Investigation of ion-pumping rhodopsins with a high-time resolution

• Project/Area Number: 18K06109
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43030:Functional biochemistry-related
• Research Institution: Nagoya Institute of Technology
• Principal Investigator: Tsunoda Satoshi 名古屋工業大学, 工学(系)研究科(研究院), 研究員 (00598857)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: ロドプシン / 電気生理学 / 光遺伝学 / イオンポンプ / 光受容体 / イオンチャネル / 微生物ロドプシン / イオン輸送 / オプトジェネテイクス / オプトジェネティクス / 生体エネルギー変換

Outline of Final Research Achievements

Ion transport properties of various microbial rhodopsin were investigated under a high-time resolved condition by combining electrophysiology with a flash laser. Single turnover reaction of sodium ion transport by KR2, a sodium pumping rhodopsin was recorded. Kinetical analysis revealed that the turnover rate was 10 times faster than reported value obtained under multiple turnover conditions, suggesting two distinct reaction pathways in the KR2 photocycle.
Moreover, ion transport of 9 different proton pumping rhodopsins were systematically tested in conventional patch-clamp recording. The pumping activity of those are pH- and membrane-voltage dependent. Data revealed that the pH at the proton exclusion side largely affect the proton transport.

Academic Significance and Societal Importance of the Research Achievements

高い時間分解能におけるイオン輸送計測から、イオン輸送の詳細な機構が明らかになった。また、合計9種類のイオンポンプロドプシンによるイオン輸送を初めて体系的に同条件で計測することで、イオン輸送に対する細胞環境の効果を検証し、ポンプ間での特性の違いを見出すことに成功した。これらの知見は光遺伝学等における光受容体を利用した生命の光操作技術や医療応用を技術応用への基盤となると期待される。

