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Biochemical Studies on the Molecular Mechanism of Neurotransmission, Centered on Nicotinic Acetylcholine Receptor (nAChR)

Research Project

Project/Area Number 63480218
Research Category

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

Allocation TypeSingle-year Grants
Research Field Neurology
Research InstitutionGifu Pharmaceutical University

Principal Investigator

HAYASHI Kyozo  Gifu Pharmaceutical University Pharmacy, Professor, 薬学部, 教授 (00029935)

Co-Investigator(Kenkyū-buntansha) ADACHI Tetsuo  Gifu Pharmaceutical University Pharmacy, Lecturer, 薬学部, 助手 (40137063)
NOMOTO Hiroshi  Gifu Pharmaceutical University Pharmacy, Lecturer, 薬学部, 助手 (80164747)
HIRANO Kazuyuki  Gifu Pharmaceutical University Pharmacy, Associate Professor, 薬学部, 助教授 (90057365)
FURUKAWA Shoei  National Institute of Neurosceience, Division of Neuroimmunology, Chief, 神経研究所, 室長 (90159129)
森 和俊  岐阜薬科大学, 薬学部, 助手 (70182194)
Project Period (FY) 1988 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1989: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1988: ¥4,200,000 (Direct Cost: ¥4,200,000)
KeywordsNicotinic Acetylcholine Receptor (nAChR) / Glycoprotein / Function / Phosphorylation / Carbohydrate Structure / 43KDa Proteins / Desensitization / Neurotransmission / 神経筋接合部 / 機能発見 / コリン性リガンド / 43kタンパク質 / 架橋形成 / 分子機構 / ヘビ神経毒 / リン酸化チロシン / 43κ蛋白 / キナーゼ
Research Abstract

Nicotinic acetylcholine receptor (nAChR) is a membrane glycoprotein which functions as transmitter receptor at vertebrate neuromuscular synapses. nAChR from the electric organ of ray consists of four kinds of subunits assembled in a molar stoichiometry of alpha_2,beta,gamma,delta. The primary structures of all these four subunits and those from some mammalian muscle nAChR have been elucidated by cloning and sequencing cDNAs or genomic DNA encoding these polypeptides.
This study was designed to elucidate the mechanism of the function of nAChR in the molecular basis using the highly purified nAChR isolated from the electric organ of Torpedo californica.
Results: Major results are summarized as follows:
1. nAChR was highly purified from the electric organ of Torpedo californica, by employing a cobrotoxin-Sepharose conjugate as an adsorbent for affinity chromatography.
2. The role of the carbohydrate moiety of nAChR in ligand-binding was examined, using peroxidate-labelled alpha-bungarotoxin. … More The bungarotoxin-binding to nAChR was inhibited by sialic acid or ovalbumin, but the removal of sialic acid or high-mannose-type oligosaccharides from nAChR resulted in no difference from the intact receptor in its bindings. These results suggested that the carbohydrate moiety of nAChR was not necessary in the binding of cholinergic ligands.
3. Protein phosphorylation is recognized as one of the major regulatory mechanism in the control of cellular metabolism. In the present study, we examined whether nAChR is phosphorylated at tyrosine residue, using anti-tyrosine antibody. The results indicated that the tyrosine residue(s) in beta- and gamma-subunits of nAChR molecule were phosphorylated in vivo. The effect of cholinergic ligandes on the extent of nAChR phosphorylation was also examined and was found to be dose dependent. These results suggest that the phosphorylation is stimulated by the conformational change of nAChR molecule by ACh-binding to the receptor and correlates with the desensitization of nAChR.
4. The nonreceptor, peripheral,nu proteins (MW: 43,000 protein; 43KDa proteins) are conspicuous components of the nAChR-rich membranes and the Torpedo electrocyte. We examined on the biochemical properties of nu_1, nu_2, and nu_3 components isolated from the peripheral nu proteins and demonstrated their properties.
Although the molecular natures of nAChR have been fairly demonstrated, the detailed mechanism of the neurotransmission still remains to be clarified. The elucidation of the three-dimensional structure of nAChR by X-ray or NMR-analysis will open the way to a better understanding of the problems. Less

