Activation of Cl- channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts

Ionic channels regulated by extracellular Ca2+ concentration ([Ca2+](o)) were examined in freshly isolated rabbit osteoclasts. K+ current was suppressed by intracellular and extracellular Cs+ ions. In this condition, high [Ca2+](o) evoked an outwardly rectifying current with a reversal potential of about -25 mV. When the concentration of extracellular Cl- ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4',4-diisothiocyanostilbene-2',2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl- channels. Cd2+ or Ni2+ caused a transient activation of the Cl- current in contrast to the sustained activation elicited by Ca2+. Intracellular 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl- current. Either when the osmolarity of extracellular medium was increased, or when 100 mu M cAMP was dissolved in the patch pipette solution, high [Ca2+](o) still elicited the Cl- current, indicating that the divalent cation- induced Cl- current was carried through Ca2+-activated Cl- channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl- current, indicating that the activation of Cl- current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl- channels under whole cell clamp. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activated the Cl- current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl- current was mediated through a G protein, and that an increase in [Ca2+](i) was critical for the activation of Cl- channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl- current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca2+-activatedCl(-) channels. (C) 1996 Wiley-Liss, Inc.