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 (DeltapH = 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 DeltapH and depolarization, and were reduced by lowering DeltapH 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 microm) 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.
Sakai
Hiromu
H
Department of Physiology, Osaka City University Graduate School of Medicine, Abeno-ku, Osaka 545-8585, Japan.
Kawawaki
Junko
J
Moriura
Yoshie
Y
Mori
Hiroyuki
H
Morihata
Hirokazu
H
Kuno
Miyuki
M
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D018942:Macrolides / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D010010:Osteoclasts / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D018408:Patch-Clamp Techniques; D006180:Proton-Translocating ATPases / Q000502:physiology*; D011522:Protons
D004791:Enzyme Inhibitors; D018942:Macrolides; D011522:Protons; D004024:Dicyclohexylcarbodiimide; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases; D002118:Calcium