1992 Fiscal Year Annual Research Report

ATP感受性K^+チャネル蛋白分子の新しい標識物質の開発

Research Project

Project/Area Number	02557039
Research Institution	Tokyo Medical and Dental University, Medical Research Institute
Principal Investigator	平岡昌和東京医科歯科大学, 難治疾患研究所, 教授 (80014281)
Co-Investigator(Kenkyū-buntansha)	堀川三郎東京医科歯科大学, 難治疾患研究所, 助手 (10127136) 中山仁北海道大学, 薬学部, 助手 (70088863)
Keywords	ATP感受性K^+チャネル蛋白 / クロマカリム / グリベンクラミド / 光アフィニティラベル法 / ATP結合能
Research Abstract	これまでの研究からK^+チャネル開口薬の作用を明らかにするとともに,グリベンクラミドのATP感受性K^+チャネル抑制作用について電気生理学的実験を行ってきた。これらの成績をもとに光アフィニティラベルあるいはアフィニティクロマト用リガンドとして,クロマカリム・ニコランジル・グリベンクラミドより合計6個の化合物を合成した。そのうち,ニコランジル化合物にはチャネル開口作用はみられず,クロマカリム化合物には開口作用がみられたが,水への溶解性が低いため再現性に乏しかった。もう一つのクロマカリム化合物につきアフィニティカラムを調製しラット脳標品中のK^+チャネル蛋白(150kDa)を精製したが,心筋標本では精製効果が顕著でなく,両組織での本K^+チャネル蛋白の性状の違いが示唆された。次にグリベンクラミド化合物による光反応性標織化合物DA-グリベンクラミド2種を合成した。これらは心筋形質膜標品にも高い結合能が確認された。この化合物にて光ラベルを行い,精製をすすめて単一バンドを示す精製標品を得た。この精製蛋白標品を単離・精製し,分析により合計120残基,全体の約20%のアミノ酸配列が解析できた。このものは既知のチャネル蛋白とは相同性がなく,別の可溶性タンパクと非常に高い類似性を示すことが判明した。この精製蛋白にはATP結合能を持つこと,抗体を用いた免疫組織科学的解析から心筋形質膜に存在することを確認した。以上の結果から,心筋ATP感受性K^+チャネルそのもの,あるいはそれを構成するサブユニットの有力な候補蛋白を同定出来たものと考えている。この精製蛋白が電気生理学的にもK^+チャネル活性を示すことを確認するために,人工脂質膜を用いての再構成実験を行っているが,未だ成功していない。併行して分子生物学的実験を行い,cDNAライブラリーと合成ヌクレオチドから目的とするcDNAクローンの検索を継続して行っている。また,別のsmall K^+チャネルの発現実験には成功した。

Research Products
(16 results)

All Other

All Publications (16 results)

[Publications] Fan Z.,Nakayamm K.,Sawanobori T.,Hiraoka M.: "Aromtic aldehydes and aromatic detones open ATP-sensitive K^+ channels en guinea-pig ventricular myocytes." Pflugers Arch.421. 409-415 (1992)
[Publications] Yamawake N.,Hirano Y.,Sawanobori T.Hiraoka M.: "Arrhythmogenic effects of isoproterenol-activated Cl^- current in guinea-pig ventricular myocytes." J.Mol.Cell.Cardiol.24. 1039-1058 (1992)
[Publications] Takanashi H.,Sawanobori T.,Hiraoka,M.et.a;.: "Ca^<2+>-activated K^+ channel is present in guinea-pig but lacking in rat hepatocyts." Jpn.J.Physiol.42. 415-430 (1992)
[Publications] Virag L.,Furukawa T.,hiraoka M.: "modulation of the effect of glibenclamide on Katp channels by ATP and ADP." Mol.Cell.Biochem. (1993)
[Publications] Hiraoka M.,Fan Z.,Furukawa T.,Nakaya-ma K.,Sawanobori T.: "Activation and reactivation of the ATP-sensiti-ve K^+ channel of the heart can be modified by drugs." Cardiovasc.Drug.Therap.(1993)
[Publications] Furukawa T.,Fan Z.Sawanobori T.,Hiraoka M.: "Modification of the adenosine 5'-triphosphate-sensitive K^+ channel by trypsinin guinea-pig ventricular myocytes." J.Physiol.(1993)
[Publications] Yamane T.,Furukawa T.Horikawa S.,Hiraoka,M.: "Fxternl ph regulates the slowly activating potassium cruuent Isk expressed en xenopus oocytes." FEBA Lett.(1993)
[Publications] Nakayama H.,Hatana-ka Y.,Yoshida Eoda K.et al.: "Photolabeled sites with a tetrodotoxin deri-vative in the domain III and IV of the electro-plax sodium channel." Biochem.Biophys.Res.Commun.184. 900-907 (1992)
[Publications] Hatanaka Y.,nakayama H.,Kanaoka Y.: "Photoaffinity labeling of the electroplax sodium channel with a photoreactive u-conotoxin caming a radioactive and chromogenic derivative." Chem.Pharm.Bull.40. 2537-2539 (1992)
[Publications] kuniyasu A.,Oka K.Ideyamada T.,Nakayama H.et al.: "Structural characterization of the cardiac calcium channel from porcine herts." J.Biochem.112. 235-242 (1992)
[Publications] Nakayama H.,Hatanaka Y.,Taki M.kanaoka Y.: "chemical and immunological characterization of binding sites for tetrodotoxin and dihydro-pyridine that are close proximity to the extermal mouth of ion pore in the sodium and calcium channels." J.protein Chem.11. 395-396 (1992)
[Publications] Nakayama H.,Yamamoto S.,Hatanaka Y.et al.: "Charactrization of saccharide moiety in the electroplax sodium channel." Chem.Pharm.Bull. 40. 3287-3291 (1993)
[Publications] Fujioka T.,hanawa T.,Nakayama H.et al.: "Preparation of anti-peptide antibodies and their application to the cardiac Na^- -channel." Peptide Chemistry. (1992)
[Publications] Chen F.O.Hirano T.Ohashi M.,Oda K.,Nakayama H.et al.: "3-Bebzitl-5-(4-hydroxyphenyl)2-[4-(1-azi-2,2,2,-trifluoroehyl)phenylacetamidol pyrazine.A photosensitive analogue of coelenteramide.Snythesis and Photolysi." Chemistry Lett.
[Publications] Nakayama H.,Shikano H.,Kanaoka Y.: "A site-directed antibody which inhibits phosphorylation of the rat brain sodium channel by cAMP-dependent protein kinase." biochim.Biophys.Acta.
[Publications] Horikawa S.,Tsukada K.: "Molecular cloning and developmental cxpression of a human kidney S-adenosylmethionine synthetase" FEBS Lett.312. 37-41 (1992)