Report

(3 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • Research Products

    (39 results)

All Other

All Publications (39 results)

  • [Publications] Yasunori Ishigaki,Takeshi Sato,Tong-Lin Song,Kyozo Hayashi: "Treatment of experimental allergic myasthenia gravis with a new immunosuppressant:15-deoxyapergualin." Adv.Neuroimmunol.540. 551-553 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 太田光熈,西谷裕,林恭三: "ヘビ神経毒の標的受容体と重症筋無力症" 最新医学. 43. 1238-1243 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 野元裕,林恭三: "神経細胞のイオンチャンネル" 病態生理. 7. 614-627 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 野元裕,林恭三: "ニコチン性アセテルコリンレセプタ-:構造と機能を中心に" 神経研究の進歩. 33. 932-939 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 野元裕,林恭三: "骨格筋:ニコチン性アセチルコリン受容体を中心に" 日本臨床. 47. 4761-4766 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 太田光熈,太田潔江,林恭三: "ニコチン性アセチルコリン受容体" 日本臨床. 47. 1213-1218 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 野元裕,太田光熈,林恭三: "ニコチン性アセチルコリン受容体" 蛋白質・核酸・酵素. 35. 1181-1194 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 林恭三,野元裕: "ニコチン性アセチルコリン受容体(nAchR)の構造と機能" 化学(印刷中).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Hiroshi Nomoto,Noriko Takahashi,Yasuhiro Nagaki,Satoshi Endo,Yoji Arata,Kyozo Hayashi: "Neuroimmunological Disease(Akihiro Igata ed.)" University of Tokyo Press, 413 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Yasunori Ishigaki,Takeshi Sato,Tong-Lin Song,Kyozo Hayashi: "Neuroimmunological Disease(Akihiro Igata ed.)" University of Tokyo Press, 413 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 林恭三,野元裕: "タンパク質.II構造と機能編(勝部幸輝ら編)" 東京化学同人, 426 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 太田光熈,林恭三: "生物トキシン" 学会出版センタ-, 227 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 林恭三,野元裕: "膜と神経・筋・シナプス-2" 喜多見書房, 465 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Mitsuhiro Ohta,Kyozo Hayashi: "Handbook of Neurotoxins(印刷中)" Marcel Dekker,

