A naturally occurring human Nedd4-2 variant displays impaired ENaC regulation in Xenopus laevis oocytes.
The epithelial Na(+) channel (ENaC) is regulated by the ubiquitin-protein ligase Nedd4-2 via interaction with ENaC PY-motifs. These PY-motifs are mutated/deleted in Liddle's syndrome, resulting in elevated Na(+) reabsorption and hypertension explained partly by impaired ENaC-Nedd4-2 interaction. We hypothesized that Nedd4-2 is a susceptibility gene for hypertension and screened 856 renal patients and healthy controls for mutations in a subset of exons of the human Nedd4-2 gene that are relevant for ENaC regulation by PCR/single-strand conformational polymorphism. Several variants were identified, and one nonsynonymous mutation (Nedd4-2-P355L) was further characterized. This mutation next to the 3' donor site of exon 15 does not affect in vitro splicing of Nedd4-2 mRNA. However, in the Xenopus oocyte expression system, Nedd4-2-P355L-dependent ENaC inhibition was weaker compared with the wild type (Nedd4-2-WT), and this difference depended on the presence of intact PY-motifs on ENaC. This could not be explained by the amount of wild type or mutant Nedd4-2 coimmunoprecipitating with ENaC. When the phosphorylation level of human Nedd4-2 Ser(448) (known to be phosphorylated by the Sgk1 kinase) was determined with a specific anti-pSer(448) antibody, we observed stronger basal phosphorylation of Nedd4-2-P355L. Both the phosphorylation level and the accompanying amiloride-sensitive Na(+) currents could be further enhanced to approximately the same levels by coexpressing Sgk1. In addition, the role of the two other putative Sgk1 phosphorylation sites (S342 and T367) appears also to be affected by the P355L mutation. The differential phosphorylation status between wild-type and mutant Nedd4-2 provides an explanation for the different potential to inhibit ENaC activity.
Fouladkou
Fatemeh
F
Division of Nephrology and Hypertension, Department of Clinical Research, University of Bern, CH-3010 Bern.
Alikhani-Koopaei
Rasoul
R
Vogt
Bruno
B
Flores
Sandra Y
SY
Malbert-Colas
Laurence
L
Lecomte
Marie-Christine
MC
Loffing
Johannes
J
Frey
Felix J
FJ
Frey
Brigitte M
BM
Staub
Olivier
O
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015536:Down-Regulation; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005260:Female; D006706:Homeostasis; D006801:Humans; D017874:Immediate-Early Proteins; D007676:Kidney Failure, Chronic / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D009865:Oocytes; D010766:Phosphorylation; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D012694:Serine / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D017874:Immediate-Early Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D015222:Sodium Channels; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus