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Molecular basis for regulation of intracellular environment and function of ion transporting proteins

Research Project

Project/Area Number 17370046
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionOsaka University

Principal Investigator

KANAZAWA Hiroshi  Osaka University, Graduate School of Science, Professor, 大学院理学研究科, 教授 (50116448)

Co-Investigator(Kenkyū-buntansha) FUKUYAMA Keiichi  Osaka University, Graduate School of Science, Professor, 大学院理学研究科, 教授 (80032283)
MITSUI Keiji  Osaka University, Graduate School of Science, Assist Professor, 大学院理学研究科, 助手 (60379279)
MATSUSHITA Masafumi  Osaka University, Graduate School of Science, Assist Professor, 大学院理学研究科, 助手 (50403100)
Project Period (FY) 2005 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥13,800,000 (Direct Cost: ¥13,800,000)
Fiscal Year 2006: ¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 2005: ¥7,300,000 (Direct Cost: ¥7,300,000)
Keywordsregulation of ion environment / membrane ion transport / Na^+ / H^+ antiporters / calcineurin homologus protein 1 / Bioenergetics ofmembrane transport / 膜イオン輸送機構 / 膜輸送の分子機構 / 蛋白質の機能解析 / 細胞内の蛋白質局在化 / 生体エネルギー共役 / 蛋白質工学 / 分子遺伝学
Research Abstract

Intracellular concentrations of ions including pH and Na^+ are maintained at certain values. Na^+/H^+ antiporters required for these regulations exit in cytoplasmic and endocytic membranes as well and play a central role. In this study we aimed to clarify ion transport mechanisms including the binding sites of the trasporting ions in the antiporters. Main results of this study are classified into the following three groups. (1) The structure and function relationship of H.pylori Na^+/H^+ antiporter NhaA (HPNhaA) has been studied by means of biochemical and molecular biological approaches. The residues involved in the ion binding and transport have been newly identified for HPNhaA and the results were published in Biochemistry ( two other manuscripts are in preparation for publication). To determine the atomic structure of NhaA, a large scale of purified HPNhaA was successfully prepared to make a crystal. Further a new approach using FRET analysis has been successfully applied to detect … More conformational changes expected for ion transporting process (JBC (2005)). (2) Three new isoforms of human Na^+/H^+ antiporter (NHE) have been identified and revealed to localize in endocytic membranes. These isoforms were clarified to function as a K^+/H^+ antiporter and localize in different compartments depending on isoform type (JBC(2005)). DT40 cells with knocked out gene for CHP which can bind to NHE1 has been established. In these cells, CHP is missing and NHE1 activity was reduced to less than 90 % of the wild type. Surprisingly NHE was degraded in these cells, indicating that CHP is essentail for stabilizing NHE. This gives us a new concept of CHP1 with NHE (Amer.J.Phys (2007)). Further we determined crystal structure of CHP1. The crystal structure suggested a binding mechanism of CHP1 wuth NHE1 (JBC(2005)). (3) We have established a new procedure to detect Nhalp activity of S. cereviciae. Application of this method gave us stoichiometry of ions (Na^+/H^+) being electrogenic (BBA(2005)). The Nhalp was found to form a dimer which is essential in functioning (BBA(2005)).
Our achievements were presented in several meeting including 2006 International Biochemistry Meeting, European Bioenergetics meeting, and so on. Less

Report

(3 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • Research Products

    (26 results)

All 2007 2006 2005 Other

All Journal Article (26 results)

  • [Journal Article] Loss of Calcineurin Homologous Protein 1 (CHP1) in chicken B lymphoma DT40 cells destabilizes Na^+/H^+ exchanger isoform 1 (NHE 1) protein2007

    • Author(s)
      Masafumi Matsushita他
    • Journal Title

      Amer. J. Phys 6月号(印刷中)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Na^+/H^+交換輸送タンパク質の作動機構と制御 : 細菌からヒトまで2007

    • Author(s)
      金澤 浩 他
    • Journal Title

      生化学 79(印刷中)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Loss of Calcineurin Homologous Protein 1 (CHP1) in chicken B lymphoma DT40 cells destabilizes Na^+/H^+ exchanger isoform 1 (NHE1) protein2007

    • Author(s)
      Masafumi Matsushita^*, Yoshie Sano^*, Shyunsuke Yokoyama^*, Tomoyo Takai^*, Hiroki Inoue^*, Keiji Mitsui^*, Kagefumi Todo^†, Hitoshi Ohmori^†, Hiroshi Kanazawa^*
    • Journal Title

      Amer.J.Phys. (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Structure and function of Na^+/H^+ antiporters : molecular basis and regulation2007

    • Author(s)
      H.Kanazawa, K.Mitsui, M.Martsuhshita
    • Journal Title

      Seikagaku (Japanese) (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Nuclear localization of the serine/threonine kinase DRAK2 is required for UV induced apoptosis.2006

    • Author(s)
      Hiroshi Kuwahara 他
    • Journal Title

      Biol. Pharm. Bullt. 29

      Pages: 225-233

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Structure-function relationship of the fifth transmembrane domain in the Na+/H+ antiporer of Helicobacter pylori : Topology and function of the residues including two consecutive essential aspartate residues2006

    • Author(s)
      Naoyuki Kuwabara, 他
    • Journal Title

      Biochemistry 45

      Pages: 14834-14842

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Nuclear localization of the serine/threonine kinase DRAK2 is required for UV induced apoptosis.2006

    • Author(s)
      Hiroshi Kuwahara, Norihiro Nakamura, Hiroshi Kanazaw
    • Journal Title

