Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (a lpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutat ions causing Liddle's syndrome alter or delete the PY motifs of beta- or ga mma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, w e investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, lik ely by acting as a competitive antagonist to endogenous Nedd4. These effect s were dependant on the PY motifs, because no Nedd4-mediated changes in cha nnel activity were observed in ENaC lacking them. The effect of Nedd4 on EN aC missing only one PY motif(of beta-ENaC), as originally described in pati ents with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surfa ce binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephro n, and hence the hypertension.