Acid-inducible proton influx currents in the plasma membrane of murine osteoclast-like cells

Acidification of the resorption pits, which is essential for dissolving bone, is produced by secretion of protons through vacuolar H+-ATPases in the plasma membrane of bone-resorbing cells, osteoclasts. Consequently, osteoclasts face highly acidic extracellular environments, where the pH gradient across the plasma membrane could generate a force driving protons into the cells. Proton influx mechanisms during the acid exposure are largely unknown, however. In this study, we investigated extracellular-acid-inducible proton influx currents in osteoclast-like cells derived from a macrophage cell line (RAW264). Decreasing extracellular pH to <5.5 induced non-ohmic inward currents. The reversal potentials depended on the pH gradients across the membrane and were independent of concentrations of Na+, Cl−, and HCO3 −, suggesting that they were carried largely by protons. The acid-inducible proton influx currents were not inhibited by amiloride, a widely used blocker for cation channels/transporters, or by 4,4'-diisothiocyanato-2,2'-stilbenesulfonate(DIDS) which blocks anion channels/transporters. Additionally, the currents were not significantly affected by V-ATPase inhibitors, bafilomycin A1 and N,N’-dicyclohexylcarbodiimide. Extracellular Ca2+ (10 mM) did not affect the currents, but 1 mM ZnCl2 decreased the currents partially. The intracellular pH in the vicinity of the plasma membrane was dropped by the acid-inducible H+ influx currents, which caused overshoot of the voltage-gated H+ channels after removal of acids. The H+ influx currents were smaller in undifferentiated, mononuclear RAW cells and were negligible in COS7 cells. These data suggest that the acid-inducible H+ influx (H+ leak) pathway may be an additional mechanism modifying the pH environments of osteoclasts upon exposure to strong acids.