title: Activation of the plasma membrane chloride channel by protein kinase C in isolated guinea-pig hepatocytes.
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.
authors.affiliation: First Department of Internal Medicine, Nippon Medical School, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Koumi
authors.forename: R
authors.initials: R
authors.lastname: Sato
authors.forename: T
authors.initials: T
authors.lastname: Aramaki
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009638:Norepinephrine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D018118:Chloride Channels; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium; D002118:Calcium; D009638:Norepinephrine