Research Products

• [Int'l Joint Research] Technion-Israel Institute of Technology(イスラエル)
• [Int'l Joint Research] Jawaharlal Nehru大学(インド)
• [Journal Article] Ion transport activity and optogenetics capability of light-driven Na+-pump KR2 (2021)
  • Author(s): Hososhima S, Kandori H, Tsunoda SP.
  • Journal Title: PLoS One. Volume: 16 Issue: 9 Pages: e0256728-e0256728
  • DOI: 10.1371/journal.pone.0256728
  • Peer Reviewed / Open Access
• [Journal Article] TAT Rhodopsin Is an Ultraviolet-Dependent Environmental pH Sensor (2021)
  • Author(s): Kataoka Chihiro、Sugimoto Teppei、Shigemura Shunta、Katayama Kota、Tsunoda Satoshi P.、Inoue Keiichi、Beja Oded、Kandori Hideki
  • Journal Title: Biochemistry Volume: 60 Issue: 12 Pages: 899-907
  • DOI: 10.1021/acs.biochem.0c00951
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Specific residues in the cytoplasmic domain modulate photocurrent kinetics of channelrhodopsin from Klebsormidium nitens (2021)
  • Author(s): Tashiro R, Sushmita K, Hososhima S, Sharma S, Kateriya S, Kandori H, Tsunoda SP.
  • Journal Title: Commun. Biol. Volume: 4 Issue: 1 Pages: 235-235
  • DOI: 10.1038/s42003-021-01755-5
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Molecular properties of new enzyme rhodopsins with PDE activity (2020)
  • Author(s): Sugiura M., Tsunoda SP., Hibi M., Kandori H
  • Journal Title: ACS Omega Volume: 5 Issue: 18 Pages: 10602-10609
  • DOI: 10.1021/acsomega.0c01113
  • Peer Reviewed / Open Access
• [Journal Article] Structural insights into the mechanism of rhodopsin phosphodiesterase (2020)
  • Author(s): Ikuta T, Shihoya W, Sugiura M, Yoshida K, Watari M, Tokano T, Yamashita K, Katayama K, Tsunoda SP, Uchihashi T, Kandori H and Nureki O.
  • Journal Title: Nat. Commun. Volume: 11 Issue: 1 Pages: 5605-5605
  • DOI: 10.1038/s41467-020-19376-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Schizorhodopsins: A novel family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps (2020)
  • Author(s): Keiichi Inoue, Satoshi P. Tsunoda, Manish Singh, Sahoko Tomida, Shoko Hososhima, Masae Konno, Ryoko Nakamura, Hiroki Watanabe, Paul-Adrian Bulzu, Horia L. Banciu, Adrian Andrei, Takayuki Uchihashi, Rohit Ghai, Oded Beja, Hideki Kandori
  • Journal Title: Science Advances Volume: 6 Issue: 15
  • DOI: 10.1126/sciadv.aaz2441
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Crystal structure of heliorhodopsin (2019)
  • Author(s): W. Shihoya, K. Inoue, M. Singh, M. Konno, S. Hososhima, K. Yamashita, K. Ikeda, A. Higuchi, T. Izume, Okazaki S, Hashimoto M, Mizutori R, Tomida S, Yamauchi Y, Abe-Yoshizumi R, K. Katayama, SP. Tsunoda, M. Shibata, Y. Furutani, A. Pushkarev, O. Béjà, T. Uchihashi, H. Kandori, O. Nureki
  • Journal Title: Nature Volume: 574 Issue: 7776 Pages: 132-136
  • DOI: 10.1038/s41586-019-1604-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Ion Channel Properties of a Cation Channelrhodopsin (2019)
  • Author(s): S. Shigemura, S. Hososhima, H. Kandori, SP. Tsunoda
  • Journal Title: Appl. Sci. Volume: 9 Issue: 17 Pages: 3440-3440
  • DOI: 10.3390/app9173440
  • Peer Reviewed / Open Access
• [Journal Article] Spectroscopic study of the transmembrane domain of a rhodopsin-phosphodiesterase fusion protein from a unicellular eukaryote (2019)
  • Author(s): M. Watari, T. Ikuta, D. Yamada, W. Shihoya, K. Yoshida, S. P. Tsunoda, O. Nureki, and H. Kandori
  • Journal Title: J. Biol. Chem Volume: 294 Issue: 10 Pages: 3432-3443
  • DOI: 10.1074/jbc.ra118.006277
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] "Light-driven sodium-pumping rhodopsin: a new concept of active transport" (2018)
