The vacuolar-type H + -ATPase (V-ATPase) in the plasma membrane of a variety of cells serves as an acid-secreting pathway, and its activity is closely related to cellular functions. Massive proton secretion often leads to electrolyte disturbances in the vicinity of the cell and may in turn affect the activity of the V-ATPase. We characterized, for the first time, the proton currents mediated by plasmalemmal V-ATPase in murine osteoclast-like cells and investigated its activity over a wide range of pH gradients across the membrane (∆pH = extracellular pH -intracellular pH). The V-ATPase currents were identified as outward H + currents and were dependent on ATP and sensitive to the inhibitors bafilomycin A 1 and N ,N -dicyclohexylcarbodiimide. Although H + was transported uphill, the electrochemical gradient for H + affected the current. The currents were increased by elevating ∆pH and depolarization, and were reduced by lowering ∆pH and hyperpolarization. Elevation of extracellular Ca 2+ (5-40 mM) diminished the currents in a dose-dependent manner and made the voltage dependence more marked. Extracellular Mg 2+ mimicked the inhibition. With 40 mM Ca 2+ , the currents decreased to < 40% at 0 mV and to < 10% at about −80 mV. Increases in the intracellular Ca 2+ (0.5-5 µM) did not affect the current. The data suggest that acid secretion through the plasmalemmal V-ATPase is regulated by a combination of the pH gradient, the membrane potential and the extracellular divalent cations. In osteoclasts, the activity-dependent accumulation of acids and Ca 2+ in the closed extracellular compartment might serve as negative feedback signals for regulating the V-ATPase.

In osteoclasts, elevation of extracellular Ca2+ is an endogenous signal that inhibits bone resorption. We recently found that an elevation of extracellular Ca2+ decreased proton extrusion through the plasma membrane vacuolar H+-ATPase (V-ATPase) rapidly. In this study we investigated mechanisms underlying this early Ca2+-sensing response, particularly in reference to the activity of the plasma membrane V-ATPase and to membrane retrieval. Whole cell clamp recordings allowed us to measure the V-ATPase currents and the cell capacitance (C(m)) simultaneously. C(m) is a measure of cell surface. Extracellular Ca2+ (2.5-40 mM) decreased C(m) and the V-ATPase current simultaneously. The decreased C(m), together with the enhanced uptake of a lipophilic dye (FM1-43), indicated that Ca2+ facilitated endocytosis. The endocytosis was blocked by dynamin inhibitors (dynasore and dynamin-inhibitory peptide), by small interfering RNA (siRNA) targeting for dynanmin-2 and also by bafilomycin A(1), a blocker of V-ATPases. The extracellular Ca2+-induced endocytosis and inhibition of the V-ATPase current were diminished by a phospholipase C inhibitor (U73122) and siRNA targeting for phospholipase C gamma2 subunit. Holding the cytosolic Ca2+ at either high (0.5-5 microM) or low levels or inhibiting calmodulin by an inhibitor (W7) or an antibody (anti-CaM) decreased the stimulated endocytosis and the inhibition of the V-ATPase current. These data suggest that extracellular Ca2+ facilitated dynamin- and V-ATPase-dependent endocytosis in association with an inhibition of the plasma membrane V-ATPase. Phospholipase C, cytosolic Ca2+, and calmodulin were involved in the signaling pathways. Membrane retrieval and the plasma membrane V-ATPase activity may cooperate during the early phase of Ca2+-sensing response in osteoclasts.

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.