| Project/Area Number |
03557005
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurophysiology and muscle physiology
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| Research Institution | Gunma University |
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Principal Investigator |
OZAWA Seiji Gunma University, School of Medicine, Professor, 医学部, 教授 (40049044)
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| Co-Investigator(Kenkyū-buntansha) |
TAKEUCHI Toshiyuki Gunma University, Institute for Endocrinology, Professor, 内分泌研究所, 教授 (00109977)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| 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)
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| Keywords | Glutamate 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 |
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| 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.
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