  • Author(s): H. Kandori*, K. Inoue, S. P. Tsunoda
  • Journal Title: Chem. Rev. Volume: in press Issue: 21 Pages: 10646-10658
  • DOI: 10.1021/acs.chemrev.7b00548
  • Peer Reviewed
• [Journal Article] Production of a Light-Gated Proton Channel by Replacing the Retinal Chromophore with Its Synthetic Vinylene Derivative. (2018)
  • Author(s): R. Takayama, A. Kaneko , T. Okitsu, S. P. Tsunoda, K. Shimono , M. Mizuno, K. Kojima, T. Tsukamoto, H. Kandori, Y. Mizutani, A. Wada, Y. Sudo
  • Journal Title: J Phys Chem Lett Volume: 9 Issue: 11 Pages: 2857-2862
  • DOI: 10.1021/acs.jpclett.8b00879
  • Peer Reviewed
• [Journal Article] A distinct abundant group of microbial rhodopsins discovered using functional metagenomics. (2018)
  • Author(s): A.Pushkarev, K. Inoue, S. Larom, J. Flores-Uribe, M. Singh, M. Konno, S. Tomida, S. Ito, R. Nakamura, S. P. Tsunoda, A. Philosof, I. Sharon, N. Yutin, E. V. Koonin, H. Kandori, O. Béjà
  • Journal Title: Nature Volume: 558 Issue: 7711 Pages: 595-599
  • DOI: 10.1038/s41586-018-0225-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Electrophysiological analysis of various proton pump rhodopsins (2021)
  • Author(s): Akari Okuyama, Shoko Hososhima, Satoshi Tsunoda, Hideki Kandori
  • Organizer: 日本生物物理学会第59回年会
  • Int'l Joint Research
• [Presentation] 様々な内向きおよび外向き プロトンポンプロドプシンへの 電気化学ポテンシャル勾配の影響 (2021)
  • Author(s): 奥山あかり・細島頌子・角田 聡・神取秀樹
  • Organizer: 日本生体エネルギー研究会第47回討論会
• [Presentation] 電気生理学的測定によるプロトンポンプロドプシンの駆動力の解明 (2021)
  • Author(s): 奥山あかり・細島頌子・角田 聡・神取秀樹
  • Organizer: 第47回生体分子科学討論会
• [Presentation] CNGチャネルを用いた高時間分解での酵素ロドプシンの機能計測系の確立 (2021)
  • Author(s): 石川和季、細島頌子、角田聡、神取秀樹
  • Organizer: 日本生物物理学会中部支部会
• [Presentation] プロトンポンプロドプシンの駆動力の比較 (2021)
  • Author(s): 奥山あかり、細島頌子、角田聡、神取秀樹
  • Organizer: 日本生物物理学会中部支部会
• [Presentation] ロドプシンフォスホジエステラーゼ（Ｒｈ― ＰＤＥ）の非対称的ｐＨ効果 (2021)
  • Author(s): 杉浦雅大、角田聡、日比正彦、神取秀樹
  • Organizer: 日本科学会 CSJ フェスタ
• [Presentation] Molecular function and structural properties of Rh-PDE (2020)
  • Author(s): Satoshi Tsunoda
  • Organizer: 第43回日本分子生物学会年会
  • Int'l Joint Research / Invited
• [Presentation] Importance of the C-terminal region in cation channelrhodopsin Ts_Rh3 (2020)
  • Author(s): Rintaro Tashiro, Kumari Sushmita, Suneel Kateriya, Hideki Kandori, Satoshi Tsunoda
  • Organizer: 日本生物物理学会第58回年会
  • Int'l Joint Research
• [Presentation] Electrophysiological study and Optogenetics application of inward-directed proton-pumping rhodopsin, NsXeR (2019)
  • Author(s): 角田聡、細島頌子、神取秀樹
  • Organizer: 日本生物物理学会第56回年会
  • Int'l Joint Research
• [Presentation] 酵素型ロドプシンの分子機構と光遺伝学への応用 (2019)
  • Author(s): 角田聡
  • Organizer: 物性研ワークショップ「レチナールタンパク質の光機能発現の物理と科学」
  • Invited
• [Presentation] 「酵素型ロドプシン：細胞内シグナル伝達を光で操る」 (2019)
  • Author(s): 角田聡
  • Organizer: 第42回日本分子生物学会年会
  • Int'l Joint Research / Invited
• [Presentation] 「光駆動型イオンポンプとイオンチャネルがイオンを輸送する仕組み」 (2018)
  • Author(s): 角田聡
  • Organizer: 大阪大学蛋白質研究所セミナー「構造情報に基づいた膜イオン輸送タンパク質の生理機能の解明に向けて」
  • Invited
• [Presentation] 「生体エネルギーを浪費する光駆動内向きプロトンポンプロドプシン」 (2018)
  • Author(s): 角田聡 神取秀樹
  • Organizer: 日本生体エネルギー研究会 第４３回討論会
• [Book] Optogenetics “Chapter 9 Molecular properties and optogenetics application of enzymerhodopsins” (2021)
  • Author(s): Tsunoda SP, Sugiura M, Kandori H
  • Publisher: Springer Nature
  • ISBN: 9789811587634
• [Remarks] 真核生物の祖先に最も近縁なアスガルド古細菌の持つ 新しい光受容タンパク質の機能を解明
  • URL: https://www.jst.go.jp/pr/announce/20200411/index.html
• [Patent(Industrial Property Rights)] 青色光応答性タンパク質及びその利用 (2021)
  • Inventor(s): 田代凛太郎、細島頌子、角田聡、神取秀樹
  • Industrial Property Rights Holder: 田代凛太郎、細島頌子、角田聡、神取秀樹
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2021-023873
  • Filing Date: 2021