Regulation of the human skeletal muscle chloride channel hClC-1 by proteinkinase C

1. The regulation of a recombinant human muscle chloride channel, hClC-1, b y protein kinase C (PKC) was investigated in human embryonic kidney (HEK 29 3) cells. 2. External application of 4 beta-phorbol esters (4 beta-PMA) reduced the i nstantaneous whole-cell current amplitude over the entire voltage range tes ted. This effect was abolished when the cells were intracellularly perfused with a specific protein kinase C inhibitor, chelerythine. Inactive 4 alpha -phorbolesters did not affect the chloride currents. We conclude that the e ffect of 4 beta-phorbol esters is mediated by protein kinase C (PKC). 3. Activation of PKC resulted in changes in macroscopic current kinetics. T he time course of current deactivation determined in the presence and absen ce of 4 beta-phorbol esters could be fitted with the sum of two exponential s and a constant value. In the presence of phorbol esters, the fast time co nstants and the minimum value of the fraction of non-deactivating current w ere increased, whereas the voltage dependence of all fractional current amp litudes remained unchanged. PKC-induced phosphorylation had only small effe cts on the voltage dependence of the relative open probability and the maxi mum absolute open probability was unaffected by treatment with 4 beta-PMA, as shown by non-stationary noise analysis. 4. The kinetic changes indicate that phosphorylation alters functional prop erties of active channels. Since the absolute open probability is not reduc ed, the observed macroscopic current reduction implies alterations of the i on permeation process. 5. Phosphorylation by PKC appears to affect ion transfer and gating process es. It is postulated that the phosphorylation site may be located at the cy toplasmic vestibule face of the pore.