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2002 Fiscal Year Final Research Report Summary

Study on Intracellular Localization and Functional Regulation of the Gastric Proton Pump.

Research Project

Project/Area Number 13672276
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biological pharmacy
Research InstitutionTOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY

Principal Investigator

ASANO Shinji  Toyama Medical and Pharmaceutical University, Life Scientific Research Center Associate professor, 生命科学実験センター, 助教授 (90167891)

Project Period (FY) 2001 – 2002
KeywordsProton pump / Structure-function relationship / Site-directed mutagenesis / Active transport / Gastric acid secretion / Inhibitor / Proteasome / Ubiqutin
Research Abstract

The gastric H^+,K^+-ATPase is the proton pump responsible for gastric acid secretion. This pump consists of the catalytic α- and non-catalytic β-subunits. In this research project, we constructed stable cell lines expressing the gastric proton pump, and studied the regulation mechanisms by site-directed mutahenesis.
(1) We constructed three kinds of stable cell lines ; the α-expressing cells, the β-expressing cells, and the α+β-expressing cells. The α-subunit was retained in the intracellular compartment, and no cell surface expression was observed in the absence of the β-subunit. On the other hand, cell surface expression of the β-subunit was observed even in the absence of the α-subunit. Cell surface expression of the α- and β-subunits were observed in the α+β-expressing cells. The α+β-expressing cells represented rubidium (^<86>Rb) and proton transport activities, which were inhibited by proton pump inhibitors.
(2) We also studied the quantity control mechanism of the gastric proton pump in the stable cell lines. The α- and (β-subunits were co-translationally inserted to the ER membrane to form the functional holoenzyme with a stoichiometry of 1 : 1. In this process, unassembled α-subunits were unstable in the cells. They were retained on the ER, modified with chains of polyubiquitin, and degraded by the proteasomes. On the other hand, unassembled β-subunits were more stable, and can travel to the cell surface by itself. We found that the number of functional proton pump, the α/β holoenzyme, was strictly regulated by ubiqutin/proteasome system.
(3) The β-subunit contains three conserved disulfide bond in the ectodomain. We studied the role of these disulfide bonds on the function of the gastric proton pump by site-directed mutagenesis. We found that each disulfide bond was important for the correct assembly between the α- and β-subunits, and their cell surface delivery, and the maintenance of H^+,K^+-ATPase activity.

  • Research Products

    (24 results)

All Other

All Publications (24 results)

  • [Publications] Kimura, T. et al.: "Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system"Biochemistry. (印刷中). (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Machen, T. et al.: "pH of TGN and recycling endosomes of H^+/K^+-ATPase-transfected HEK 293 cells : Implications for pH regulation in the secretory pathway"American Journal of Physiology. (印刷中). (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kimura, T. et al.: "Mutational study on the roles of disulfide bonds in the β-subunit of gastric H^+, K^+-ATPase"Journal of Biological Chemistry. 277. 20671-20677 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kimura, T. et al.: "Stable expression of gastric proton pump activity at cell surface"Journal of Biochemistry. 131. 923-932 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tabuchi, Y. et al.: "Development and characterization of conditionally immortalized gastric epithelial cell lines from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene"Cell Structure Function. 27. 71-79 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tabuchi, Y. et al.: "Identification of genes responsive to sodium butyrate in colonic epithelial cells"Biochem. Biophys. Res. Commun.. 293. 1287-1294 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Asano, S. et al.: "Mutational Analysis of Gastric Proton Pump, α-and β-Subunit""Mechanism and Consequences of Proton Transport"(Kluwer Academic Publishers). 59-70 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takahashi, Y. et al.: "Expression of ATP1AL1, a non-gastric proton pump, in human colorectum"Japanese Journal of Physiology. 52. 317-321 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sakai, H. et al.: "Molecular and pharmacological properties of inwardly rectifying K^+ channels of human lung cancer cells"European Journal of Pharmacology. 435. 125-133 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Asano, S. et al.: "Alanine-scanning mutagenesis of the sixth transmembarne segment of gastric H^+, K^+-ATPase"Journal of Biological Chemistry. 276. 31265-31273 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tabuchi, Y. et al.: "Cibenzoline, an ATP-sensitive K^+ channel blocker, binds to the K^+ -binding site from the cytoplasmic side of gastric H^+, K^+-ATPase"British Journal of Pharmacology. 134. 1655-1662 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sakai, H. et al.: "Is rabbit CLCA1 related to the basolateral Ca^<2+> -dependent Cl^-channel of gastric parietal cells?"Japanese Journal of Physiology. 51. 121-125 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 浅野真司: "消化管上皮組織機能とイオンチャネル"医学のあゆみ. 201. 856-860 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kimura, T. et al.: "Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system."Biochemistry (in press).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Machen, T. et al.: "pH of TGN and recycling endosomes of H^+/K^+-ATPase-transfected HEK 293 cells : Implications for pH regulation in the secretory pathway."American Journal of Physiology (in press).

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kimura, T. et al.: "Mutational study on the roles of disulfide bonds in the β-subunit of gastric H^+,K^+-ATPase."Journal of Biological Chemistry. 277. 20671-20677 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kimura, T. et al.: "Stable expression of gastric proton pump activity at cell surface."Journal of Biochemistry. 131. 923-932 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tabuchi, Y. et al.: "Development and characterization of conditionally Immortalized gastric epithelial cell lines from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene."Cell Structure Function. 27. 71-79 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tabuchi, Y. et al.: "Identification of genes responsive to sodium butyrate in cplonic epithelial cells."Biochemical Biophysical Research Communication. 293. 1287-1294 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Takahashi, Y. et al.: "Expression of ATP1AL1, a non-gastric proton pump in human colorectum."Japanese Journal of Physiology. 52. 317-321 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sakai, H. et al.: "Molecular and pharmacological properties of inwardly rectiiying K^+ channels of human lung cancer cells."European Journal of Pharmacology. 435. 125-133 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Asano, S. et al.: "Alanine-scanning mutagenesis of the sixth trans-membrane segment of gastric H^+,K^+-ATPase."Journal of Biological Chemistry. 276. 31265-31273 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tabuchi, Y. et al.: "Cibenzoline, an ATP-sensitive K^+ channel blocker, binds to the K^+-binding site from the cytoplasmic side of gastric H^+,K^+-ATPase."British Journal of pharmacology. 134. 1655-1662 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sakai, H. et al.: "Is rabbit CLCA1 related to the basolateral Ca^<2+>-dependent Cl^- channel of gastric parietal cells ?"Japanese Journal of Physiology. 51. 121-125 (2001)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2004-04-14  

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