Regulation of the human skeletal muscle chloride channel hClC-1 by protein kinase C.
1. The regulation of a recombinant human muscle chloride channel, hClC-1, by protein kinase C (PKC) was investigated in human embryonic kidney (HEK 293) cells. 2. External application of 4beta-phorbol esters (4beta-PMA) reduced the instantaneous whole-cell current amplitude over the entire voltage range tested. This effect was abolished when the cells were intracellularly perfused with a specific protein kinase C inhibitor, chelerythine. Inactive 4alpha-phorbolesters did not affect the chloride currents. We conclude that the effect of 4beta-phorbol esters is mediated by protein kinase C (PKC). 3. Activation of PKC resulted in changes in macroscopic current kinetics. The time course of current deactivation determined in the presence and absence of 4beta-phorbol esters could be fitted with the sum of two exponentials and a constant value. In the presence of phorbol esters, the fast time constants and the minimum value of the fraction of non-deactivating current were increased, whereas the voltage dependence of all fractional current amplitudes remained unchanged. PKC-induced phosphorylation had only small effects on the voltage dependence of the relative open probability and the maximum absolute open probability was unaffected by treatment with 4beta-PMA, as shown by non-stationary noise analysis. 4. The kinetic changes indicate that phosphorylation alters functional properties of active channels. Since the absolute open probability is not reduced, the observed macroscopic current reduction implies alterations of the ion permeation process. 5. Phosphorylation by PKC appears to affect ion transfer and gating processes. It is postulated that the phosphorylation site may be located at the cytoplasmic vestibule face of the pore.
Rosenbohm
A
A
Abteilung Allgemeine Physiologie, Universitat Ulm, Oberer Eselsberg, D-89069 Ulm, Germany.
Rüdel
R
R
Fahlke
C
C
D000470:Alkaloids; D053119:Benzophenanthridines; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D004558:Electric Stimulation; D004594:Electrophysiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D009124:Muscle Proteins / Q000187:drug effects / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010617:Phenanthridines / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; C086321:CLC-1 channel; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D009124:Muscle Proteins; D010617:Phenanthridines; D011994:Recombinant Proteins; C016299:chelerythrine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate