ACTIVATION OF THE PLASMA-MEMBRANE CHLORIDE CHANNEL BY PROTEIN-KINASE-C IN ISOLATED GUINEA-PIG HEPATOCYTES

1. To assess the nature of the underlying mechanism of noradrenaline-i nduced increase of Cl- conductances in hepatocytes, macroscopic and un itary currents through noradrenaline-induced Cl- channels were examine d in enzymatically isolated guinea-pig hepatocytes using whole-cell, c ell-attached and excised inside-out configurations of the patch-clamp technique. 2. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+](1)) was set at 0.1 mu M, bath application o f noradrenaline activated the time-independent membrane currents under whole-cell voltage-clamp conditions. The current was similarly activa ted by phorbol ester (PMA), an activator of protein kinase C (PKC), wh ile a specific protein kinase Cf inhibitor, H-9, reversed PMA activati on of the current. The inactive phorbol ester, 4 alpha-phorbol 12-myri state, 13-acetate (alpha PMA), failed to activate the channel. 3. The reversal potential of the PMA-activated current shifted by similar to 60 mV per 10-fold change in the external Cl- concentration, indicating that the current was Cl- selective. Bath application of 4,4'-diisothi ocyanatostilbene-2,2'-disulphonic acid (DIDS) partially inhibited both the noradrenaline- and PMA-induced currents. 4. In single channel rec ordings from cell-attached patches, bath application of noradrenaline or PMA induced unitary current activity, the averaged slope conductanc e of which was 10.1 +/- 1.5 pS (mean +/- S.D.; n = 12) in the noradren aline-induced current and 9.7 +/- 1.3 pS (n = 7) in the PMA-induced cu rrent. The open time distribution was moderately well fitted by a sing le exponential function with mean open lifetime of 88 5 +/- 10 6 ms (n = 10), while at least two exponentials were required to fit the close d time distributions with a time constant for the fast component of 24 .4 +/- 5.8 ms (n = 10) and for the slow component of 316.9 +/- 49.2 ms (n = 10). 5. Bath application of purified PKC to excised inside-out p atches activated the channel. The PKC selective inhibitor, PKC(19-36), and DIDS inhibited the PKC-activated channel. 6. These results sugges t that PKC can phosphorylate the channel protein or a related structur e leading to the activation of Cl- channels in guinea-pig hepatocytes.