Molecular mechanism of the substrate selectivity in Voltage-sensing phosphatase

• Project/Area Number: 25860163
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: General physiology
• Research Institution: Osaka Medical College (2015); Osaka University (2013-2014)
• Principal Investigator: SAKATA Souhei 大阪医科大学, 医学部, 講師 (40528006)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2014: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2013: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: 膜電位 / イノシトールリン脂質 / 非天然アミノ酸 / 電位依存性ホスファターゼ / 脱リン酸化酵素

Outline of Final Research Achievements

Voltage-sensing phosphatase, VSP, has an activity to dephosphorylate several kinds of phosphoinositides in a voltage-dependent manner. Our recent study has shown that VSP mainly dephosphoryltaes PI(3,4,5)P3 in low membrane voltages but dephosphorylates PI(3,4)P2 in high voltages. However, little is know about the molecular mechanisms how the membrane voltage regulates the substrate specificity of VSP. In this work, I introduced a fluorescent label using the method of the genetic incorporation of a fluorescent unnatural amino acid to reveal the conformational change of the catalytic region which is responsible for the dephosphorylation activity. Results showed that Cα2 loop located in the catalytic region plays key roles in the substrate specificity in VSP.

Research Products

• [Journal Article] Gating mechanisms of voltage-gated proton channels. (2015)
  • Author(s): Okamura Y, Fujiwara Y, Sakata S.
  • Journal Title: Annu Rev Biochem. Volume: 84 Issue: 1 Pages: 685-709
  • DOI: 10.1146/annurev-biochem-060614-034307
  • Peer Reviewed
• [Journal Article] Functional diversity of voltage-sensing phosphatases (VSPs) in two urodele amphibians (2014)
  • Author(s): Mutua J, Jinno Y, Sakata S, Okochi Y, Ueno S, Tsutsui H, Kawai T, Iwao Y, Okamura Y.
  • Journal Title: Physiological Reports Volume: 2 Issue: 7 Pages: 1-13
  • DOI: 10.14814/phy2.12061
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Phosphatase activity of the voltage-sensing phosphatase, VSP, shows graded dependence on the extent of activation of the voltage sensor (2014)
  • Author(s): Sakata S & Okamura Y
  • Journal Title: J. Physiol. Volume: 592(Pt 5) Issue: 5 Pages: 899-914
  • DOI: 10.1113/jphysiol.2013.263640
  • Peer Reviewed / Open Access
• [Journal Article] A Kir3.4 mutation causes Andersen–Tawil syndrome by an inhibitory effect on Kir2.1. (2014)
  • Author(s): Kokunai Y, Nakata T, Furuta M, Sakata S, Kimura H, Aiba T, Yoshinaga M, Osaki Y, Nakamori M, Itoh H, Sato T, Kubota T, Kadota K, Shindo K, Mochizuki H, Shimizu W, Horie M, Okamura Y, Ohno K, Takahashi MP
  • Journal Title: Neurology Volume: 82 Issue: 12 Pages: 1058-64
  • DOI: 10.1212/wnl.0000000000000239
  • Peer Reviewed / Open Access
• [Journal Article] X-ray crystal structure of voltage-gated proton channel (2014)
  • Author(s): Takeshita K, Sakata S, Yamashita E, Fujiwara Y, Kawanabe A, Kurokawa T, Okochi Y, Matsuda M, Narita H, Okamura Y & Nakagawa A
  • Journal Title: Nat. Strucl. & Mol. Biol. Volume: (4) Issue: 4 Pages: 352-7
  • DOI: 10.1038/nsmb.2783
  • Peer Reviewed / Open Access
• [Journal Article] Potential role of voltage-sensing phosphatases in regulation of cell structure through the production of PI(3,4)P₂ (2013)
  • Author(s): Yamaguchi S, Kurokawa T, Taira I, Aoki N, Sakata S, Okamura Y & Homma KJ
  • Journal Title: J. Cell Physiol. Volume: 229 Issue: 4 Pages: 422
  • DOI: 10.1002/jcp.24463
  • URL: https://pure.teikyo.jp/en/publications/20824bef-a19c-4d50-b34a-91c3897b4187
  • Peer Reviewed / Open Access
• [Presentation] ゼブラフィッシュの神経筋接合部を用いたシナプスホメオスタシスの分子メカニズムの研究 (2016)
  • Author(s): Souhei Sakata , Meghan Mott, Victor Mari Luna, Kimberly Epley and Fumihito Ono
  • Organizer: 第９３回日本生理学会大会
  • Place of Presentation: 札幌
  • Year and Date: 2016-03-22
• [Presentation] 蛍光を持つ非天然アミノ酸を利用した電位依存性ホスファターゼの酵素ドメインの電位依存的な構造変化の検出 (2015)
  • Author(s): 坂田宗平、岡村康司
  • Organizer: 第92回日本生理学会大会
  • Place of Presentation: 神戸
  • Year and Date: 2015-03-21
• [Presentation] Chimeric study between sea urchin and mouse orthologs of voltage-gated proton channel revealed the critical sites for the activation speed (2014)
  • Author(s): Souhei Sakata, Nana Miyawaki, Tatsuki Kurokawa, Nurdan Oezkucur, Yasushi Okamura
  • Organizer: 第９１回日本生理学会大会
  • Place of Presentation: 鹿児島大学 郡元キャンパス
• [Presentation] 電位依存性ホスファターゼ 噓倀 の酵素活性は、電位センサーの活性化レベルに 応じて段階的に発揮される (2013)
  • Author(s): 坂田宗平、岡村康司
  • Organizer: 第106回近畿生理学談話会
  • Place of Presentation: 奈良県立医科大学
• [Remarks] 大阪大学医学系研究科統合生理学
  • URL: http://www.med.osaka-u.ac.jp/pub/phys2/okamura/