Kinase regulation of hENaC mediated through a region in the COOH-terminal portion of the alpha-subunit.

In an effort to gain insight into how kinases might regulate epithelial Na(+) channel (ENaC) activity, we expressed human ENaC (hENaC) in Xenopus oocytes and examined the effect of agents that modulate the activity of some kinases. Activation of protein kinase C (PKC) by phorbol ester increased the activity of ENaC, but only in oocytes with a baseline current of <2,000 nA. Inhibitors of protein kinases produced varying effects. Chelerythrine, an inhibitor of PKC, produced a significant inhibition of ENaC current, but calphostin C, another PKC inhibitor, had no effect. The PKA/protein kinase G inhibitor H-8 had no effect, whereas the p38 mitogen-activated protein kinase inhibitor, SB-203580 had a significant inhibitory effect. Staurosporine, a nonspecific kinase inhibitor, was the most potent tested. It inhibited ENaC currents in both oocytes and in M-1 cells, a model for the collecting duct. Site-directed mutagenesis revealed that the staurosporine effect did not require an intact COOH terminus of either the beta- or gamma-hENaC subunit. However, an intact COOH terminus of the alpha-subunit was required for this effect. These results suggest that an integrated kinase network regulates ENaC activity through an action that requires a portion of the alpha-subunit.