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1991 Fiscal Year Final Research Report Summary

Reconstitution of Ion Channels from Cardiac Sarcolemmal Membranes

Research Project

Project/Area Number 01460269
Research Category

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

Allocation TypeSingle-year Grants
Research Field 生物物性学
Research InstitutionKyushu University

Principal Investigator

KIRINO Yutaka  Kyushu University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (10012668)

Co-Investigator(Kenkyū-buntansha) HIRASHIMA Naohide  Kyushu University Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助手 (10192296)
ANZAI Kazunori  Kyushu University Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助手 (70128643)
Project Period (FY) 1989 – 1991
Keywordsion channels / reconstitution / planar bilayer / cardiac sarcolemmal membranes
Research Abstract

Preparation method for sarcolemmal membrane vesicles from porcine cardiac myocytes in high purity was established. In order to evaluate the purity of the sarcolemmal membranes, it is useful to measure marker enzyme activity such as Na^+, K^+-ATPase activity as a whole by unmasking the latent activities. For the unmasking reagent, saponin was found to be very effective and convenient.
Dihydropyridine receptor complex was purified in about 2000-fold by the use of immune affinity chromatography with a gel attaching a monoclonal antibody against alpha_2delta subunit complex of L-PM Ca^<2+> channel from rabbit skeletal muscles. alpha_1, alpha_2, and delta subunit were detected in SDS-PAGE analysis, while other subunit such as beta and gamma were not detected. The procedure established in this research takes shorter time than the procedures reported before. It may thus make us possible to reconstitute the purified subunit into proteoliposomes or planar bilayer membranes with keeping their activities in natural.
The sarcolemmal vesicles were fused to planar lipid bilayers and ion channel activities were measured. In the solution containing 100 mM Ba^<2+>, lion-inactivating channels were detected at negative membrane potentials. One of the channel was identified as B-QW Ca^<2+> channel, which had been reported in the patch clamp experiment. In the solution containing 600 mM Na^+, a cation channel with a conductance of 10 pS between -80 to 0 mV was detected. The charmer also allows K^+ to pass through, and the permeability ratio between Na^+ and K^+ was unity. The channel therefore is a new non-selective cation channel, which has not been reported before.

  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] Kazunori ANZAI,Hitomi KADONO,Mitsuyo HAMASUNA,Sannamu LEE,Haruhiko AOYAGI,Yutaka KIRINO: "Ion channel activity of synthetic basic peptides in planar lipid bilayers" J.Pharmacobio-Dyn.12. s-122 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kazunori ANZAI,Masami YOSHIDA,Yutaka KIRINO: "Change in intravesicular volume of liposomes by freeze-thaw treatment as studied by the ESR stopped-flow technique" Biochim.Biophys.Acta. 1021. 21-26 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.AGAWA,S.LEE,S.ONO,H.AOYAGI,M.OHNO,T.TANIGUCHI,K.ANZAI,Y.KIRINO: "Effect of amphipathic α-hferical model peptides posessing various numbers of amino acid residues on phospholipid membranes and their biological activity" Peptide Chemistry. 1989. 145-148 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yutaka KIRINO,Naohide HIRASHIMA,Yoshiro SAITO: "Patch-clamp measurements of ion channels in biomembrane vesicles reconstituted into giant proteoliposomes by freeze-thawing without use of detergent" Methods Neurosci.4. 374-383 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] kazunori ANZAI,Hitomi KADONO,Mitsuyo HAMASUNA,Sannammu LEE,Haruhiko AOYAGI,Yutaka KIRINO: "Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides" Biochim.Biophys.Acta. 1064. 256-266 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Naohide HIRASHIMA,Hitoshi ISHIBASHI,Yutaka KIRINO: "Comparative electrophysiological study of reconstituted membrane preparations of the potassium channel of the rabbit skeletal muscle sarsoplasmic reticulum" Biochim.Biophys.Acta. 1067. 235-240 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yukio AGAWA,S.LEE,S.ONO,H.AOYAGI,M.OHNO,T.TANIGUCHI,K.ANZAI,Yutaka KIRINO: "Interaction with phospholipid bilayers,ion channel formation,and antimicrobial activity of basic amphipathic α-helical model peptide having various chain length" J.Biol.Chem.266. 20218-20222 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kazunori ANZAI,Hiroshi,TOKUMARU,Naoki SOEJIMA,ken'ichi HATANAKA,Yutaka KIRINO: "Preparation and characterization of cardiac sarcolemmal vesicles from porcine ventricular muscles"

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hiroshi TOKUMARU,Kazunori ANZAI,Yutaka KIRINO: "Purification of the cardiac 1,4-dihydropyridine receptor using immunoaffinity chromatography with a monoclonal antibody against the α_2δ subunit of the skeletal muscle dihydropyridine receptor"