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Kazuto Ohkura, Seiji Inoue, Kiyoshi Ikeda, and Kyozo Hayashi: "Aminoacid sequences of four cytotoxins (cytotoxins I, II, III and IV) purified from the venom of the Thailand cobra, Naja naja siamensis." Biochem.Biophys.Acta, 954(2), 148-153 (1988).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Jean-Marc Grognet, Andre Menez, Alex Drake, Kyozo Hayashi, Ian E.G. Morrison, Robert C.Hider: "Circular dichroic spectra of elapid cardiotoxins." Eur.J.Biochem., 172(2), 383-388 (1988).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Mitsuhiro Ohta, Kiyoe Ohta, Fumiyo Mori, Kyozo Hayashi, and Hiroshi Nishitani: "Patients with myasthenia gravis and thymoma have anti-skeletal muscle and anti-acetylcholine receptor antibodies simultaneously." J.Clin.Biochem.Nutr., 6(1), 65-75 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Toshiya Endo, Masanao Oya, Kyozo Hayashi, and Tatsuo Miyazawa: "Step-wise thermal denaturation of cobratoxin, a snake venom neurotoxin from Naja naja atra---A proton nuclear magnetic resonance study." J.Protein Chem., 8(4), 575-581 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Toshiya Endo, Masanao Oya, Francois J.Joubert, Kyozo Hayashi, and Tatsuo Miyazawa: "State of functionally essential Trp-29 in snake venom neurotoxins---A proton nuclear magnetic resonance study." J.Protein Chem., 8(4), 583-588 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Ryoichi Kase, Hisaya Kitagawa, Kyozo Hayashi, Kenjiro Tanoue, and Fuyuhiko Inagami: "Neutralizing monoclonal antibody specific for alpha-bungarotoxin---Preparation and characterization of the antibody and localization of antigenic region of alpha-bungarotoxin." FEBS Lett., 254(1,2), 106-110 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Seiji Inoue, Koji Okumura, Michiyo Tsujino, Kazuto Ohkura, Kiyoshi Ikeda, Masayuki Takechi, Yasuo Tanaka, and Kyozo Hayashi: "Amino acid sequences of cytotoxin-like basic proteins derived from cobra venoms." FEBS Lett., 257(2), 319-323 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Mitsuhiro Ohta, Kiyoe Ohta, Nobuyuki Itoh, Masayuki Kurobe, Kyozo Hayashi, and Hiroshi Nishitani: "Anti-skeletal muscle antibodies in the sera from myasthenic patients with thymoma---Identification of antimyosin, actomysin, actin and alpha-actinin antibodies by a solid-phase radioimmunoassay and a Western blotting analysis." Clin.Chem.Acta.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Mitsuhiro Ohta, Kiyoe Ohta, Nobuyuki Itoh, Hiroshi Nishitani, Hirokazu Hara, Kyozo Hayashi: "An improved radioimmunoassay for anti-acetylcholine receptor antibody---Application for the detection of extremely low antibody titers in sera from myasthenia gravis." Neurology.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Mitsuhiro Ohta, Kiyoe Ohta, Fumiyo Mori, Nobuyuki Itoh, Hiroshi Nishitani, and Kyozo Hayashi: "An improved radioimmunoassay for anti-acetylcholine receptor antibody---Application for the detection of extremely low antibody titers in sera from myasthenia gravis." Clin.Chem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 野元裕,林恭三: "ニコチン性アセチルコリンセプタ-:構造と機能を中心に" 神経研究の進歩. 33. 932-939 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 野元裕,林恭三: "骨格筋:アセチルコリン受容体を中心に" 日本臨床. 47. 4761-4766 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 太田光熈,太田潔江,林恭三: "ニコチン性アセチルコリン受容体" 日本臨床. 47. 1213-1218 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 野元裕,太田光熈,林恭三: "ニコチン性アセチルコリン受容体" 蛋白質・核酸・酵素. 35. 1181-1194 (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] 林恭三,野元裕: "ニコチン性アセチルコリン受容体(nAChR)の構造と機能" 化学.

    • Related Report
      1989 Annual Research Report
  • [Publications] 林恭三,野元裕: "膜と神経・筋・シナプスー2" 多見書房, 465 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Mitsuhiro Ohta,Kyozo Hayashi: "Handbook of Neurotoxins" Marcel Dekker,

    • Related Report
      1989 Annual Research Report
  • [Publications] J.M.Grognet,;A.Menez,;A.Drake,;K.Hayashi,;I.E.G.Morrison,;R.C.Hider.: European Journal of Biochemistry. 172. 383-388 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] K.Ohkura,;S.Inoue,;K.Ikeda,;K.Hayashi.: Biochim.Biophys.Acta. 954. 148-153 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] H.Nomoto,;N.Takahashi,;Y.Nagaki,;S.Endo,;Y.Arata,;K.Hayashi.: Neuroimmunological Diseases. 241-246 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Y.Ishigaki,;T.Sato,;Tong-Lin Song,;K.Hayashi.: Neuroimmunological Diseases. 403-406 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] T.Endo,;M.Oya,;F.J.Joubert,;K.Hayashi,;T.Miyazawa.: Journal of Protein Chemistry.

    • Related Report
      1988 Annual Research Report
  • [Publications] T.Endo,;M.Oya,;K.Hayashi,;T.Miyazawa: Journal of Protein Chemistry.

    • Related Report
      1988 Annual Research Report
  • [Publications] 林恭三、野元裕: "タンパク質(II) 構造と機能編(勝部幸穂他編)--アセチルコリン受容体" 化学同人, 319-344 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] 太田光煕、林恭三: "生物トキシン--ヘビ毒" 学会出版センター, 30-48 (1988)

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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