CHARACTERIZATION OF THE CALCIUM-ACTIVATED CHLORIDE CHANNEL IN ISOLATED GUINEA-PIG HEPATOCYTES

Macroscopic and unitary currents through Ca2+-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+](i)) was set at 1 mu M (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by similar to 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+](i) and was inactivated in Ca2+-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatastilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the dosed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = fl). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+](i). The relationship between [Ca2+](i) and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 mu M. These results suggest that guinea-pig hepatocytes possess Ca2+ activated Cl- channels.