Characterization of molecular mechanism of electrogenic transporters accompanying ion movement

• Project/Area Number: 23590062
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: Toho University
• Principal Investigator: MIYAUCHI Seiji 東邦大学, 薬学部, 教授 (30202352)
• Co-Investigator(Kenkyū-buntansha): KIKUKAWA Takashi 北海道大学, 生端生命科学研究科(研究院), 講師 (20281842)
• Project Period (FY): 2011 – 2013
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2012: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2011: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: トランスポーター / 分子弁 / オリゴペプチドトランスポーター / 光駆動性イオンポンプ / 水素結合ネットワーク / pKaシフト / 輸送サイクル

Research Abstract

To clarify the molecular mechanism of the electrogenic transporter accompanying ion movement, we precisely determined the transport of halorhodopsin, a light-driven Cl- pump (pHR) and human oligopeptide transporter (hPEPT1).
(1) The crystal structure (PDB:3QBG) of pHR clearly shows that the amino acid residue, Ser 130 is located in the vicinity of the protonated Schiff base and forms the hydrogen bond to the Schiff vase. Based on the Cl- transport activities of mutant Ser130, it has turned out that Ser130 coordinates with PBS and functions as a molecular valve to hinder the internal leak, backflux of Cl- during anion-transport cycle.
(2) We demonstrated that mutation of Ser302 located in the vicinity of His57 in PEPT1 caused the alkaline shift of the optimum pH in the transport activity. This shift demonstrates that the hydroxyl group of Ser302 forms the hydrogen-bonding with the imidazole of His57, which might keep the uptake activity optimum in the range of physiological intestinal pH.

Research Products

• [Journal Article] Thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry (2013)
  • Author(s): Hayashi, S., Tamogami, J., Kikukawa, T., Okamoto, H., Shimono, K., Miyauchi, S., Demura, M., Nara, T., and Kamo, N.
  • Journal Title: Biophys Chem Volume: 172 Pages: 61-67
  • Peer Reviewed
• [Journal Article] A study of the interaction of drugs with liposomes with isothermal titration calorimetry (2013)
  • Author(s): Osanai H., Ikehara T., Miyauchi S., Shimono K., Tamogami J., Nara T., Kamo N.
  • Journal Title: J Biophys Chem Volume: 4 Pages: 11-21
  • Peer Reviewed
• [Journal Article] Salt bridge in the conserved His-Asp cluster in Gloeobacter rhodopsin contributes to trimer formation (2013)
  • Author(s): Tsukamoto, T., Kikukawa, T., Kurata, T., Jung, K. H., Kamo, N., and Demura, M.
  • Journal Title: F EBS Lett Volume: 587 Pages: 322-327
  • NAID: 120005228320
  • Peer Reviewed
• [Journal Article] A study of the interaction of drugs with liposomes with isothermal titration calorimetry (2013)
  • Author(s): Osanai H, Ikehara T, Miyauchi S1, Shimono K, Tamogami J, Nara T, Kamo N
  • Journal Title: Journal of Biophysical Chemistry Volume: 4 Issue: 01 Pages: 11-21
  • DOI: 10.4236/jbpc.2013.41002
  • Peer Reviewed
• [Journal Article] Thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry (2013)
  • Author(s): Hayashi, Saori
  • Journal Title: Biophysical Chemistry Volume: 172 Pages: 61-67
  • DOI: 10.1016/j.bpc.2013.01.001
  • Peer Reviewed
• [Journal Article] Photoinduced proton release in proteorhodopsin at low pH : the possibility of a decrease in the pK(a) of Asp227 (2012)
  • Author(s): Tamogami, J., Kikukawa, T., Nara, T., Shimono, K., Demura, M., and Kamo, N.
  • Journal Title: Biochemistry Volume: 51 Pages: 9290-9301
  • Peer Reviewed
• [Journal Article] Influence of halide binding on the hydrogen bonding network in the active site of Salinibacter sensory rhodopsin I (2012)
  • Author(s): Reissig, L., Iwata, T., Kikukawa, T., Demura, M., Kamo, N., Kandori, H., and Sudo, Y.
