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Developing a reconstructing system that compensates for a missing link in the regulation of L-type Ca channels in cardiac myocytes

Research Project

Project/Area Number 14370013
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field General physiology
Research InstitutionHIROSHIMA UNIVERSITY

Principal Investigator

YAMAOKA Kaoru  Hiroshima University, Graduate School of Biomedical Sciences, Associate Prof., 大学院・医歯薬学総合研究科, 助教授 (10200586)

Co-Investigator(Kenkyū-buntansha) 木下 英司  広島大学, 大学院・医歯薬学総合研究科, 助手 (80304418)
Project Period (FY) 2002 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2004: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2003: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2002: ¥4,800,000 (Direct Cost: ¥4,800,000)
KeywordsL-type Ca channel / Phosphorylation / rat / Adenovirus / Ion-selectivity / gene transfer / アデノウイルス / イオン透過性 / ランタン / Naチャネル / イオンフィルター
Research Abstract

The channel activity is augmented by phsophorylation through cAMP-dependent kinase : maximum activation of phosphorylation can increase peak amplitude of Ca^<2+> currents up to 5-6 folds of the control current. The fact that inability to reconstruct the regulation of L-type Ca channels through cAMP-dependent phosphorylation by transducing L-type Ca channel genes in heterologous expression system hampers the development of elucidating molecular mechanisms in the regulation of L-type Ca channels. We assume that heterologous expression systems lack a regulatory factor in controlling the activity of L-type Ca channels from cardiac origin which should exist in native cardiac cells. Thus, our strategy to fully reconstruct the regulation of L-type Ca channel is transducing L-type Ca channel genes into cardiac myocyte itself. In order to achieve these objects, we have two problems to overcome, 1) differentiating function of artificially introduced Ca channels from those originally expressed in … More cardiac myocytes, 2) obtaining functional virus vectors containing L-type Ca channel gene. For the first one, we tested the ion selectivity of mutated L-type Ca channels, such as EEKA or DEKA type of which the portion of the ion selective filter was changed from EEEE of wild type. The results indicated that both mutants have 10 times higher permeability of Na^+ against Cs^+. Ba^<2+> permeability was abolished in DEKA type, while EEKA type has 56 times higher permeability of Ba^<2+> against Cs^+. DEKA type acquired resistance to La^<3+> block. With using this Na^+-selective Ca channel mutants, we would be able to selectively record currents from transduced L-type Ca channels in cardiac myocytes in the presence of tetrodotoxin. For the second one, we could insert Ca_V 1.2 gene in the adenovirus vector that lacks E1/E3 region. Unfortunately, we were unable to amplify virus particles in HEK293 cells due to the size of insert (8kb). A recent report of Ganeasn AN et al.indicated that they could successfully transduce Ca_V1.2 gene in cardiac myocytes by constructing adenovirus vector that lacks fiber gene in addition to E1/E3 region (BBRC 749-754,2005). We are hoping to introduce their technology for further development of our Ca channel study. Less

Report

(4 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (14 results)

All 2005 2004 2003 Other

All Journal Article (12 results) Publications (2 results)

  • [Journal Article] Na^+ channel pharmacology and molecular mechanisms of gating.2005

    • Author(s)
      Yamaoka K, Vogel SM, Seyama I
    • Journal Title

      Current Pharmaceutical Design (in press)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Na^+ channel pharmacology and molecular mechanisms of gating.2005

    • Author(s)
      Yamaoka K, Vogel SM, Seyama I
    • Journal Title

      Curr Pharm Des (in press)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Na^+ channel pharmacology and molecular mechanisms of gating.2005

    • Author(s)
      Yamaoka K et al.
    • Journal Title

      Current Pharmaceutical Design (in press)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Identification of a non-selective cation channel current in myometrial cells isolated from pregnant rats.2004

    • Author(s)
      Miyohsi H, Yamaoka K, Garfield RE, Ohama K
    • Journal Title

      Pfliugers Archiv European Journal of Physiology 447

      Pages: 457-464

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A quantitative and comparative study of the effects of a synthetic ciguatoxin CTX3C on the kinetic properties of voltage-dependent sodium channels.2004

    • Author(s)
      Yamaoka K, Inoue M, Miyahara H, Miyazaki K, Hirama M
    • Journal Title

      British Journal of Pharmacology 142

      Pages: 879-889

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Identification of a non-selective cation channel current in myometrial cells isolated from pregnant rats.2004

    • Author(s)
      Miyoshi H, Yamaoka K, Garfield RE, Ohama K
    • Journal Title

      Pflugers Arch 447

      Pages: 457-464

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A quantitative and comparative study of the effects of a synthetic ciguatoxin CTX3C on the kinetic properties of voltage-dependent sodium channels.2004

    • Author(s)
      Yamaoka K, Inoue M, Miyahara H, Miyazaki K, Hirama M
    • Journal Title

      Br J Pharmacol 142

      Pages: 879-886

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A quantitative and comparative study of the effects of a synthetic ciguatoxin CTX3C on the kinetic properties of voltage-dependent sodium channels.2004

    • Author(s)
      Yamaoka K et al.
    • Journal Title

      British Journal of Pharmacology 142・5

      Pages: 879-889

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Distinct sites regulating grayanotoxin binding and unbinding to D4S6 of Na_v 1.4 sodium channel as revealed by improved estimation of toxin sensitivity.2003

    • Author(s)
      Maejima H, Kinoshita E, Seyama I, Yamaoka K
    • Journal Title

      Journal of Biological Chemistry 278

      Pages: 9464-9471

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Regulation of L-type Ca^<2+> channels in the heart : overview of recent advancers.2003

    • Author(s)
      Yamaoka K, Kameyama M
    • Journal Title

      Molecular and Cellular Biochemistry 253

      Pages: 3-13

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Distinct sites regulating grayanotoxin binding and unbinding to D4S6 of Na_V1.4 sodium channel as revealed by improved estimation of toxin sensitivity.2003

    • Author(s)
      Maejima H, Kinoshita E, Seyama I, Yamaoka K
    • Journal Title

      J Biol Chem 278

      Pages: 9464-9471

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Regulatioin of L-type Ca^<2+> channels in the heart : overview of recent advances.2003

    • Author(s)
      Yamaoka K, Kameyama M
    • Journal Title

      Mol Cell Biochem 253

      Pages: 3-13

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] Yamaoka K, Kameyama M: "Regulation of L-type Ca^<2+> channels in the heart : overview of recent advancers"Molecular and Cellular Biochemistry. 253(1-2). 3-13 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] K Yamaoka, M Kameyama: "Regulation of L-type Ca^<2+> channels in the heart : overview of recent advancers"Molecular and Cellular Biochemistry. (In press). (2003)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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