High-resolution single particle analysis of intercellular channels

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K07266
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Nagoya University
• Principal Investigator: Oshima Atsunori 名古屋大学, 細胞生理学研究センター, 教授 (80456847)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: ギャップ結合チャネル / クライオ電子顕微鏡 / 高分解能構造解析 / イネキシン / 単粒子解析

Outline of Final Research Achievements

We obtain the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. The INX-6 gap junction channel comprises hexadecameric subunits in an open state. The INX-6 structure is highly similar to the human connexin-26 structure, despite the lack of significant sequence similarity. We also have a hemichannel structures of INX-6 in a lipid nanodisc showing that flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted N-terminal deletion mutant reveal that lipid-mediated N-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution.Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into how the large-size pore of gap junction hemichannels can be completely closed in a lipid bilayer.

Free Research Field

構造生物学

Academic Significance and Societal Importance of the Research Achievements

本研究で明らかにしたINX-6の構造解析から、新規の膜タンパク質複合体の原子構造の決定に、結晶化を必要としないクライオ電子顕微鏡単粒子解析法が有効であることを示した。無脊椎動物と脊椎動物ではアミノ酸配列の相同性の低いタンパク質から共通点の多いチャネル構造が形成されることを明らかにした。クライオ電子顕微鏡法は、創薬ターゲットとして大きな割合を占める膜タンパク質の構造研究に今後も役立てられることが期待される。