Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage.
Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or disrupt a C-terminal PY motif in the epithelial Na+ channel (ENaC). Previous work indicates that these mutations increase expression of ENaC at the cell surface by disrupting its binding to Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. However, it remains uncertain whether this mechanism alone is responsible; increased activity of ENaC channels could also contribute to excessive Na+ transport in Liddle's syndrome. ENaC activity is controlled in part by its cleavage state; proteolytic cleavage produces channels with a high open-state probability, whereas uncleaved channels are inactive. Here, we found that Liddle's syndrome mutations have two distinct effects of ENaC surface expression, both of which contribute to increased Na+ transport. First, these mutations increased ENaC expression at the cell surface; second, they increased the fraction of ENaC at the cell surface that was cleaved (active). This disproportionate increase in cleavage was reproduced by expression of a dominant-negative Nedd4-2 or mutation of ENaC ubiquitination sites, interventions that disrupt ENaC endocytosis and lysosomal degradation. Conversely, overexpression of Nedd4-2 had the opposite effect, decreasing the fraction of cleaved ENaC at the cell surface. Thus, the data not only suggest that Nedd4-2 regulates epithelial Na+ transport in part by controlling the relative expression of cleaved and uncleaved ENaC at the cell surface but also provide a mechanism by which Liddle's syndrome mutations alter ENaC activity.
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242, USA.
Kristin K
KK
Knight
Diane R
DR
Olson
Ruifeng
R
Zhou
Peter M
PM
Snyder
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D056827:Endosomal Sorting Complexes Required for Transport; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D053503:Epithelial Sodium Channels; D006801:Humans; D006973:Hypertension / Q000201:enzymology* / Q000235:genetics*; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000032:analysis / Q000378:metabolism*; D013577:Syndrome; D014357:Trypsin / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D025801:Ubiquitin; D012964:Sodium; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D014357:Trypsin