Helicobacter pylori infection. J Clin Pathol 49:309-12
Wingo, C S; Smolka, A J (1995) Function and structure of H-K-ATPase in the kidney. Am J Physiol 269:F1-16
Callaghan, J M; Tan, S S; Khan, M A et al. (1995) Renal expression of the gene encoding the gastric H(+)-K(+)-ATPase beta-subunit. Am J Physiol 268:F363-74

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