  • Journal Title: Biochemistry Volume: 51 Pages: 8802-8813
  • Peer Reviewed
• [Journal Article] Homotrimer formation and dissociation of pharaonis halorhodopsin in detergent system (2012)
  • Author(s): Tsukamoto, T., Sasaki, T., Fujimoto, K. J., Kikukawa, T., Kamiya, M., Aizawa, T., Kawano, K., Kamo, N., and Demura, M.
  • Journal Title: Biophys J Volume: 102 Pages: 2906-2915
  • Peer Reviewed
• [Journal Article] Expression of salinarum halorhodopsin in Escherichia coli cells : solubilization in the presence of retinal yields the natural state (2011)
  • Author(s): Yamashita, Y., Kikukawa, T., Tsukamoto, T., Kamiya, M., Aizawa, T., Kawano, K., Miyauchi, S., Kamo, N., and Demura, M.
  • Journal Title: Biochim Biophys Acta Volume: 1808 Pages: 2905-2912
  • NAID: 120003762628
  • Peer Reviewed
• [Journal Article] Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga (2011)
  • Author(s): Wada, T., Shimono, K., Kikukawa, T., Hato, M., Shinya, N., Kim, S. Y., Kimura-Someya, T., Shirouzu, M., Tamogami, J., Miyauchi, S., Jung, K. H., Kamo, N., and Yokoyama, S.
  • Journal Title: J Mol Biol Volume: 411 Pages: 986-998
  • Peer Reviewed
• [Journal Article] Photochemistry of Acetabularia rhodopsin II from a marine plant, Acetabularia acetabulum (2011)
  • Author(s): Kikukawa, T., Shimono, K., Tamogami, J., Miyauchi, S., Kim, S. Y., Kimura- Someya, T., Shirouzu, M., Jung, K. H., Yokoyama, S., and Kamo, N.
  • Journal Title: Biochemistry Volume: 50 Pages: 8888-8898
  • Peer Reviewed
• [Journal Article] Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (phoborhodopsin) from Natronobacterium pharaonis (2011)
  • Author(s): Tateishi, Y., Abe, T., Tamogami, J., Nakao, Y., Kikukawa, T., Kamo, N., and Unno, M.
  • Journal Title: Biochemistry Volume: 50 Pages: 2135-2143
  • Peer Reviewed
• [Journal Article] Photochemistry of acetabularia rhodopsin II from a marine plant, Aceta bularia acetabulum (2011)
  • Author(s): Kikukawa Takashi
  • Journal Title: Biochemistry Volume: 50 Issue: 41 Pages: 8888-8898
  • DOI: 10.1021/bi2009932
  • Peer Reviewed
• [Journal Article] Expression of salinarum halorhodopsin in Escherichia coli cells : Solubilization in the presence of retinal yields the natural state (2011)
  • Author(s): Yamashita Yasutaka
  • Journal Title: Biochimica et Biophysica Acta-Biomembranes Volume: 1808 Issue: 12 Pages: 2905-2912
  • DOI: 10.1016/j.bbamem.2011.08.035
  • Peer Reviewed
• [Presentation] アフリカツメガエル卵母細胞発現系を用いたヒト・ナトリウム依存性胆汁酸輸送担体(hNTCP)の機能解析 (2014)
  • Author(s): 増田雅行,下野和実,真坂亙,宮内正二
  • Organizer: 第134年会日本薬学会
  • Place of Presentation: 日熊本市総合体育館,熊本
  • Year and Date: 2014-03-30
• [Presentation] The protonation state of a crucial residue, His57 in human oligopeptide transporter (hPEPT1) regulates the transport activity? (2013)
