Mutual conversion of ion-pump and ion-channel rhodopsins by amino acid replacement

• Project/Area Number: 18K14658
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 43040:Biophysics-related
• Research Institution: Hokkaido University
• Principal Investigator: Tsukamoto Takashi 北海道大学, 先端生命科学研究院, 助教 (30744271)
• Project Period (FY): 2018-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: 光受容体 / ロドプシン / イオンチャネル / イオンポンプ / 構造変化 / 速度論的解析 / 機能変換 / フラッシュフォトリシス / オプトジェネティクス

Outline of Final Research Achievements

The goal of this study was to identify what the determinant factor is for the different ion transport mechanisms, pump and channel. Here, we used anion pump and anion channel rhodopsins as examples, which are a family of photoreceptor proteins in microbes. We mainly focused on the anion channel rhodopsins in this study and explored novel ones and investigated their mechanisms for the anion channel activity. We developed a simple measuring technique for the anion transport activity. Because of this, we successfully identified some anion channel rhodopsins with unique anion transport properties. In addition, we reported for the first time the anion binding in the initial state and the functional mechanism which was strongly affected by the anion concentration.

Academic Significance and Societal Importance of the Research Achievements

微生物の光受容体タンパク質・ロドプシンは、共通のタンパク質構造と活性化反応をもつにもかかわらず、『ポンプ』と『チャネル』という全く異なる方式で基質イオンを輸送する。それぞれの輸送機構はよく理解されてきたが、輸送方式を決定する要因は明らかではない。これを解明するためには、アミノ酸変異によるポンプ⇔チャネル相互機能変換が有効である。
本研究は、ポンプをポンプたらしめる、チャネルをチャネルたらしめるロドプシンの精巧な動作原理の理解につながる。本研究成果を応用し、オプトジェネティクス（光による神経細胞活動の操作技術）に利用可能な分子開発に展開できる。

Research Products

• [Journal Article] Quantitation of the neural silencing activity of anion channelrhodopsins in Caenorhabditis elegans and their applicability for long-term illumination (2019)
  • Author(s): Yamanashi T, Maki M, Kojima K, Shibukawa A, Tsukamoto T, Chowdhury S, Yamanaka A, Takagi S, Sudo Y
  • Journal Title: Sci. Rep. Volume: 9(1) Issue: 1 Pages: 7863-7863
  • DOI: 10.1038/s41598-019-44308-x
  • NAID: 120006733902
  • Peer Reviewed / Open Access
• [Journal Article] Functional importance of the oligomer formation of the cyanobacterial H+ pump Gloeobacter rhodopsin (2019)
  • Author(s): Iizuka Azusa、Kajimoto Kousuke、Fujisawa Tomotsumi、Tsukamoto Takashi、Aizawa Tomoyasu、Kamo Naoki、Jung Kwang-Hwan、Unno Masashi、Demura Makoto、Kikukawa Takashi
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 10711-10711
  • DOI: 10.1038/s41598-019-47178-5
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl- concentrations (2018)
  • Author(s): Tsukamoto Takashi、Kikuchi Chihiro、Suzuki Hiromu、Aizawa Tomoyasu、Kikukawa Takashi、Demura Makoto
  • Journal Title: Scientific Reports Volume: 8 Issue: 1 Pages: 13445-13445
  • DOI: 10.1038/s41598-018-31742-6
  • Peer Reviewed / Open Access
• [Journal Article] High thermal stability of oligomeric assemblies of Thermophilic rhodopsin in a lipid environment. (2018)
  • Author(s): Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, and Yasuhisa Mizutani
  • Journal Title: J. Phys. Chem. B Volume: 122 Issue: 27 Pages: 6945-6953
  • DOI: 10.1021/acs.jpcb.8b04894
  • 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
• [Presentation] Replacements of “H+ donor” residues in the light-driven H+-pump rhodopsins (2019)
  • Author(s): Kikukawa, T., Sasaki, S., Nishiya, K., Tsukamoto, T., Aizazwa, T.,Demura, M., Tamogami, J.
  • Organizer: 17th International Congress on Photobiology & 18th Congress of the European Society for Photobiology
  • Int'l Joint Research
• [Presentation] Time-resolved detection of Na+ uptake and release reactions of Na+-pumping rhodopsin (2019)
  • Author(s): Murabe, K., Kato, T., Tsukamoto, T., Aizawa, T., Demura, T., Kikukawa, T.
  • Organizer: The 9th Asia and Oceania Conference on Photobiology
  • Int'l Joint Research
• [Presentation] Importance of His166 for Cl- pump activity of Mastigocladopsis repens halorhodopsin (2019)
  • Author(s): Iwama, K., Watanabe, Y., Tsukamoto, T., Aizawa, T., Demura, M., Kikukawa, T.
  • Organizer: 日本生物物理学会第57回年会
• [Presentation] Inversion of Proton Transport for Thermophilic Rhodopsin by Neutralizing the Secondary Counterion Asp229 (2019)
  • Author(s): Kiyoshima, M., Kikukawa, T., Aizawa, T., Demura, M., Sudo, Y., Tsukamoto, T.
  • Organizer: 日本生物物理学会第57回年会
• [Presentation] Comparative Study for Anion Transport Activity of Anion Channelrhodopsins by Using a Simple pH Electrode Method (2019)
  • Author(s): Kikuchi, C., Aizawa, T., Takashi Kikukawa, T., Demura, M., Tsukamoto, T.
  • Organizer: 日本生物物理学会第57回年会
• [Presentation] An Anion Channelrhodopsin with a Naturally Super-Slow Photocycle (2019)
  • Author(s): Tsukamoto, T., Kitahara, T., Kurane, H., Kikuchi, C., Aizawa, T., Kikukawa, T., Demura, M.
  • Organizer: 日本生物物理学会第57回年会
• [Presentation] Substrate anion concentration significantly affects the fast channel closing of Proteomonas sulcata anion channelrhodopsin-1 (2018)
  • Author(s): Takashi Tsukamoto、Chihiro Kikuchi、Hiromu Suzuki、Tomoyasu Aizawa、Takashi Kikukawa、Makoto Demura
  • Organizer: 日本生物物理学会第56回年会
• [Presentation] Anion concentration dependency on the photocycle of PsuACR1: Implications for the impairment of its fast channel function (2018)
  • Author(s): Takashi Tsukamoto、Chihiro Kikuchi、Hiromu Suzuki、Tomoyasu Aizawa、Takashi Kikukawa、Makoto Demura
  • Organizer: 18th International Conference on Retinal Proteins
  • Int'l Joint Research
• [Presentation] Photochemical Properties of Microbial Rhodopsin Pumps and Channels (2018)
  • Author(s): Takashi Tsukamoto
  • Organizer: Asia Pasific Society for Biology and Medical Sciences
  • Int'l Joint Research / Invited
• [Remarks] 研究室ホームページ
  • URL: https://tenure-track.cris.hokudai.ac.jp/lab/tsukamoto/
• [Remarks] 北海道大学プレスリリース
  • URL: https://www.hokudai.ac.jp/news/180912_pr.pdf