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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043138-08
Application #
2882772
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Hamilton, Frank A
Project Start
1990-08-01
Project End
2001-02-28
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
8
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Lapierre, Lynne A; Avant, Kenya M; Caldwell, Catherine M et al. (2007) Characterization of immunoisolated human gastric parietal cells tubulovesicles: identification of regulators of apical recycling. Am J Physiol Gastrointest Liver Physiol 292:G1249-62
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

Showing the most recent 10 out of 14 publications