The voltage-gated H ؉ channel is a powerful H ؉ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H ؉ channel operated on activation of protein kinase C together with cell acidosis. Introduction: Hϩ is a key signaling ion in bone resorption. In addition to H ϩ pumps and exchangers, osteoclasts are equipped with H ϩ conductive pathways to compensate rapidly for pH imbalance. The H ϩ channel is distinct in its strong H ϩ extrusion ability and voltage-dependent gatings. Methods: To investigate how and when the H ϩ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H ϩ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). Results and Conclusions:Whole cell recordings clearly showed that the H ϩ current was enhanced by increasing the pH gradient across the plasma membrane (⌬pH), indicating that the H ϩ channel changed its activity by sensing ⌬pH. The reversal potential (V rev ) was a valuable tool for the real-time monitoring of ⌬pH in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 M) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of V rev , suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (Iratio) were determined. The use of Iratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q10s for the proton currents were evaluated from the Iratios. Q10 exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q10. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q10 was decomposed into Q10 of the channel and of the access resistances. Finally, the Q10 for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.

An outwardly rectifying Cl− (ORCl) current of murine osteoclasts was activated by hypotonic stimulation. The current was characterized by rapid activation, little inactivation, strong outward rectification, blockage by DIDS and permeability to organic acids (pyruvate and glutamate). The hypotonically activated ORCl current was inhibited by intracellular dialysis with an ATP‐free pipette solution, but not by replacement of ATP with a poorly hydrolysable ATP analogue adenosine 5′‐O‐(3‐thiotriphosphate). The current amplitude was reduced when intracellular alkalinity increased over the pH range 6.6–8.0. Intracellular application of cytochalasin D occasionally activated the ORCl current without hypotonic stress, but inhibited activation of the ORCl current by hypotonic stimulation. The hypotonically activated ORCl current was unaffected by a non‐actin‐depolymerizing cytochalasin, chaetoglobosin C, but partially inhibited by deoxyribonuclease I. Removal of extracellular Ca2+ inhibited activation of the ORCl current by hypotonic shock, but did not reduce the current once activated. The hypotonically activated ORCl current was partially decreased by intracellular dialysis with 20 mm EGTA. With 10 mm Ca2+ in the extracellular medium, the ORCl current was activated in response to more minor decreases in osmolarity than with 1 mm Ca2+. The increased sensitivity to hypotonicity was mimicked by increasing the intracellular Ca2+ level (pCa 6.5). These results suggest that hypotonic stimulation and a rise in the extracellular Ca2+ level synergistically activate the ORCl channel of murine osteoclasts, and that the activating process is modified by multiple intracellular factors (pH, ATP and actin cytoskeletal organization).

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.