Junko Kawawaki

Regulatory mechanisms and physiological relevance of a voltage-gated H+ channel in murine osteoclasts: phorbol myristate acetate induces cell acidosis and the channel activation.

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.

Temporal fluctuations of voltage-gated proton currents in rat spinal microglia via pH-dependent and -independent mechanisms

Voltage-gated proton (H(+)) channels are unique mechanisms to extrude a massive amount of H(+), and are proposed to regulate intracellular pH of microglia during respiratory bursts. Temporal variations of the H(+) current were studied in rat spinal microglia cultivated on the glial cell layer using the voltage-ramp protocol. Repetitive applications of the large and long-lasting depolarization decreased the amplitudes of the H(+) current transiently and reversibly. This decrease was accompanied by a shift of the reversal potential to a more positive direction, indicating that a drop in the transmembrane pH gradient (delta pH) by the H(+) efflux through the channel reduced the current. The decline of the H(+) current during depolarizations was also observed in a rat microglial cell line (GMI-R1). An increase in the extracellular buffer suppressed the reduction of the current, suggesting that H(+) secreted into the extracellular space contributed to the drop in delta pH. On the other hand, the amplitudes of the H(+) current often fluctuated greatly at intervals of 5-20 min without changes in delta pH. These results suggest that the H(+) current of microglia is tuned via both delta pH-dependent and -independent mechanisms, which may regulate both microglial behavior and the pH environments of the surrounding neural tissue.

Phospholipase C-dependent Ca2+-sensing pathways leading to endocytosis and inhibition of the plasma membrane vacuolar H+-ATPase in 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 (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.

pH dependence and inhibition by extracellular calcium of proton currents via plasmalemmal vacuolar‐type H+‐ATPase in murine osteoclasts

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.

Increases in intracellular pH facilitate endocytosis and decrease availability of voltage-gated proton channels in osteoclasts and microglia

•  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.

Inhibition of voltage-gated proton channels by local anaesthetics in GMI-R1 rat microglia.

Non‐technical summary  Lidocaine and bupivacaine are the most commonly used local anaesthetics in clinical practices such as neuraxial anaesthesia and local infiltration. They are known to suppress phagocytosis and the production of reactive oxygen species in immune cells. Voltage‐gated proton channels are abundantly expressed in immune cells, including microglia, and play crucial roles in sustaining phagocytosis. We show that both lidocaine and bupivacaine increase the intracellular pH of microglia by their weak base properties and, consequently, inhibit proton channels. This is a novel mechanism underlying actions of local anaesthetics. Our results also indicate that the proton channel is a useful tool for monitoring the behaviours of lidocaine and bupivacaine across the cellular membrane.

A heterogeneous electrophysiological profile of bone marrow-derived mast cells

Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba2+, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K+, thereby indicating that K+ mediated the IR currents. The negative conductance region was seen at potentials positive to EK. The OR currents did not apparently depend on the extracellular K+ concentration, but were reduced by lowering the extracellular Cl− concentration. The OR currents were partially blocked by 1 mm Ba2+, and were further blocked by a Cl− channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl− concentrations, suggesting that Cl− was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K+ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K+ channel and the outwardly rectifying Cl− channel seem to contribute to the regulation of the membrane potential.

Electrogenicity of Vacuolar H+-ATPases at the Plasma Membrane of 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.