Increased activity of the epithelial sodium channel (ENaC) in the respiratory airways contributes to the pathophysiology of cystic fibrosis (CF) a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Each subunit has two transmembrane domains a large extracellular loop and cytosolic N- and C-termini. AEB071 The recently published crystal structure of the related acid-sensing ion channel ASIC1 suggests that ENaC is probably a heterotrimer (Canessa 2007 Jasti 2007; Stockand 2008). The genetic disease cystic fibrosis (CF) is usually caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Riordan 1989). The CFTR gene encodes a Cl? channel found in the apical membrane of many epithelial cells (Sheppard & Welsh 1999 Thus CF epithelia have a reduced apical Cl? conductance. Some patients may have all the classical symptoms of CF while others have milder or even atypical disease manifestations. The CF phenotype is usually a continuum of symptoms and cannot be easily defined in two distinct disease categories (common atypical CF) (De Boeck 2006). The large variability of CF symptoms suggests that CF modifier genes exist. Interestingly the genes encoding AEB071 the β- and γ-subunits of ENaC have been reported to be potential modifier genes in CF (Stanke 2006). Moreover missense mutations in the β-subunit of ENaC causing abnormal channel function have been identified in two patients with CF-like syndromes without CFTR mutations (Sheridan 2005). In addition the tumour necrosis factor α (TNFα) receptor located next to the gene encoding the α-subunit of ENaC has been suggested as a potential modifier gene (Stanke 2006). In respiratory epithelia a fine balance between Cl? secretion via CFTR and Na+ absorption ENaC is necessary to LEG2 antibody maintain an appropriate airway surface liquid (ASL) volume which is usually important for pulmonary mucus clearance. CF airway epithelia are characterized by a combined defect of accelerated Na+ transport and the failure to secrete Cl? (Boucher 2004 2007 These abnormal ion transport properties are thought to cause ASL volume depletion leading to ‘thickened’ mucus plaques and plugs which become the nidus for bacterial infections. In some patients with CF(-like) disease a mutation cannot be identified on both CFTR alleles. Mutations in the genes encoding ENaC may potentially explain disease in these atypical CF patients. Indeed sodium hyper-absorption through ENaC is usually thought to contribute to CF pathophysiology (Boucher 2004 2007 and mice overexpressing βENaC in the airways present with CF-like pulmonary disease (Mall 2004; Zhou 2008). In a recent study we investigated the possibility that atypical CF patients might harbour mutations in their ENaC genes (Azad 2009). αW493R-ENaC one of the identified mutations stimulated ENaC currents when expressed in oocytes. The size of this gain-of-function effect was AEB071 similar to that described for an hereditary form of salt-sensitive arterial hypertension (Rossier & Schild AEB071 2008 The latter mutations affect a PY-motif in the C-termini of the β- or γ-subunit of ENaC. The PY-motif is critical for the conversation of the channel with the regulatory protein Nedd4-2 which promotes channel internalization and degradation. Mutations affecting the PY-motifs lead to higher channel activity at the plasma membrane due to a rise in cell surface expression and an impaired Na+-dependent feedback inhibition of the mutant channel (Staub & Verrey 2005 AEB071 In contrast to the mutations causing Liddle’s syndrome the αW493R mutation is usually localized in the extracellular loop of the α-subunit. Thus the gain-of-function effect of this mutation is likely to be mediated by a different mechanism. The aim of the present study was to investigate the mechanisms underlying the gain-of-function phenotype of the mutant channel. Methods Plasmids Full length cDNAs for the wild-type α- β- and γ-subunits and for the mutant αW493R subunit of human ENaC were in pcDNA3.1. The T663 polymorphism was present in the α-subunit unless stated otherwise. Linearized plasmids were used as templates for cRNA synthesis using either T7 (αβγ-hENaC αW493R-hENaC and αβγ-rENaC) or SP6 (β-FLAG-rENaC) RNA polymerases (mMessage mMachine Ambion Austin TX USA). To minimize the risk of expression artefacts that may arise from differences in cRNA quality cRNAs for wild-type and mutant ENaC were synthesized in parallel and the experiments were performed using at least two different batches of cRNA. The αW493R αW493A αW493C and αW493E mutants were.