1分子計測法によるチャネルタンパクの構造揺らぎ・機能揺らぎ相関の研究

• Project Area: Molecular Science of Fluctuations toward Biological Functions
• Project/Area Number: 21107518
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: The Institute of Physical and Chemical Research (2010); Osaka University (2009)
• Principal Investigator: 井出 徹 独立行政法人理化学研究所, 細胞動態計測研究開発グループ, 研究員 (60231148)
• Project Period (FY): 2009 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥7,020,000 (Direct Cost: ¥5,400,000、Indirect Cost: ¥1,620,000); Fiscal Year 2010: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000); Fiscal Year 2009: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000)
• Keywords: ゆらぎ / 1分子計測 / 1分子操作 / イオンチャネル / KcsA / 人工脂質膜 / 構造機能相関

Research Abstract

チャネルタンパクは生体膜を貫通するタンパクで、その中心にイオンを透過させる細孔を持つ。特定の刺激に応じて、細孔を開閉させることにより膜の透過性を制御している。単一チャネル電流計測技術と蛍光標識による1分子イメージング法を組み合わせれば、分子内運動による機能変化を従来法に較べてより直接的に示すことが出来るものと期待されている。この方法を用いて、チャネルタンパクの構造ゆらぎと機能の関係を見出すことが目的である。
細菌のKチャネル(KcsA)のCys置換変異体を数種類作成し蛍光標識を行った。巨視的な蛍光計測の結果、これらのうち幾つかは、チャネルの開確率が低い(pH7)条件と高い(pH4)条件下で蛍光強度に著しい差を生じさせることが分かった。この結果からゲーティングに伴いタンパクの構造が大きく変わることを見いだした。ところが、タンパク1分子で同じ計測を行うと単一チャネル電流に見られる速い揺らぎは見えず、ゆっくりとした揺らぎが観測された。即ち、構造揺らぎと機能揺らぎが同期していない。これは、構造と機能が1対1に対応しない(機能に対応しない構造が存在する)可能性を示している。揺らぐ「状態」が機能に対応しているのかも知れない。
上記の構造変化を確認するために、チャネルタンパクの1分子操作を行った。人工膜に組み込んだチャネルタンパクの細胞内領域を膜方向に操作することにより、活性が著しく上昇することを確認した。中性溶液中ではKcsAチャネルは殆ど活性を示さないが、ガラス微小探針等で操作することにより、酸性(pH4)条件下に匹敵する程の活性を持たせることが出来た。これは、上記蛍光計測の結果を確認するとともに新たな計測手法としても注目される。

