Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.
Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000494:pharmacology*; D034281:Dynamins; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D020558:GTP Phosphohydrolases / Q000276:immunology; D006801:Humans; D006973:Hypertension / Q000235:genetics* / Q000378:metabolism; D008025:Ligases; D000075702:Nedd4 Ubiquitin Protein Ligases; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship; D013577:Syndrome; D014357:Trypsin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000276:immunology; D029867:Xenopus Proteins; D000099271:Prostasin