The PY motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from subapical pool.
The epithelial-Na(+)-channel (alphabetagammaENaC) regulates kidney salt-transport and blood pressure. Each ENaC subunit contains a PY motif (PPxY) and its mutation in beta/gammaENaC causes Liddle syndrome, a hereditary hypertension. These (extended) PY motifs (PP(616)xY(618)xxL(621)) serve as binding sites for the ubiquitin ligase Nedd4-2, which decreases cell-surface expression of ENaC by unknown route(s). Using polarized kidney epithelia [Madin-Darby canine kidney I (MDCK-I)] cells stably expressing extracellularly myc-tagged wild type (WT) or PY-motif mutants of betaENaC (P616A, Y618A or L621A, with WT-alphagammaENaC), and live-imaging plus enzyme-linked immunosorbent assay (ELISA)-type assays to analyze routes/rates of ENaC internalization/recycling, we show here that cell-surface half-life of all PY mutants was fourfold longer than WT-ENaC (approximately 120 versus 30 minutes), reflecting primarily reduced channel internalization but also attenuated replenishment of cell-surface ENaC from a large subapical pool. The Y618A mutant revealed more severe internalization and replenishment defects than the other PY mutants. Internalized WT-ENaC was detected in sorting/recycling and late endosomes/lysosomes, while the Y618A mutant accumulated in the former. Nedd4-2 ubiquitinated ENaC at the apical membrane causing channel internalization and degradation. Cyclic AMP (cAMP) accelerated mobilization of subapical ENaC to the cell surface and long-term ENaC recycling, but only mobilization, not recycling, was inhibited in the PY mutants. These results suggest that the ENaC PY motifs (and Nedd4-2) primarily regulate channel internalization but also affect cAMP-dependent replenishment, providing important insight into the Liddle syndrome defects.
Lu
Chen
C
Program in Cell Biology, The Hospital for Sick Children, Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5G 1X8.
Pribanic
Sandra
S
Debonneville
Anne
A
Jiang
Chong
C
Rotin
Daniela
D
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D006973:Hypertension / Q000235:genetics; D007700:Kinetics; D018613:Microscopy, Confocal; D008871:Microvilli / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013577:Syndrome; D013873:Thionucleotides / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors; D053503:Epithelial Sodium Channels; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D025801:Ubiquitin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D015056:1-Methyl-3-isobutylxanthine