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.