Cl- interference with the epithelial Na+ channel ENaC

J Biol Chem. 2005 Sep 9;280(36):31587-94. doi: 10.1074/jbc.M504347200. Epub 2005 Jul 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Cells, Cultured
  • Chlorides / chemistry*
  • Chlorides / physiology*
  • Cystic Fibrosis Transmembrane Conductance Regulator / chemistry
  • Cystic Fibrosis Transmembrane Conductance Regulator / physiology*
  • Epithelial Sodium Channels
  • Humans
  • Mice
  • Mutation
  • Oocytes / metabolism
  • Patch-Clamp Techniques
  • Rats
  • Sodium / metabolism
  • Sodium Channel Blockers / chemistry*
  • Sodium Channels / genetics
  • Sodium Channels / physiology*
  • Xenopus

Substances

  • CFTR protein, human
  • Chlorides
  • Epithelial Sodium Channels
  • Sodium Channel Blockers
  • Sodium Channels
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • Adenosine Triphosphate
  • Sodium