Analysis of dynamic structural rearrangements of functioning ion channels by fluorescent unnatural amino acid

2019 Fiscal Year Annual Research Report

• Project/Area Number: 17H04021
• Research Institution: National Institute for Physiological Sciences
• Principal Investigator: 久保 義弘 生理学研究所, 分子細胞生理研究領域, 教授 (80211887)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: ATP受容体チャネル / 蛍光非天然アミノ酸 / 動的構造変化

Outline of Annual Research Achievements

ATP受容体チャネルP2X2の膜電位依存的動的構造変化の解明を目的として、蛍光非天然アミノ酸 (fUAA)の分子内導入を用いたVoltage clamp fluorometry法による解析を進めた。第2膜貫通部位のAla337の位置で膜電位依存的な蛍光強度の変化が観察された。その蛍光強度の変化は膜電位全域でリニアであること等から、fUAAが強い電場内に位置しそのelectrochromic な性質による変化と考えられた。構造上、ATP存在下では、第1膜貫通部位の Phe44がAla337の近傍に位置する。よって、Ala337とPhe44の相互作用が、その場が強い電場にあるため活性が膜電位依存的となることが示唆された。ATP存在下でのPhe44の膜電位依存的動きが焦点となるが、この位置にfUAAを導入した分子は機能を欠失した。そこでPhe44の近傍を対象に、遷移金属イオンを導入する新しい光学的解析法の導入にも取り組んだ。
Two Pore Na+チャネル TPC3についても、fUAAの導入による構造変化解析を行った。併せて、第2リピートの膜電位センサーに導入したCys残基のCys修飾剤による修飾速度の解析を行い、第1リピートに結合する Phosphoinositides が減少すると修飾が遅くなる、すなわち膜電位センサーの動きが低下することを観察した。これが、Phosphoinositidesの結合による活性化誘導の分子機構であることが示唆された。
GIRKチャネルの機能調節機構については、抗ヒスタミン薬が機能を阻害することを見出し、網羅的な点変異体の解析により、その結合部位がポアヘリックスにあることを見出した。さらに、分子ドッキング様式の解析を行い、ポア構造とPIP2の結合の両方に影響を与えうる新規の結合様式であることを明らかにした。

Research Progress Status

令和元年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和元年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] Weizmann Institute of Science(イスラエル)
  • Country Name: ISRAEL
  • Counterpart Institution: Weizmann Institute of Science
• [Int'l Joint Research] Sussex University(英国)
  • Country Name: UNITED KINGDOM
  • Counterpart Institution: Sussex University
• [Int'l Joint Research] University of California Berkeley(米国)
  • Country Name: U.S.A.
  • Counterpart Institution: University of California Berkeley
• [Int'l Joint Research] McGill University(カナダ)
  • Country Name: CANADA
  • Counterpart Institution: McGill University
• [Journal Article] Non-sedating antihistamines block G-protein-gated inwardly rectifying K+ channels. (2019)
  • Author(s): Chen IS, Liu C, Tateyama M, Karbat I, Uesugi M, Reuveny E, Kubo Y
  • Journal Title: Br J Pharmacol Volume: 176 Pages: 3161-3179
  • DOI: 10.1111/bph.14717
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Phosphoinositides modulate the voltage dependence of two-pore channel 3 (2019)
  • Author(s): Shimomura T, Kubo Y
  • Journal Title: J Gen Physiol Volume: 151 Pages: 986-1006
  • DOI: 10.1085/jgp.201812285
  • Peer Reviewed
• [Journal Article] Congenital goitrous hypothyroidism is caused by dysfunction of the iodide transporter SLC26A7 (2019)
  • Author(s): Ishii J, Suzuki A, Kimura T, Tateyama M, Tanaka T, Yazawa T, Arimasu Y, Chen IS, Aoyama K, Kubo Y, Saitoh S, Mizuno H, Kamma H
  • Journal Title: Commun Biol Volume: 2 Pages: 270
  • DOI: 10.1038/s42003-019-0503-6
  • Peer Reviewed
• [Presentation] Inhibitory mechanisms of G-protein-gated inwardly rectifying K+ channel by antihistamines (2020)
  • Author(s): Chen IS, Liu C, Tateyama M, Karbat I, Uesugi M, Reuveny E, Kubo Y
  • Organizer: Biophysical Society 64th Annual Meeting
  • Int'l Joint Research
• [Presentation] PI(3,4)P2-dependent modulation of voltage dependence in Two-Pore Chanel 3 (2020)
  • Author(s): Shimomura T, Kubo Y
  • Organizer: Biophysical Society 64th Annual Meeting
  • Int'l Joint Research
• [Presentation] Relative voltage sensor activation kinetics determines the effects of sensor neutralization in voltage-gated sodium channels (2020)
  • Author(s): Brake, N, Manchino A, Yan Y, Shimomura T, Kubo Y, Bowie D, Khadra A
  • Organizer: Biophysical Society 64th Annual Meeting
• [Presentation] PI(3,4)P2- and voltage-dependent gating of two-pore Na+ channel 3 (2020)
  • Author(s): Shimomura T, Hirazawa K, Kubo Y
  • Organizer: The 97th Annual Meeting of the Physiological Society of Japan
• [Presentation] Chen IS, Kubo Y (2020)
  • Author(s): Roles of the pore helix in the regulation mechanisms of gating of GIRK channels
  • Organizer: The 97th Annual Meeting of the Physiological Society of Japan
• [Presentation] Analysis of the structural dynamics of Two-pore Na+ channel 3 (2020)
  • Author(s): Hirazawa K, Shimomura T, Kubo Y
  • Organizer: The 97th Annual Meeting of the Physiological Society of Japan
• [Presentation] Activation of the THIK-2 channels by Gi/o and Gq coupled receptors (2020)
  • Author(s): Tateyama M, Kubo Y
  • Organizer: The 97th Annual Meeting of the Physiological Society of Japan
• [Presentation] Physiological roles of Prrt3, an orphan metabotropic receptor: Comprehensive behavioral test battery analysis using homozgous full gene knock-out mice derived from flox mice (2020)
  • 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
• [Presentation] ATP- and voltage-dependent gating of P2X2 receptor analyzed by voltage-clamp fluorometry (2020)
  • Author(s): Andriani RT, Kubo Y
  • Organizer: The 97th Annual Meeting of the Physiological Society of Japan
• [Presentation] Modulation of ion channel function by Sigma-1 receptor, a multimodal membrane protein (2020)
  • Author(s): Liu C, Chen IS, Murrell-Lagnado R, Kubo Y
  • Organizer: The 97th Annual Meeting of the Physiological Society of Japan
• [Presentation] ATP-and voltage-dependent gating of P2X2 receptor analyzed by voltage-clamp fluorometry (2019)
  • Author(s): Andriani RT, Kubo Y
  • Organizer: The 7th International Ion Channel Conference
  • Int'l Joint Research
• [Presentation] Phosphoinositides modulate the voltage dependence of Two-Pore Channel 3 (2019)
  • Author(s): Kubo Y
  • Organizer: The 7th International Ion Channel Conference
  • Int'l Joint Research / Invited
• [Presentation] Phosphoinositides modulate the voltage dependence in Two-Pore Na+ Channel 3 (2019)
  • Author(s): Kubo Y
  • Organizer: The Ion Channel Modulation Symposium
  • Int'l Joint Research / Invited