  • Author(s): Miyauchi Seiji
  • Organizer: The Asian Federation for Pharmaceutical Sciences (AFPS) 2013
  • Place of Presentation: Ramada Plaza, Jeju, South Korea
  • Year and Date: 2013-11-22
  • Invited
• [Presentation] Ser302 in Human Oligopeptide Transporter (hPEPT1) Regulates the Uptake Activity through a Hydrogen-bonding Interaction with the Crucial Residue, His57 (2013)
  • Author(s): Miyauchi Seiji, Shimono Kazumi, Nara Toshifumi
  • Organizer: 2013 American Association of Pharmaceutical Scientists Annual Meeting and Exposition
  • Place of Presentation: Gonzalez Convention Center, San Antonio, USA
  • Year and Date: 2013-11-13
• [Presentation] Na^+ dependent opioid peptide transporter as a molecular target for drug development (2013)
  • Author(s): Miyauchi Seiji
  • Organizer: The First International Conference of Sugiyama Laboratory : Drug Discovery, Development and Optimization of ADME Properties
  • Place of Presentation: Hibiya Convention Hall, Tokyo, Japan
  • Year and Date: 2013-09-26
  • Invited
• [Presentation] 無細胞タンパク質合成系を利用したヒト銅トランスポーター(hCtr1)の大量合成 (2012)
  • Author(s): 下野和実,水谷賢人,横山茂之,宮内正二
  • Organizer: 第7回トランスポーター研究会
  • Place of Presentation: 日京都大学農学部(京都府)
  • Year and Date: 2012-06-09
• [Presentation] ヒトオリゴペプチド輸送担体(hPEPT1)の活性中心であるHis57とSer302の相互作用 (2012)
  • Author(s): 米田真一郎,宮内正二,佐々木将太朗,加茂直樹,下野和実
  • Organizer: 日本薬剤学会,第27年会
  • Place of Presentation: 神戸国際会議場(兵庫県)
  • Year and Date: 2012-05-24
• [Presentation] Human sodium-coupled monocarboxylate transporter (SMCT): Its physiological functions (2012)
  • Author(s): Miyauchi, Seiji
  • Organizer: 28^hAnnual Research Conference in Pharmaceutical Sciences
  • Place of Presentation: Chulalongkorn University, Bangkok, Thailand
  • Year and Date: 2012-01-20
  • Invited
• [Presentation] Sodium-dependent monocarboxylate transporter (2012)
  • Author(s): S. Miyauchi
  • Organizer: 28thAnnual Research Conference in Pharmaceutical Sciences(招待講演)
  • Place of Presentation: Chulalongkorn University, Bangkok
• [Presentation] H^+/オリゴペプチド共輸送担体(hPEPT1)の活性中心His57近傍における水素結合とその役割 (2011)
  • Author(s): 宮内正二,米田真一郎,佐々木将太郎,下野和実
  • Organizer: 第33回生体膜と薬物の相互作用シンポジウム
  • Place of Presentation: 岡山大学創立五十周年記念館(岡山大学津島キャンパス,岡山市)
  • Year and Date: 2011-11-25
• [Presentation] Characterization of human Na^+/taurocholate (TCA) cotransporting polypeptide (hNTCP) with an electrophysiological technique (2011)
  • Author(s): Miyauchi Seiji, Shimono Kazumi, Nara Toshifumi, Kamo Naoki
  • Organizer: American Association of Pharmaceutical Scientists (AAPS) Annual Meeting
  • Place of Presentation: Walter E. Washington Convention Center (Washington DC)
  • Year and Date: 2011-10-25
