Cl– Interference with the Epithelial Na+ Channel ENaC

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl – channel that also controls the activity of other membrane transport proteins, such as the epithelial Na + channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl – ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl – channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl – channel CLC-0 caused inhibition of amiloride-sensitive Na + currents after cAMP-dependent stimulation and in the presence of a 100 m m bath Cl – concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl – concentration was increased in Xenopus oocytes in the presence of a high bath Cl – concentration, which inhibited ENaC without changing surface expression of αβγENaC. In contrast, a 5 m m bath Cl – concentration reduced the cytosolic Cl – concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl – into oocytes and in inside/out macropatches by exposure to high cytosolic Cl – concentrations. The effect of Cl – was mimicked by Br – , , and I – . Inhibition by Cl – was reduced in trimeric channels with a truncated COOH terminus of βENaC and γENaC, and it was no longer detected in dimeric αΔCβ ENaC channels. Deletion of the NH 2 terminus of α-, β-, or γENaC, mutations in the NH 2 -terminal phosphatidylinositol bisphosphate-binding domain of βENaC and γEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl – -dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of βENaC for Cl – -dependent inhibition of the Na + channel, which, however, may only be part of a complex regulation of ENaC by CFTR.