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masaya SHIGEMORI,Masahiro HIBINO,Kazuhiko KINOSHITA,Jr Hiroyasu ITOH,Kuniaki NAGAYAMA,Akira IKEGAMI: "Electroporation of cell membrane under a pulsed laser fluorescence microscope" Dynamic behavior of Macromolecules.Colloids,Liquid Crystals and Biological Systems by Optical and Electrooptical Methods(T.Watanabe,Ed.). 311-316 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 桐野 豊: "骨格筋の細胞内カルシウム動態" 「日本臨床」、別冊特集:骨格筋疾患. 47. 41-48 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 桐野 豊: "“電子スピン共鳴"、新基礎生化学実験法第5巻「高次構造・状態分析」(松橋通生、中嶋暉躬 編)" 丸善, 20 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.KINOSHITA,Jr,HIBINO,H.ITO,M.SHIGEMORI,K.HIRANO Y.KIRINO,T.HAYAKAWA: "Handbook of electroporation and electrofusion (D.C.Chang,B.M.Chassy,J.A.Saunders,E.E.Sowers,Eds.)" Academic Press, 581 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 桐野 豊,小沢 俊彦: "エッセンスESRー基礎と実験ー" 廣川書店, 169 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 桐野 豊: "“機能再構成(イオンチャネル)"、「レセプタ-」(井村 裕夫,岡 哲雄,芳賀 達也,岸本 英爾 編)" 朝倉書店,

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 桐野 豊: "「薬物の構造解析と医薬品開発」(荒田洋治 編)" 廣川書店,

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 桐野 豊: "「生体成分の分析科学」(中嶋 暉躬,吉岡 正則 編)" 廣川書店,

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 桐野 豊: "「物理化学」(清水 博,桐野 豊 編)" 南江堂,

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kazunori Anzai, Hitomi Kadono, Mitsuyo Hamasuna, Sannamu Lee, Haruhiko Aoyagi, and Yutaka Kirino: " "Ion channel activity of synthetic basic peptides in planar lipid bilayers"" J. Pharmacobio-Dyn.12. s-122 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kazunori Anzai, Masami Yoshida, and yutaka Kirino: " "Change in intravesicular volume of liposomes by freeze-thaw treatment as studied by the ESR stopped-flow technique"" Biochem. Biophys. Acta. 1021. 21-26 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yukio Agawa, Sannamu Lee, Shin Ono, Haruhiko Aoyagi, Motonori Ohno, Takako Taniguchi, Kazunori Anzai, and Yutaka Kirino: " "Effect of amphipathic alpha-helical model peptides posessing various numbers of amino acid residues on phospholipid membranes and their biological activity"" Peptide Chemistry. 1989. 145-148 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yutaka Kirino, Naohide hirashima, and Yoshiro Saito: " "Patch-clamp measurement of ion channels in biomembrane vesicles reconstituted into giant proteoliposomes by freeze-thawing without use of detergent"" Methods Neurosci. 4. 374-383 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kazunori Anzai, Hitomi Kadono, Mitsuyo Hamasuna, Sannam Lee, Haruhiko Aoyagi, and Yutaka Kirino: " "Formation of ion channels by synthetic basic peptides in planar lipid bilayer membrane" Biochem. Biophys. Acta. 1064. 256-266 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Naohide Hirashima, Hitoshi Ishibashi, and Yutaka Kirino: " "Comparative electrophysiological study of reconstituted membrane preparations of the potassium channel of the rabbit skeletal muscle sarcoplasmic reticulum"" Biochem. Biophys. Acta. 1067. 235-240 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yukio Agawa, Sannamu Lee, Shin Ono, Haruhiko Aoyagi, Motonori Ohno, Takako Taniguchi, Kazunori Anzai, and Yutaka Kirino: " "Interaction with phospholipid bilayers, ion channel formation, and antimicrobial activity of basic amphipathic alpha-herical model peptides of various chain lengths"" J. Biol. Chem.266. 20218-20222 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kazunori Anzai, Hiroshi Tokumaru, Naoki Soejima, Kenichi Hatanaka, and Yutaka Kirino: " "Preparation and characterization of cardiac sarcolemmal vesicles from porcine ventricular muscles""

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hiroshi Tokumaru, Kazunori Anzai, Teruo Abe, and Yutaka Kirino: " "Purification of the cardiac 1, 4-dihydropyridine receptor using immunoaffinity chromatography with a monoclonal antibody against the alpha_2delta subunit of the skeletal muscle dihydropyridine receptor""

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1993-03-16  

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