Analysis of dynamic structural rearrangements of functioning ion channels by fluorescent unnatural amino acid
| Project/Area Number |
17H04021
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | National Institute for Physiological Sciences |
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Principal Investigator |
KUBO YOSHIHIRO 生理学研究所, 分子細胞生理研究領域, 教授 (80211887)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Project Status |
Completed (Fiscal Year 2019)
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| Budget Amount *help |
¥17,940,000 (Direct Cost: ¥13,800,000、Indirect Cost: ¥4,140,000)
Fiscal Year 2019: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2018: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2017: ¥7,150,000 (Direct Cost: ¥5,500,000、Indirect Cost: ¥1,650,000)
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| Keywords | ATP受容体チャネル / 蛍光非天然アミノ酸 / 動的構造変化 / 生体分子 / イオンチャネル |
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| Outline of Final Research Achievements |
ATP receptor channel P2X2 shows voltage-dependent gating, in spite of the absence of canonical voltage-sensor in the molecule. We approached the molecular mechanisms by voltage-clamp fluorometry analysis using unnatural Fluorescent Amino Acid (fUAA). When fUAA was introduced at Ala337 by mutagenesis in the transmembrane domain 2 (TM2), voltage dependent fluorescence change was observed. As the change was linear in the recorded voltage range, it was shown that it is not due to conformational change of P2X2 protein but due to an electrochromic effect of fUAA, and that 337 position is located at a converged electric field. As it is known Phe44 in TM1 is close to Ala337 in the presence of ATP, and as we showed the importance of the interaction between Ala337 and Phe44 for activation by mutagenesis, it was suggested that the interaction between them in the strong electric field is voltage-dependent and that it underlies the voltage-dependent gating of P2X2.
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| Academic Significance and Societal Importance of the Research Achievements |
ATP受容体チャネルP2X2は、典型的な膜電位センサーが無いにも関わらず、未知のメカニズムにより膜電位依存的ゲーティングを示す。本研究では、この根源的な謎にアプローチした。本研究の特徴として、非天然蛍光アミノ酸を分子内に導入し、膜電位固定下で蛍光測光を行い、膜電位依存的構造変化等を捉える革新的な方法論を用いた点があげられる。本研究で得られた知見、および確立した方法論は、今後、膜タンパク質研究に広く敷衍しうる可能性を有する。
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Report
(4 results)
Research Products
(39 results)
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[Journal Article] Ivermectin activates GIRK channels in a PIP2 -dependent, Gβγ-independent manner and an amino acid residue at the slide helix governs the activation.2017
Author(s)
Chen, I.S., Tateyama, M., Fukata, Y., Uesugi, M., & Kubo, Y.
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Journal Title
Journal of Physiology
Volume: 595
Issue: 17
Pages: 5895-5912
DOI
Related Report
Peer Reviewed
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[Presentation] Physiological roles of Prrt3, an orphan metabotropic receptor: Comprehensive behavioral test battery analysis using homozgous full gene knock-out mice derived from flox mice2020
Author(s)
Yamamoto T, Hattori S, Zhou L, Natsume R, Konno K, Chen IS, Watanabe M, Sakimura K, Miyakawa T, Kubo Y
Organizer
The 97th Annual Meeting of the Physiological Society of Japan
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