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 oocyte s and examined the effect of agents that modulate the activity of some kina ses. Activation of protein kinase C (PKC) by phorbol ester increased the ac tivity of ENaC, but only in oocytes with a baseline current of <2,000 nA. I nhibitors of protein kinases produced varying effects. Chelerythrine, an in hibitor of PKC, produced a significant inhibition of ENaC current, but calp hostin C, another PKC inhibitor, had no effect. The PKA/protein kinase G in hibitor H-8 had no effect, whereas the p38 mitogen-activated protein kinase inhibitor, SE-203580 had a significant inhibitory effect. Staurosporine, a nonspecific kinase inhibitor, was the most potent tested. It inhibited ENa C currents in both oocytes and in M-l cells, a model for the collecting duc t. 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 th is effect. These results suggest that an integrated kinase network regulate s ENaC activity through an action that requires a portion of the alpha-subu nit.