Research Products

• [Journal Article] Rearrangements in the KcsA cytoplasmic domain underlie its gating. (2010)
  • Author(s): Hirano Minako
  • Journal Title: J Biol Chem Volume: 285 Pages: 3777-3783
  • Peer Reviewed
• [Journal Article] Single channel properties of lysenin measured in artificial lipid bilayers and their applications to biomolecule detection. (2010)
  • Author(s): Takaaki Aoki
  • Journal Title: Proc Jpn Acad Ser B Phys Biol Sci Volume: 86 Pages: 920-925
  • Peer Reviewed
• [Journal Article] Simultaneous optical and electrical recording of single drug bonding to single ion channel proteins. (2010)
  • Author(s): Ide T.
  • Journal Title: ChemPhysChem Volume: 11 Pages: 3408-3411
  • Peer Reviewed
• [Journal Article] Rearrangements in the KcsA cytoplasmic domain underlie its gating (2010)
  • Author(s): Minako Hirano, Yuko Takeuchi, Takaaki Aoki, Toshio Yanagida, Toru Ide
  • Journal Title: Journal of Biological Chemistry 285 Pages: 3777-3783
  • Peer Reviewed
• [Journal Article] Current recordings of ion channel proteins immobilized on resinbeads (2009)
  • Author(s): Hirano, M., Takeuchi, Y., Aoki, T., Yanagida, T., Ide, T.
  • Journal Title: Analytical Chemistry 81(8) Pages: 3151-3154
  • Peer Reviewed
• [Journal Article] Simultaneous optical and electrical single channel recordings on a PEG-glass
  • Author(s): Toru Ide, Yuko Takeuchi, Hiroyuki Noji, Kazuhito Tabata
  • Journal Title: Langmuir (印刷中)
  • Peer Reviewed
• [Presentation] The role of the cytoplasmic domain in pH-dependent gating by the KcsA channel (2011)
  • Author(s): Minako Hirano
  • Organizer: Biophysical Society 55th Annual Meeting
  • Place of Presentation: Baltimore, Maryland US
  • Year and Date: 2011-03-07
• [Presentation] The role of the cytoplasmic domain in pH-dependent gating by the KcsA channel (2010)
  • Author(s): Minako Hirano
  • Organizer: Molecular Science of Fluctuations toward Biological Functions, The 4rd International Symposium
  • Place of Presentation: 滋賀県大津市
  • Year and Date: 2010-12-01
• [Presentation] Direct manipulation of a single potassium channel gate with an atomic force microscope probe (2010)
  • Author(s): Toru Ide
  • Organizer: Molecular Science of Fluctuations toward Biological Functions, The 4rd International Symposium
  • Place of Presentation: 滋賀県大津市
  • Year and Date: 2010-12-01
• [Presentation] The role of the cytoplasmic domain in pH-dependent gating by the KcsA channel (2010)
  • Author(s): Minako Hirano
  • Organizer: GCOE/Structural Biology Research Center International Symposium
  • Place of Presentation: 愛知県名古屋市
  • Year and Date: 2010-11-23
• [Presentation] The role of the cytoplasmic domain in pH-dependent gating by the KcsA channel (2010)
  • Author(s): 平野美奈子
  • Organizer: 第48回 日本生物物理学会年会
  • Place of Presentation: 宮城県仙台市
  • Year and Date: 2010-09-22
• [Presentation] 可視1分子計測によるチャネルたんぱく質の分子内運動計測 (2010)
  • Author(s): 井出徹
  • Organizer: 第30回表面科学学術講演会・第51回真空に関する連合講演会合同講演会
  • Place of Presentation: 大阪府吹田市
  • Year and Date: 2010-09-04
• [Presentation] Kチャネルゲートの1分子操作 (2010)
  • Author(s): 井出徹
  • Organizer: 第10回日本蛋白質科学会年会 蛋白質1分子内部運動と機能発現
  • Place of Presentation: 北海道札幌市
  • Year and Date: 2010-06-17
• [Presentation] Simultaneous optical and electrical recording of single drug bindings to single ion channel proteins (2009)
  • Author(s): T.Ide, M.Hirano
  • Organizer: Molecular Science of Fluctuations toward Biological Functions, 3rd International Symposium
  • Place of Presentation: Nagoya University
  • Year and Date: 2009-12-20
• [Presentation] Rearrangements in the KcsA cytoplasmic domain underlie its gating (2009)
  • Author(s): M.Hirano, Y.Takeuchi, T.Aoki, T.Yanagida, T.Ide
  • Organizer: Molecular Science of Fluctuations toward Biological Functions, 3rd International Symposium
  • Place of Presentation: Nagoya University
  • Year and Date: 2009-12-20
• [Presentation] Artificial lipid bilayers for ion channel studies (2009)
  • Author(s): Toru Ide
  • Organizer: Innovative nanoscience of supermolecular motor proteins working in biomembranes.
  • Place of Presentation: Kyoto University (Inamori Hall)
  • Year and Date: 2009-09-08
• [Presentation] Rearrangements in the KcsA cytoplasmic domain underlie its gating (2009)
  • Author(s): M.Hirano, Y.Takeuchi, T.Aoki, T.Yanagida, T.Ide
  • Organizer: Innovative nanoscience of supermolecular motor proteins working in biomembranes.
  • Place of Presentation: Kyoto University (Inamori Hall)
  • Year and Date: 2009-09-08
• [Presentation] Gating mechanism analysis in voltage sensitive potassium channels (2009)
  • Author(s): Y.Takeuchi, M.Hirano, T.Aoki, T. Yanagida, T.Ide
  • Organizer: Innovative nanoscience of supermolecular motor proteins working in biomembranes.
  • Place of Presentation: Kyoto University (Inamori Hall)
  • Year and Date: 2009-09-08
• [Presentation] Single Antibiotics Channel Current Measurement (2009)
  • Author(s): Takaaki Aoki, Minako Hirano, Yuko Takeuchi, Toshihide Kobayashi, Toshio Yanagida, Toru Ide
  • Organizer: Innovative nanoscience of supermolecular motor proteins working in biomembranes.
  • Place of Presentation: Kyoto University (Inamori Hall)
  • Year and Date: 2009-09-08
• [Presentation] Gating mechanism analysis in voltage sensitive potassium channels (2009)
  • Author(s): Y.Takeuchi, M.Hirano, T.Aoki, T.Yanagida, T.Ide
  • Organizer: 5th Handai Nanoscience and Nanotechnology International Symposium
  • Place of Presentation: 大阪大学・銀杏会館
• [Presentation] Rearrangements in the KcsA cytoplasmic domain underlie its gating (2009)
  • Author(s): M.Hirano, Y.Takeuchi, T.Aoki, T.Yanagida, T.Ide
  • Organizer: 5th Handai Nanoscience and Nanotechnology International Symposium
  • Place of Presentation: 大阪大学・銀杏会館
• [Presentation] Rearrangements in the KcsA cytoplasmic domain underlie its gating (2009)
  • Author(s): M.Hirano, Y.Takeuchi, T.Aoki, T.Yanagida, T.Ide
  • Organizer: 日本生物物理学会第47回年会
  • Place of Presentation: アクティーとくしま
• [Book] "Simultaneous optical and electrical recording of single drug bindings to single ion channel proteins." in Cell Signaling Reactions : Single-Molecular Kinetic Analysis (Ed.by Y.Sako and M.Ueda) (2011)
  • Author(s): Toru Ide
  • Total Pages: 14
  • Publisher: Springer