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

电压门控质子（Hv）通道是一种有效的H^+外排途径，也是一种高度灵敏的实时pH监测器。本研究主要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，在跨膜的pH梯度下被钳制。通过高浓度（>100 mM）的pH缓冲液。在没有主要离子的情况下，膜电位以10 s的间隔波动<10 ms to >。这种振荡消失的锌，一个阻断剂的Hv通道。Hv通道的活性通过温度升高和乳酸酸中毒（pH 6.8）而增加。广泛变化的电流幅度建议存在的动态招聘机制的HV通道。我们首次在破骨细胞中成功地电生理学记录了通过液泡型H^+-ATP酶的外向H^+电流，在破骨细胞中，H^+泵在质膜上是丰富的。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，电流幅度远小于Hv通道的电流幅度，并且没有大的波动，这意味着这两种H^+分泌膜分子受到不同的募集机制的调节。为了进一步研究H^+信号，我们还设计了一种跳跃式方法，并测试了基于ATP干扰的病理细胞模型。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。

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.