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. 1995 Sep 1;487(Pt 2):379–394. doi: 10.1113/jphysiol.1995.sp020887

Activation of the plasma membrane chloride channel by protein kinase C in isolated guinea-pig hepatocytes.

S Koumi 1, R Sato 1, T Aramaki 1
PMCID: PMC1156580  PMID: 8558471

Abstract

1. To assess the nature of the underlying mechanism of noradrenaline-induced increase of Cl- conductances in hepatocytes, macroscopic and unitary currents through noradrenaline-induced Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, cell-attached and excised inside-out configurations of the patch-clamp technique. 2. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 0.1 microM, bath application of noradrenaline activated the time-independent membrane currents under whole-cell voltage-clamp conditions. The current was similarly activated by phorbol ester (PMA), an activator of protein kinase C (PKC), while a specific protein kinase C inhibitor, H-9, reversed PMA activation of the current. The inactive phorbol ester, 4 alpha-phorbol 12-myristate, 13-acetate (alpha PMA), failed to activate the channel. 3. The reversal potential of the PMA-activated current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration, indicating that the current was Cl- selective. Bath application of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) partially inhibited both the noradrenaline- and PMA-induced currents. 4. In single channel recordings from cell-attached patches, bath application of noradrenaline or PMA induced unitary current activity, the averaged slope conductance of which was 10.1 +/- 1.5 pS (mean +/- S.D.; n = 12) in the noradrenaline-induced current and 9.7 +/- 1.3 pS (n = 7) in the PMA-induced current. The open time distribution was moderately well fitted by a single exponential function with mean open lifetime of 88.5 +/- 10.6 ms (n = 10), while at least two exponentials were required to fit the closed 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 patches activated the channel. The PKC selective inhibitor, PKC(19-36), and DIDS inhibited the PKC-activated channel. 6. These results suggest that PKC can phosphorylate the channel protein or a related structure leading to the activation of Cl- channels in guinea-pig hepatocytes.

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