The PY Motif of ENaC, Mutated in Liddle Syndrome, Regulates Channel Internalization, Sorting and Mobilization from Subapical Pool
2007; Wiley; Volume: 8; Issue: 9 Linguagem: Inglês
10.1111/j.1600-0854.2007.00602.x
ISSN1600-0854
AutoresChen Lu, Sandra Pribanić, Anne Debonneville, Chong Jiang, Daniela Rotin,
Tópico(s)Pancreatic function and diabetes
ResumoThe epithelial‐Na + ‐channel (αβγENaC) regulates kidney salt‐transport and blood pressure. Each ENaC subunit contains a PY motif (PPxY) and its mutation in β/γENaC 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 βENaC (P616A, Y618A or L621A, with WT‐αγENaC), 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 (∼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.
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