Cl- interference with the epithelial Na+ channel ENaC

Abstract

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 mm 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 mM 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-, NO-3, 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 NH2 terminus of α-, β-, or γENaC, mutations in the NH2-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 carbosyl 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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.