title: 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 alpha beta gammaENaC. 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-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, 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 betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.
authors: Institut für Physiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors: Tanja
authors: T
authors: Bachhuber
authors: Jens
authors: J
authors: König
authors: Thilo
authors: T
authors: Voelcker
authors: Bettina
authors: B
authors: Mürle
authors: Rainer
authors: R
authors: Schreiber
authors: Karl
authors: K
authors: Kunzelmann
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000737:chemistry* / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000502:physiology*; D053503:Epithelial Sodium Channels; D006801:Humans; D051379:Mice; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D012964:Sodium / Q000378:metabolism; D026941:Sodium Channel Blockers / Q000737:chemistry*; D015222:Sodium Channels / Q000235:genetics / Q000502:physiology*; D014981:Xenopus
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D053503:Epithelial Sodium Channels; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D012964:Sodium