Roles of proton channels in proton signaling : pH-clamp and analysis of proton current oscillation

• Project/Area Number: 16590168
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: General physiology
• Research Institution: Osaka City University
• Principal Investigator: KUNO Miyuki Osaka City University, Graduate School of Medicine Department of Physiology, Associate Professor, 大学院・医学研究科, 助教授 (00145773)
• Project Period (FY): 2004 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
• Keywords: proton channel / pH regulation / oscillation / proton pump / temperature-sensitivity / recruitment / osteoclast / microglia / ゆらぎ / Q_<10> / 膜電位 / プロトンチャネル

Research Abstract

Voltage-gated proton (Hv) channels serve as an efficient H^+ efflux pathway and also as a highly-sensitive real-time pH-monitor. This study was focused on exploiting their unique properties to investigate dynamics and mechanisms of proton-signaling system. We measured the. membrane potential in microglia and osteoclasts, under the pH gradient across the membrane was clamped. by high concentrations (>100 mM) of pH buffers. In the absence of major ions, the membrane potential fluctuated at intervals from <10 ms to > 10 s. This oscillation disappeared by Zn, a blocker for Hv channels. Activity of the Hv channels was increased by a rise in temperature and by lactic acidosis (pH 6.8). Widely-varying current-amplitudes suggested a presence of dynamic recruitment mechanisms for Hv channels. We succeeded electrophysiological recordings of outward H^+ currents through the vacuolar-type H^+-ATPase for the first time in osteoclasts in which the H^+ pump is rich at the plasma membrane. The V-ATPase-mediated H^+ currents were affected by the electrochemical gradients for pH and voltages. The current amplitude was much smaller than that of the Hv channels, however, and lacked large fluctuation, implicating that the two H^+-secreting membrane molecules are regulated by different recruiting mechanisms. We also devised an osmotic-jump method and tested pathological cell models based on ATP-disturbances for further study of H^+ signaling. These findings suggest that the Hv channel is responsible for H^+ signal oscillation and may contribute to regulation of various cellular responses.

