Key points• Voltage-gated H + channels help to compensate for the pH and voltage disturbances generated by production of reactive oxygen species.• In this study, we investigated how changes in the intracellular pH levels control H + channel activity in macrophage-lineage cells, osteoclasts and microglia.• An increase in intracellular pH decreased the numbers of H + channels available at the plasma membrane through facilitation of dynamin-dependent endocytic internalization.• This inhibitory regulation mechanism for H + channels is novel.• The results help us to understand better the significance of the intracellular pH levels in membrane dynamics and H + channel availability, which, in turn, may modulate natural immunity.Abstract Voltage-gated proton channels (H + channels) are highly proton-selective transmembrane pathways. Although the primary determinants for activation are the pH and voltage gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pH i ) in regulating the availability of H + channels in osteoclasts and microglia. In whole-cell clamp recordings, the pH i was elevated after exposure to NH 4 Cl and returned to the control level after washout. However, the H + channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H + channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage dependence for activation, were unchanged, the decreases in the H + channel conductance were probably due to the decreases in H + channels available at the plasma membrane. The decreases in the H + channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH 4 Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pH i increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H + channels, pH i increases by these H + -transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H + channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pH i increases in osteoclasts and microglia decrease the numbers of H + channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.

Acidification of the resorption pits, which is essential for dissolving bone, is produced by secretion of protons through vacuolar H(+)-ATPases in the plasma membrane of bone-resorbing cells, osteoclasts. Consequently, osteoclasts face highly acidic extracellular environments, where the pH gradient across the plasma membrane could generate a force driving protons into the cells. Proton influx mechanisms during the acid exposure are largely unknown, however. In this study, we investigated extracellular-acid-inducible proton influx currents in osteoclast-like cells derived from a macrophage cell line (RAW264). Decreasing extracellular pH to <5.5 induced non-ohmic inward currents. The reversal potentials depended on the pH gradients across the membrane and were independent of concentrations of Na(+), Cl(-), and HCO3 (-), suggesting that they were carried largely by protons. The acid-inducible proton influx currents were not inhibited by amiloride, a widely used blocker for cation channels/transporters, or by 4,4'-diisothiocyanato-2,2'-stilbenesulfonate(DIDS) which blocks anion channels/transporters. Additionally, the currents were not significantly affected by V-ATPase inhibitors, bafilomycin A1 and N,N'-dicyclohexylcarbodiimide. Extracellular Ca(2+) (10 mM) did not affect the currents, but 1 mM ZnCl2 decreased the currents partially. The intracellular pH in the vicinity of the plasma membrane was dropped by the acid-inducible H(+) influx currents, which caused overshoot of the voltage-gated H(+) channels after removal of acids. The H(+) influx currents were smaller in undifferentiated, mononuclear RAW cells and were negligible in COS7 cells. These data suggest that the acid-inducible H(+) influx (H(+) leak) pathway may be an additional mechanism modifying the pH environments of osteoclasts upon exposure to strong acids.

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