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

质子通道在质子信号传导中的作用：pH钳和质子电流振荡分析

• 批准号: 16590168
• 负责人: KUNO Miyuki
• 金额: $ 2.24万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590168/
• 关键词: 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

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.

电压门控质子（Hv）通道是一种高效的H^+外排途径，也是一种高灵敏度的实时ph监测仪。本研究的重点是利用其独特的性质来研究质子信号系统的动力学和机制。我们测量了。小胶质细胞和破骨细胞的膜电位，在pH梯度下跨膜夹住。高浓度（bbb100 mM） pH缓冲液。在没有主离子的情况下，膜电位波动的间隔从< 10ms到bb10s不等。这种振荡消失的锌，阻滞剂的Hv通道。温度升高和乳酸性酸中毒（pH值6.8）增加了Hv通道的活性。大范围变化的电流振幅表明高压通道存在动态招募机制。我们首次在细胞质膜上富含H^+泵的破骨细胞中成功地通过液泡型H^+- atp酶记录了向外的H^+电流。v - atp酶介导的H^+电流受pH和电压的电化学梯度的影响。然而，电流振幅远小于Hv通道，且缺乏大的波动，这表明两种分泌H^+的膜分子受不同的招募机制调节。我们还设计了一种渗透跳跃方法，并基于atp干扰测试了病理细胞模型，以进一步研究H^+信号。这些发现表明Hv通道负责H^+信号振荡，并可能有助于调节各种细胞反应。

项目成果

パッチクランプ装置

膜片钳装置

• 发表时间: 2004

Proton currents through the plasmalemmal vacuolar-type H^+ -ATPase in osteoclast.

质子电流通过破骨细胞中的质膜液泡型 H+-ATP 酶。

• 发表时间: 2006
• 期刊: Biophysical Journal Abstract
• 作者: Sakai;H.
• 通讯作者: H.

Coupled K+-water flux through the HERG potassium channel measured by an osmotic pulse method

• DOI: 10.1085/jgp.200509377
• 发表时间: 2005-11-01
• 期刊: JOURNAL OF GENERAL PHYSIOLOGY
• 影响因子: 3.8
• 作者: Ando, H;Kuno, M;Oiki, S
• 通讯作者: Oiki, S

The Peripheral Nervous System 2. (in Japanese)

周围神经系统2.（日语）

• 发表时间: 2005
• 期刊: Structure, Function and Materials of the Human Body (Nihon Iji Shinpou-sha) 9(2)
• 作者: Kuno;M.;Ando;K.;Sugihara;I.;Akita;K.
• 通讯作者: K.

人体の正常構造と機能:IX神経系(2)

人体正常结构和功能：IX神经系统（2）

• 发表时间: 2005
• 作者: Noguchi;T.;Watanabe;K.;Kim HJ;久野 みゆき
• 通讯作者: 久野 みゆき