Research Products

• [Journal Article] Unregulated insulin secretion by pancreatic b-cells in hyperinsulinism/hyperammonemia syndrome Role of glutamate dehydrogenase, ATP-sensitive potassiumu channel, and nonselective cation channel (2006)
  • Author(s): Kawajiri, M.
  • Journal Title: Pediatric Res. 59(3) Pages: 359-354
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Proton currents through the plasmalemmal vacuolar-type H^+ -ATPase in osteoclast. (2006)
  • Author(s): Sakai, H.
  • Journal Title: Biophysical Journal Abstract
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Voltage-and pH-dependence of proton flux through the plasmalemmal vacuolar-type H^+-ATPase in osteoclasts (2006)
  • Author(s): Sakai, H.
  • Journal Title: J.Physiol.Sci. 56
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Unregulated insulin secretion by pancreatic b-cells in hyperinsulinism/hyperammonemia syndrome : Role of glutamate dehydrogenase, ATP-sensitive potassium channel, and nonselective cation channel. (2006)
  • Author(s): Kawajiri, M., Okano, Y., Kuno, M., Hase, Y., Inada, H., Tashiro, F., Miyazaki, J., Yamono, Y.
  • Journal Title: Pediatric Res. 59(3) Pages: 359-364
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Proton currents through the plasmalemmal vacuolar-type H^+-ATPase in osteoclast. (2006)
  • Author(s): Sakai, H., Kawawaki, J., Moriura, Y., Mori, H., Morihata, H., Kuno M.
  • Journal Title: Biophysical Journal Abstract 283a
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Voltage- and pH-dependence of proton flux through the plasmalemmal vacuolar-type H^+-ATPase in Osteoclasts. (2006)
  • Author(s): Sakai, H., Kawawaki, J., Moriura, Y., Mori, H., Hirokazu, M., Kuno, M.
  • Journal Title: J.Physiol.Sci. 56(Suppl.)
  • NAID: 130007038999
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Unique temperature dependence of the voltage-gated proton channel : Two-phase behavior with a variable transition temperature (2005)
  • Author(s): Kuno, M.
  • Journal Title: Jpn.J.Physiol. 55
  • NAID: 130005447955
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A novel method for the streaming potential under the whole-cell patch-clamp configuration. (2005)
  • Author(s): Ando, H.
  • Journal Title: Jpn.J.Physiol. 55
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Coupled K^+-water flux through the HERG potassium channel measured by an osmotic pulse method (2005)
  • Author(s): Ando, H.
  • Journal Title: J.Gen.Physiol. 126 Pages: 529-538
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Unique Temperature Dependence of the Voltage-Gated Proton Channel : Two-Phase Behavior with a Variable Transition Temperature. (2005)
  • Author(s): Kuno, M., Morihata, H., Mori, H., Kawawaki, J., Sakai, H., Shimizu H., Oiki, S.
  • Journal Title: Jpn.J.Physiol. 55(Supple)
  • NAID: 130005447955
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Coupled K^+-water flux through the HERG potassium channel measured by an osmotic pulse method. (2005)
  • Author(s): Ando, H., Kuno, M., Shimizu, H., Muramatsu, I., Oiki, S.
  • Journal Title: J.Gen.Physiol. 126 Pages: 529-538
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The Peripheral Nervous System 2. (in Japanese) (2005)
  • Author(s): Kuno, M., Ando, K., Sugihara, I., Akita, K.
  • Journal Title: Structure, Function and Materials of the Human Body (Nihon Iji Shinpou-sha) 9(2) Pages: 1-93
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Membrane potential serves as a principal regulator of H^+ secretion via the plasmalemmal, vacuolar-type H^+-ATPase in osteoclasts. (2005)
  • Author(s): Sakai, H., Mori, H., Morihata, H., Kawawaki, J., Kimura, M., Shioi, A., Kuno, M.
  • Journal Title: Biophysical J. 88(1)(Supple)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Unique temperature dependence of the voltage-gated proton channel : Two-phase behavior with a variable transition temperature (2005)
  • Author(s): Kuno, A
  • Journal Title: Jpn.J.Physiol. 55
  • NAID: 130005447955
• [Journal Article] Membrane potential serves as a principal regulator of H^+ secretion via the plasmalemmal, vacuolar-type H^+-ATPase in osteoclasts. (2005)
  • Author(s): Sakai H.
  • Journal Title: Biophysical J.(Abstract of 49^<th> Annual Meeting of Biophysical Society) (未定)
• [Journal Article] Effects of general anesthetics on P2X_7 receptors in rat microglia. (in Japanese). (2004)
  • Author(s): Nakanishi, M., Mori, T., Nishikawa, K., Kuno, M., Asada, A.
  • Journal Title: Shinkei Masui Shuchu Chiryou (in Japanese) 8 Pages: 82-83
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Activation of voltage-gated proton channels and calcium mobilization upon acute and prolonged cell acidosis in rat microglia. (2004)
  • Author(s): Morihata, H., Mori, H., Sakai, H., Kawawaki, J., Sawada, M., Tsutada, T., Kuno, M.
  • Journal Title: Biophys.J. 86,(1), Supple Part 2
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Electrophysiological analysis of voltage-dependent regulation of H^+ secretion in COS cells expressing the membrane sector (V_0) of osteoclast vacuolar type H^+-ATPase. (2004)
  • Author(s): Sakai, H., Mori, H., Morihata, H., Kawawaki, J., Shioi, A., Kuno, M.
  • Journal Title: Jpn.J.Physiol. 54 (Supple)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] pH homeostasis of microglia : The voltage-gated proton channel serves as a pH regulator upon cell acidosis. (2004)
  • Author(s): Kuno, M., Morihata, H., Kawawaki, J., Sakai, H., Sawada, M., Tsutada, T., Mori, H.
  • Journal Title: Neuroscience Res. 50 (Suppl1)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Electrophysiological analysis of voltage-dependent regulation of H^+ secretion in COS cells expressing the membrane sector (V_0) of osteoclast vacuolar type H^+-ATPase. (2004)
  • Author(s): Sakai, H.
  • Journal Title: Jpn.J.Physiol. 54
• [Journal Article] Activation of voltage-gated proton channels of rat microglia upon acute and prolonged cell acidosis. (2004)
  • Author(s): Morihata H.
  • Journal Title: Jpn.J.Physiol. 54
• [Journal Article] pH homeostasis of microglia : The voltage-gated proton channel serves as a pH regulator upon cell acidosis. (2004)
  • Author(s): Kuno M.
  • Journal Title: Neuroscience Res. 50
• [Journal Article] ラットミクログリアP2X7受容体に対する全身麻酔薬の作用 (2004)
  • Author(s): 仲西未佳
  • Journal Title: 神経麻酔・集中治療(プロシーディング) Pages: 82-83
• [Journal Article] 膜電位依存性プロトンチャネルのユニークな温度特性:2相性(高Q10-低Q10)応答と多様な転移温度
  • Author(s): 久野みゆき
  • Journal Title: 第82回生理学会大会発表予定 (未定)
  • NAID: 130005447955
• [Book] 人体の正常構造と機能:IX神経系(2) (2005)
  • Author(s): 久野 みゆき
  • Total Pages: 93
  • Publisher: 日本医事新報社
  • Description: 「研究成果報告書概要(和文)」より
• [Patent(Industrial Property Rights)] パッチクランプ装置 (2004)
  • Inventor(s): 老木 茂稔, 久野みゆき, 安部可伸
  • Industrial Property Rights Holder: 老木 茂稔, 久野みゆき, 安部可伸
  • Filing Date: 2004-03-30
  • Description: 「研究成果報告書概要(和文)」より