Aims: Liddle's syndrome is a hereditary form of salt-sensitive hypertension caused by gain-of-function mutations of the epithelial sodium channel (ENaC). The mutations usually affect a PY-motif in the C terminus of the b- or g-subunit of the channel and impair Nedd4-2 mediated channel internalization and degradation. The aim of the present study was to functionally characterize a novel mutation (aP642A) in the PY-motif of the a-subunit of ENaC identified in a patient with putative Liddle's syndrome. Methods: Wild-type (wt) abgENaC or mutant aP642AbgENaC expressed in Xenopus laevis oocytes were studied by measuring amiloride (2 mM) sensitive whole-cell currents (DI_ami) with two-electrode voltage-clamp. Channel surface expression was quantified with a chemiluminescence based assay and the introduction of a FLAG reporter epitope in the extracellular loop of the b-subunit. n indicates individual number of oocytes, N indicates number of batches of oocytes. Results: After incubation in a low sodium (9 mM) medium DI_ami was about 1.4-fold larger in mutant (n=163) than in wt ENaC expressing oocytes (n=166, N=14, p<0.001). Activation of sodium feedback inhibition by incubating the oocytes in a high sodium (96 mM) medium increased the stimulatory effect of the mutation to 2.4-fold (mutant: n=81, wt: n=83, N=7, p<0.001). Surface expression of mutant ENaC (n=71) was 1.9-fold higher than that of wt ENaC (n=72, N=3, p<0.001) and was paralleled by a 2.1-fold increase of DI_ami in matched control oocytes (mutant: n=24, wt: n=24, N=3, p<0.001). Proteolytic ENaC activation by chymotrypsin (2 mM) stimulated DI_ami of mutant and wt ENaC to a similar extent (mutant: 2.8-fold, n=26; wt: 2.9-fold, n=27, N=3). Conclusion: The novel aP642A mutation stimulates ENaC function in a similar manner as previously described ENaC mutations of patients with Liddle's syndrome. Thus, the aP642A mutation is likely to cause Liddle's syndrome.