1992 Fiscal Year Annual Research Report

ATP感受性K^+チャネル蛋白分子の新しい標識物質の開発

Principal Investigator

平岡 昌和 東京医科歯科大学, 難治疾患研究所, 教授 (80014281)

Research Products

[Publications] Fan Z.,Nakayamm K.,Sawanobori T.,Hiraoka M.: "Aromtic aldehydes and aromatic detones open ATP-sensitive K^+ channels en guinea-pig ventricular myocytes." Pflugers Arch.421. 409-415 (1992)

[Publications] Yamawake N.,Hirano Y.,Sawanobori T.Hiraoka M.: "Arrhythmogenic effects of isoproterenol-activated Cl^- current in guinea-pig ventricular myocytes." J.Mol.Cell.Cardiol.24. 1039-1058 (1992)

[Publications] Takanashi H.,Sawanobori T.,Hiraoka,M.et.a;.: "Ca^<2+>-activated K^+ channel is present in guinea-pig but lacking in rat hepatocyts." Jpn.J.Physiol.42. 415-430 (1992)

[Publications] Virag L.,Furukawa T.,hiraoka M.: "modulation of the effect of glibenclamide on Katp channels by ATP and ADP." Mol.Cell.Biochem. (1993)

[Publications] Hiraoka M.,Fan Z.,Furukawa T.,Nakaya-ma K.,Sawanobori T.: "Activation and reactivation of the ATP-sensiti-ve K^+ channel of the heart can be modified by drugs." Cardiovasc.Drug.Therap.(1993)

[Publications] Furukawa T.,Fan Z.Sawanobori T.,Hiraoka M.: "Modification of the adenosine 5'-triphosphate-sensitive K^+ channel by trypsinin guinea-pig ventricular myocytes." J.Physiol.(1993)

[Publications] Yamane T.,Furukawa T.Horikawa S.,Hiraoka,M.: "Fxternl ph regulates the slowly activating potassium cruuent Isk expressed en xenopus oocytes." FEBA Lett.(1993)

[Publications] Nakayama H.,Hatana-ka Y.,Yoshida Eoda K.et al.: "Photolabeled sites with a tetrodotoxin deri-vative in the domain III and IV of the electro-plax sodium channel." Biochem.Biophys.Res.Commun.184. 900-907 (1992)

[Publications] Hatanaka Y.,nakayama H.,Kanaoka Y.: "Photoaffinity labeling of the electroplax sodium channel with a photoreactive u-conotoxin caming a radioactive and chromogenic derivative." Chem.Pharm.Bull.40. 2537-2539 (1992)

[Publications] kuniyasu A.,Oka K.Ideyamada T.,Nakayama H.et al.: "Structural characterization of the cardiac calcium channel from porcine herts." J.Biochem.112. 235-242 (1992)

[Publications] Nakayama H.,Hatanaka Y.,Taki M.kanaoka Y.: "chemical and immunological characterization of binding sites for tetrodotoxin and dihydro-pyridine that are close proximity to the extermal mouth of ion pore in the sodium and calcium channels." J.protein Chem.11. 395-396 (1992)

[Publications] Nakayama H.,Yamamoto S.,Hatanaka Y.et al.: "Charactrization of saccharide moiety in the electroplax sodium channel." Chem.Pharm.Bull. 40. 3287-3291 (1993)

[Publications] Fujioka T.,hanawa T.,Nakayama H.et al.: "Preparation of anti-peptide antibodies and their application to the cardiac Na^- -channel." Peptide Chemistry. (1992)

[Publications] Chen F.O.Hirano T.Ohashi M.,Oda K.,Nakayama H.et al.: "3-Bebzitl-5-(4-hydroxyphenyl)2-[4-(1-azi-2,2,2,-trifluoroehyl)phenylacetamidol pyrazine.A photosensitive analogue of coelenteramide.Snythesis and Photolysi." Chemistry Lett.

[Publications] Nakayama H.,Shikano H.,Kanaoka Y.: "A site-directed antibody which inhibits phosphorylation of the rat brain sodium channel by cAMP-dependent protein kinase." biochim.Biophys.Acta.

[Publications] Horikawa S.,Tsukada K.: "Molecular cloning and developmental cxpression of a human kidney S-adenosylmethionine synthetase" FEBS Lett.312. 37-41 (1992)

平岡昌和東京医科歯科大学, 難治疾患研究所, 教授 (80014281)