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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

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 (pHi) in regulating the availability of H+ channels in osteoclasts and microglia. In whole-cell clamp recordings, the pHi was elevated after exposure to NH4Cl 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 NH4Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H+ channels, pHi 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 pHi increases in osteoclasts and microglia decrease the numbers of H+ channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.

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 (ΔpH = 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 A1 and N,N′-dicyclohexylcarbodiimide. Although H+ was transported uphill, the electrochemical gradient for H+ affected the current. The currents were increased by elevating ΔpH and depolarization, and were reduced by lowering ΔpH and hyperpolarization. Elevation of extracellular Ca2+ (5–40 mm) diminished the currents in a dose-dependent manner and made the voltage dependence more marked. Extracellular Mg2+ mimicked the inhibition. With 40 mm Ca2+, the currents decreased to < 40% at 0 mV and to < 10% at about −80 mV. Increases in the intracellular Ca2+ (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 Ca2+ in the closed extracellular compartment might serve as negative feedback signals for regulating the V-ATPase.

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 (pHi) in regulating the availability of H(+) channels in osteoclasts and microglia. In whole-cell clamp recordings, the pHi was elevated after exposure to NH4Cl 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 NH4Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H(+) channels, pHi 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 pHi increases in osteoclasts and microglia decrease the numbers of H(+) channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.

The vacuolar-type H+-ATPase (V-ATPase) is an electrogenic H+ pump that is distributed widely in living organisms. Voltage-gated proton channels (H+ channels) are found in many types of cells including phagocytes. Although their transport mechanisms are distinct, both depend on pH and voltage and share the outcomes, intracellular alkalinization (extracellular acidification) and hyperpolarization. So far, V-ATPases and H+ channels have been studied separately in different cells, using the different approaches under different conditions. V-ATPases and H+ channels are expressed in the plasma membrane of osteoclasts, multinuclear bone-resorbing cells, which allowed us to investigate the two electrogenic H+ transfer mechanisms quantitatively under the same conditions. The current-voltage relationships of the V-ATPases are linear and outward currents were maintained even when the extracellular space was more acidic than the cell inside. Protons flowed through the V-ATPase continuously. In contrast, H+ channels were silent at voltages lower than the threshold. However, once it opened, the H+ channel current surpassed the V-ATPase current. Consequently, the profile of H+ efflux was composed of two phases, one dominated by the V-ATPase and the other, by the H+ channel. Inhibition of V-ATPases by bafilomycin was often accompanied by decreases in the H+ channel current. These results suggested that the V-ATPase and the H+ channel work over different ranges of pH- and voltage-gradients across the membrane in single cells and co-regulate the pH environments of osteoclasts.

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 ( Cm) simultaneously. Cm is a measure of cell surface. Extracellular Ca2+ (2.5–40 mM) decreased Cm and the V-ATPase current simultaneously. The decreased Cm, 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 A1, 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 γ2 subunit. Holding the cytosolic Ca2+ at either high (0.5–5 μM) 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.

The vacuolar-type H+-ATPase (V-ATPase) is an electrogenic H+ pump that is distributed widely in living organisms. Electrogenicity contributes to the proton motive force, but its precise evaluation is difficult mostly because V-ATPases are abundant at the intracellular membranes of acidic vesicles where fluxes of counter ions and H+-leakage could not be fully controlled. In osteoclasts, bone-resorbing cells, V-ATPases are recruited to the plasma membrane (the ruffled membrane) by exocytotic fusion of lysozomes. The electogenicity of the plasma membrane V-ATPases was evaluated under the whole-cell current-clamp recordings in the absence of Na+, K+ and Cl-, where H+ was a major determinant of the membrane potentials. Under pHo/pHi 7.3/5.5 (ΔpH 1.8), the membrane potential varied greatly among cells, from −250 - −7 mV. Bafilomycin, a specific blocker for V-ATPases, depolarized cells by several to 220 mV. The depolarization was dependent on the amplitudes of the V-ATPase currents and eliminated by replacing intracellular ATP by ADP. The V-ATPase-mediated potential was reduced by decreasing ΔpH and disappeared at near ΔpH −2.5. As the whole-cell leak conductance was ∼0.23 nS (the area resistance of the plasma membrane: 6 x 105 Ω cm2), 10 pA of V-ATPase currents could yield the membrane potential of ∼ 40 mV. Small ΔpH-dependent potential due to H+ leakage currents remained in the presence of bafilomycin. Potentials generated by V-ATPases and H+ leak are thus identified successfully. The ruffled membrane could provides a useful model for investigating H+ fluxes across the membrane energized by V-ATPases.

Vacuolar-type H+-ATPases (V-ATPases) are widely distributed at intracellular membranes from yeast to mammalians and could transport H+ across membranes against negative pH gradients. In osteoclasts, V-ATPases are recruited to the plasma membrane by exocytotic fusion of lysozomal membranes to form the ruffled membrane facing to the bone surface and play an important role in acid secretion. Ca2+ released from bone tissue is accumulated in the closed extracellular compartment, which in turn inhibits osteoclast functions. V-ATPases at the ruffled membrane would be exposed to this high level of extracellular Ca2+. We recently succeeded to record proton currents of the plasma membrane V-ATPase in murine osteoclasts using the conventional whole-cell clamp technique, and found that the pump current was decreased by increased extracellular Ca2+. In this study, we investigated whether endocytotic/exocytotic processes were involved in the Ca2+-induced inhibition of the V-ATPase by measuring the H+ current and the cell capacitance (Cm) simultaneously. Cm is a reliable monitor for surface area. Extracellular Ca2+ (5–40 mM) decreased the H+ current and the Cm simultaneously. The Ca2+-induced inhibitions were dose-dependent and were mimicked by Mg2+. The decrease in the Cm by 40 mM Ca2+ was only fractional, corresponding to ∼10% of the control (∼150 pF), but the decrease in the V-ATPase current was by 70–80%. Bafilomycin A1, inhibited the Ca2+-induced decrease in the Cm, indicating that V-ATPase-rich membrane may be targeted for endocytosis. These data suggest that extracellular Ca2+ facilitates internalization of the V-ATPase through endocytosis, which may be a mechanism for Ca2+-induced inhibition of osteoclastic bone resorption.