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–, , 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 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.
This work was supported by Deutsche Forschungsgemeinschaft Grant Ku756/7-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.