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

Abstract

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.