1991 Fiscal Year Final Research Report Summary
Molecular genetical analysis on structure and function of GABA_A and GABA_B receptors
| Project/Area Number |
01480143
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General pharmacology
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| Research Institution | Kyoto Prefectural University of Medicine |
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Principal Investigator |
KIRIYAMA Kinya Kyoto Pref. Univ. of Med., Dept. of Pharmacla. Instructor, 医学部, 教授 (20079734)
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| Co-Investigator(Kenkyū-buntansha) |
HIROUCHI Masaaki Kyoto Pref. Univ. of Med., Dept. of Pharmacol. Instructor, 医科部, 助手 (70181196)
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| Project Period (FY) |
1989 – 1991
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| Keywords | GABA_A receptor / GABA_B receptor / Purification / cDNA cloning / Primary structure / Monoclonal antibody / Reconstitution / Functional expression |
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| Research Abstract |
Molecular genetical and molecular pharmacological studies on cerebral GABA_A and GABA_B receptors have been employed and the following results were obtained.
1) The alpha_1-subunits of human brain GABA_A receptor consists of 456 amino acid residues and its sequence is similar to that of bovine brain. 2) The single mRNA (4.6Kb) for GABA_A receptor alpha^<>1-subunit is expressed in human brain. 3) The alpha_1-subunit of GABA_A receptor in the human brain possesses exon-intron organization. 4) The alpha_1-subunit of GABA_A receptor in the human brain consists of four transmembrane domains. 5) The microinjection of mRNA for GABA_A receptor alpha_1-subunit from the human brain induces the expression of functionally relevant GABA_A receptor. 6) The GABA_A receptor alpha_1-subunit in the mouse and rat brains is expressed from two mRNAs (4.1 and-4.6K) by an alternative splicing. 7) The 13, 000 fold purification of GABA_B receptor is achieved by baclofen-affinity column chromatography. 8) The GABA_B receptor is negatively coupled with adenylate cyclas, inositol phosphate turnover and calcium channel via Gi or Go protein and these couplings can be eliminated by solubilization of the receptor. 9) The GABA_B receptor is a glycoprotein having the molecular weight of 80 Kda. 10) The GABA_A receptor can be down-regulated by a prolonged exposure to its agonist and this downregulation is caused by the suppression of the expression of mRNA. 11) Both purified GABA_A and GABA_B receptors can be functionally reconstituted on phospholipid vesicles. 12) Monoclonal antibodies for GABA_A and GABA_B receptors (GA-I : Ige, GB-I : IgM) are obtained by the injection of respective receptor protein to Balb/C mouse and by the subsequent screening of specific clones. 13) The derect actions of various drugs on GABA_A and GABA_B receptors can be analyzed using purified and reconstituted each receptor protein.
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