      Biol.Pharm.Built. 29

      Pages: 225-233

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Structure-function relationship of the fifth transmembrane domain in the Near antiporer of Helicobacter pylori : Topology and function of the residues including two consecutive essential aspartate residues.2006

    • Author(s)
      Naoyuki Kuwabara, Hiroki Inoue, Yumi, Tsuboi, Keiji Mitsui, Masafumi Matsushita, Hiroshi Kanazawa
    • Journal Title

      Biochemistry 45

      Pages: 14834-14842

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Nuclear localization of the serine/threonine kinase DRAK2 is required for UV induced apoptosis.2006

    • Author(s)
      Hiroshi Kuwahara
    • Journal Title

      Biol. Pharm. Bullt. 29

      Pages: 225-233

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Structure-function relationship of the fifth transmembrane domain in the Na^+/H^+ antiporer of Helicobacter pylori : Topology and function of the residues including two consecutive essential aspartate residues.2006

    • Author(s)
      Naoyuki Kuwabara
    • Journal Title

      Biochemistry 45

      Pages: 14834-14842

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Nuclear localization of the serine/threonine kinase DRAK2 is required for UV induced2006

    • Author(s)
      Hiroshi Kuwahara 他
    • Journal Title

      Biol.Pharm.Bullt 280

      Pages: 225-233

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Four Na^+/H^+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation.2005

    • Author(s)
      NorihiroNakamura, 他
    • Journal Title

      J. Biol. Chem. 280

      Pages: 1561-1572

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Characterization of the ion transport activity of budding yeast Na^+/H^+ antiporter, Nhalp, on the secretory vesicles2005

    • Author(s)
      Ryuichi Ohgaki他
    • Journal Title

      Biochim. Biophys. Act. 1712

      Pages: 185-196

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Structural Characterization of Calcineurin B Homologous Protein 12005

    • Author(s)
      Youichi Naoe他
    • Journal Title

      J. Biol. Chem. 280

      Pages: 32372-32378

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Detection of an oligomer formation and conformational change of Na^+/H^+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer analysis2005

    • Author(s)
      Akira Karasawa他
    • Journal Title

      J. Biol. Chem 1720

      Pages: 125-136

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Oligomerization of the Saccharomyces cerevisiae Na^+/H^+ antiporter Nhalp : Implications for its antiporter activity2005

    • Author(s)
      Keiji Mitsui他
    • Journal Title

      Biophys Biochim Act. Biomembrane 1720

      Pages: 125-136

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Four Na^+/H^+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation.2005

    • Author(s)
      Norihiro Nakamura, Shingo Tanaka, Yoshinori Teko, Hiroshi Kanazawa
    • Journal Title

      J.Biol.Chem. 280

      Pages: 1561-1572

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Characterization of the ion transport activity of budding yeast Na^+/H^+ antiporter, Nhalp, on the secretory vesicles2005

    • Author(s)
      Ryuichi Ohgaki, Norihiro Nakamura, Keiji Mitsui, Hiroshi Kanazawa
    • Journal Title

      Biochem.Biophys.Act Biomembrane 1712

      Pages: 185-196

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Structural Characterizationof Calcineurin B Homologous Protein 12005

    • Author(s)
      Youichi Naoe, Kyouhei Arita, Hiroshi Hashimoto, Hiroshi Kanazawa, Mamoru Sato, Toshiyuki Shimizu
    • Journal Title

      J.Biol.Chem. 280

      Pages: 32372-32378

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Detection of an oligomer formation and conformational change of Na^+/H^+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer analysis2005

    • Author(s)
      Karasawa, Yumi Tsuboi, Hiroki Inoue, Rie Kinoshita, Norihiro Nakamura, Hiroshi Kanazawa
    • Journal Title

      J.Biol.Chem. 280

      Pages: 41900-41911

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Oligomerization of the Saccharomyces cerevisiae antiporter Nhalp : Implications for its antiporter activity2005

    • Author(s)
      Keiji Mitsui, Hidetomo Yasui, Norihiro Nakamura, Hiroshi Kanazawa
    • Journal Title

      Biophys Biochim Act.Biomembrane 1720

      Pages: 125-136

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Four Na^+/H^+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation2005

    • Author(s)
      Norihiro Nakamura 他
    • Journal Title

      J.Biol.Chem. 280

      Pages: 1561-1572

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Characterization of the ion transport activity of budding yeast Na^+/H^+ antiporter, Nha1p, on the secretory vesicles2005

    • Author(s)
      Ryuichi Ohgaki 他
    • Journal Title

      Biochem.Biophys.Act Biomembrane 1712

      Pages: 185-196

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Structural Characterization of Calcineurin B Homologous Protein 12005

    • Author(s)
      Youichi Naoe 他
    • Journal Title

      J.Biol.Chem. 280

      Pages: 32372-32378

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Detection of an oligomer formation and conformational change of Na^+/H^+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer analysis2005

    • Author(s)
      Akira Karasawa 他
    • Journal Title

      J.Biol.Chem. 280

      Pages: 41900-41911

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Loss of Calcineurin Homologous Protein 1 (CHP1) in chicken B lymphoma DT40 cells destabilizes Na^+/H^+ exchanger isoform 1 (NHE1) protein

    • Author(s)
      Masafumi Matsushita
    • Journal Title

      Amer.J.Phys. (印刷中)

    • Related Report
      2006 Annual Research Report

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Published: 2005-04-01   Modified: 2016-04-21  

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