Ionic channels regulated by extracellular Ca2+ concentration ([Ca"],) were examined in freshly isolated rabbit osteoclasts. K+ current was suppressed by intracellular and extracellular Cs' ions. In this condition, high [Ca2+lo evoked an outwardly rectifying current with a reversal potential of about -25 mV. When theconcentration of extracellular CI-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 CI-channels. Cd" or Ni2+ caused a transient activation of the CI-current in contrast to the sustained activation elicited by Ca". lntracellular 20 mM ethylene glycol-bis(Paminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the divalent cation-induced CI-current. Either when the osmolarity of extracellular medium was increased, or when 100 pM CAMP was dissolved in the patch pipette solution, high [Ca2+lO still elicited the CIcurrent, indicating that the divalent cationinduced CI-current was carried through Ca2+-activated CI-channels. Under perforated whole cell clamp extracellular divalent cations evoked the CI-current, indicating that the activation of CI-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 volurnesensitive CI-channels under whole cell clamp. lntracellular application of guanosine 5'-0-(3-thiotriphosphate) (GTPyS) activated the CI-current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of CI-current was mediated through a G protein, and that an increase in [Ca2+], was critical for the activation of CI-channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the CI-current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of CaL+-activated CI-channels. o 1996 WiIey-Liss, Inc 0 1996 WILEY-LISS, INC. not be simply explained by the inhibition of the inwardly rectifying K' current, but suggests that other ionic channels also contribute to the membrane depolarization. Several classes of ionic channels have been reported in osteoclasts in addition to the inwardly rectifymg K' channels (Ravesloot et al., 1989;Sims et al., 1991;Kelly et al., 1992Kelly et al., , 1994)). In the present study, therefore, we examined whether or not ionic channels other than inwardly rectifymg K' channels were altered by [Ca2+], in freshly isolated rabbit osteoclasts.
MATERIALS AND METHODS
Materials
GTP, adenosine 5'-triphosphate (ATP), 4,4'-diisothiocyanostilbene-2,2'disulphonic acid (DIDS), pertussis