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An attempt to establish clonal cell strains expressing glutamate receptor channels.

Research Project

Project/Area Number 03557005
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Neurophysiology and muscle physiology
Research InstitutionGunma University

Principal Investigator

OZAWA Seiji  Gunma University, School of Medicine, Professor, 医学部, 教授 (40049044)

Co-Investigator(Kenkyū-buntansha) TAKEUCHI Toshiyuki  Gunma University, Institute for Endocrinology, Professor, 内分泌研究所, 教授 (00109977)
Project Period (FY) 1991 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥10,400,000 (Direct Cost: ¥10,400,000)
Fiscal Year 1992: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1991: ¥7,000,000 (Direct Cost: ¥7,000,000)
KeywordsGlutamate receptor / AMPA / Kainate / Expression vector / Transfection / Clonal cell strain / Patch-clamp / Fura-2 / cDNA / 発現プロモーター / fura-2 / cDNA発現
Research Abstract

GluR1-GluR3 cDNAs encoding non-NMDA glutamate receptor subunits have been isolated from the rat brain (Boulter al., Science, 249:1033, 1990). Either homomeric or heteromeric receptors composed of these subunits respond to not only AMPA and quisqualate, but also kainate. The purpose of the present study is to establish clonal cell strains that express the functional channels composed of these subunits in order to facilitate biochemical, physilogical and pharmacological studies of glutamate receptors.
GluR1 and GluR3 cDNAs were kindly provided by Dr. Jim Boulter (The Salk Institute, USA). We have confirmed that AMPA, kainate and glutamate induce current responses in Xenopus oocytes injected with in vitro transcribed RNA from either GluR1 or GluR3 cDNA. GluR1 or GluR3 cDNA was first cloned into pKan 2 vector with the neomycine-resistant gene. This vector containing either GluR1 or GluR3 cDNA in the sense direction was then subcloned into a pcDL-sR alpha 296 expression vector. This expression vector construct was transfected into the rat glioma C6 cells by electroporation. These C6 cells were placed under the neomycin selection. The selected C6 cells were subjected to radioligand binding assay for the non-NMDA receptor. The specific binding of [^3H] AMPA was detected in these cells. Next, we examined whether functional non-NMDA receptors were expressed in these cells. Whole-cell patch-clamp studies indicated that AMPA kainate and glutamate failed to induce electrophysiological responses. Since homomeric GluR1 or GluR3 receptors are known to be highly permeable to Ca^<2+>, we also tried to detect an increase in cytosolic Ca^<2+> concentration in response to these agonists with the use of fura-2 microfluorometry. However, these agonists failed to change the cytosolic Ca^<2+> concentration in these cells. Thus, more work is needed to establish clonal cell strains expressing functional GluR receptor channels.

Report

(3 results)
  • 1992 Annual Research Report   Final Research Report Summary
  • 1991 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] 小澤 瀞司: "中枢ニューロンのグルタミン酸受容体チャネル" 膜. 16(5). 258-269 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] 小澤 瀞司: "グルタミン酸レセプターCa^<2+>透過性" 実験医学. 10(6). 611-618 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] TSUZUKI,K.et al: "Agonist-and subunit-dependent potentiation of glutamate receptors by a nootropic drug aniracetam." Molecular Brain Research. 16. 105-110 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] 小澤 瀞司: "培養海馬ニューロンのグルタミン酸受容体チャネル" 神経精神薬理. 15(2). 109-117 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] OZAWA,S.: "Glutamate receptor channels in hippocampal neurons." Japanese Journal of Physiology.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Tsuzuki, K. et al.: "Agonist- and subunit-dependent potentiation of glutamate receptors by a nootropic drug aniracetam." Molecular Brain Research. 16. 105-110 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Ozawa, S.: "Glutamate receptor channels in hippocampal neurons." Japanese Journal of Physiology.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Tsuzuki,K.et al.: "Agonist-and subunit-dependent potentiation of glutamate receptors by a nootropic drug aniracetam." Molecular Brain Research. 16. 105-110 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 小澤 瀞司: "培養海馬ニューロンのグルタミン酸受容体チャネル" 神経精神薬理. 15(2). 109-117 (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Ozawa,S.: "Glutamate receptor channels in hippocampal neurons." Japanese Journal of Physiology.

    • Related Report
      1992 Annual Research Report
  • [Publications] Keisuke Tsuzuki et al.: "Agonist- and subunit-dependent potentiation of glutamate receptors by a nootropic drug aniracetam." Molecular Brain Research.

    • Related Report
      1991 Annual Research Report
  • [Publications] Seiji Ozawa et al.: "Two distinct types of responses to kainate and AMPA in cultured hippocampal neurons.In Excitatory Amino Acids (Vol.9).ed.by R.P.Simon." Thieme Medical Publishers,Inc.New York, 7 (1992)

    • Related Report
      1991 Annual Research Report

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Published: 1991-04-01   Modified: 2016-04-21  

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