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

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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.