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 wa s suppressed by intracellular and extracellular Cs+ ions. In this cond ition, high [Ca2+](o) evoked an outwardly rectifying current with a re versal potential of about -25 mV. When the concentration of extracellu lar Cl- ions was altered, the reversal potential of the outwardly rect ifying current shifted as predicted by the Nernst equation. 4',4-diiso thiocyanostilbene-2',2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was car ried 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-induce d Cl- current. Either when the osmolarity of extracellular medium was increased, or when 100 mu M cAMP was dissolved in the patch pipette so lution, high [Ca2+](o) still elicited the Cl- current, indicating that the divalent cation- induced Cl- current was carried through Ca2+-act ivated Cl- channels. Under perforated whole cell clamp extracellular d ivalent cations evoked the Cl- current, indicating that the activation of Cl- current did not arise from possible leakage of divalent cation s from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensi tive Cl- channels under whole cell clamp. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activated the Cl- c urrent and it was inhibited by intracellular 20 mM EGTA, suggesting th at 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), caus ed an irreversible activation of the Cl- current, suggesting that prot ein phosphatase 1 or 2A was involved in the regulation of Ca2+-activat edCl(-) channels. (C) 1996 Wiley-Liss, Inc.