• [Presentation] H^+/オリゴペプチド共輸送担体(hPEPT1)におけるHis57-Ser302水素結合による活性調節 (2011)
  • Author(s): 佐々木将太郎,下野和実,藤澤祐紀,奈良敏文,加茂直樹,宮内正二
  • Organizer: 第6回トランスポーター研究会年会
  • Place of Presentation: 東北大学片平さくらホール(仙台)
  • Year and Date: 2011-06-11
• [Presentation] H+/オリゴペプチド共輸送担体(hPEPT1)におけるHis57-Ser302水素結合による活性調節 (2011)
  • Author(s): 佐々木将太郎，下野和実，藤澤祐紀，奈良敏文，加茂直樹，宮内正二
  • Organizer: 第6回トランスポーター研究会年会
  • Place of Presentation: 東北大学 片平さくらホール（仙台）
• [Presentation] 等温滴定型熱量計を用いた大腸菌由来 H+/オリゴペプチド共輸送担体（YdgR）の基質結合の熱力学的解析 (2011)
  • Author(s): 下野和実，浅倉祐未，宮内正二
  • Organizer: 第6回トランスポーター研究会年会
  • Place of Presentation: 東北大学 片平さくらホール（仙台）
• [Presentation] Characterization of human Na+/taurocholate (TCA) cotransporting polypeptide (hNTCP) with an (2011)
  • Author(s): S. Miyauchi, K. Shimono, T. Nara, N. Kamo
  • Organizer: American Association of Pharmaceutical Scientists (AAPS) Annual Meeting
  • Place of Presentation: Walter E. Washington Convention Center (Washington DC)
• [Presentation] H+/オリゴペプチド共輸送担体(hPEPT1)の活性中心His57近傍における水素結合とその役割 (2011)
  • Author(s): 宮内正二，米田真一郎，佐々木将太郎，下野和実
  • Organizer: 第３３回生体膜と薬物の相互作用シンポジウム
  • Place of Presentation: 岡山大学創立五十周年記念館（岡山大学津島キャンパス，岡山市）
• [Presentation] Na+ dependent opioid peptide transporter as a molecular target for drug development
  • Author(s): Miyauchi Seiji
  • Organizer: The First International Conference of Sugiyama Laboratory: Drug Discovery, Development and Optimization of ADME Properties
  • Place of Presentation: Hibiya Convention Hall, Tokyo, Japan
  • Invited
• [Presentation] Ser302 in Human Oligopeptide Transporter (hPEPT1) Regulates the Uptake Activity through a Hydrogen-bonding Interaction with the Crucial Residue, His57
  • Author(s): Miyauchi Seiji, Shimono Kazumi, Nara Toshifumi
  • Organizer: 2013 American Association of Pharmaceutical Scientists Annual Meeting and Exposition
  • Place of Presentation: Gonzalez Convention Center, San Antonio, USA
• [Presentation] アフリカツメガエル卵母細胞発現系を用いたヒト・ナトリウム依存性胆汁酸輸送担体(hNTCP)の機能解析
  • Author(s): 増田雅行，下野和実，真坂 亙，宮内正二
  • Organizer: 第１３４年会日本薬学会
  • Place of Presentation: 熊本市総合体育館，熊本
• [Presentation] ヒトオリゴペプチド輸送担体(hPEPT1)の活性中心であるHis57 とSer302 の相互作用
  • Author(s): 米田真一郎，宮内正二，佐々木将太朗，加茂直樹，下野和実
  • Organizer: 日本薬剤学会，第27年会
  • Place of Presentation: 神戸国際会議場（兵庫県）
• [Presentation] 無細胞タンパク質合成系を利用したヒト銅トランスポーター（hCtr1）の大量合成
  • Author(s): 下野和実，水谷賢人，横山茂之，宮内正二
  • Organizer: 第7回トランスポーター研究会
  • Place of Presentation: 京都大学農学部（京都府）
• [Book] 「薬剤学実験法マニュアルII」日本薬剤学会出版委員会編,第3章(薬物吸収)1節(経口投与)3項「In vivo実験」 (2014)
  • Author(s): 宮内正二
  • Publisher: 南江堂
• [Book] 薬剤学実験法マニュアルII (2014)
  • Author(s): 宮内正二
  • Total Pages: 7
  • Publisher: 南江堂
• [Book] 「パートナー薬剤学」寺田勝英,伊藤智夫編,第4章5節「薬物の排泄」 (2012)
  • Author(s): 宮内正二
  • Publisher: 南江堂