ENaC Degradation in A6 Cells by the Ubiquitin-Proteosome Proteolytic Pathway

• Published in: The Journal of biological chemistry Vol. 276; no. 16; pp. 12903 - 12910
• Main Authors: Malik, B, Schlanger, L, Al-Khalili, O, Bao, H F, Yue, G, Price, S R, Mitch, W E, Eaton, D C
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 20-04-2001
• Subjects: Amiloride; Amino Acid Sequence; Animals; Antibody Specificity; Cell Line; Cell Membrane - drug effects; Cell Membrane - physiology; Cysteine Endopeptidases - metabolism; Cysteine Proteinase Inhibitors - pharmacology; Epithelial Sodium Channels; Freshwater; Humans; Kinetics; Leupeptins - pharmacology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Models, Biological; Molecular Sequence Data; Multienzyme Complexes - metabolism; Nedd4 protein; Nephrons; Peptide Fragments - chemistry; Peptide Fragments - immunology; Proteasome Endopeptidase Complex; Protein Subunits; Proteosomes; Sodium Channels - chemistry; Sodium Channels - genetics; Sodium Channels - metabolism; Ubiquitins - metabolism; Urothelium - cytology; Urothelium - physiology; Xenopus; Xenopus laevis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M010626200

Amiloride-sensitive epithelial Na + channels (ENaC) are responsible for trans-epithelial Na + transport in the kidney, lung, and colon. The channel consists of three subunits (α, β, γ) each containing a proline rich
region (PP X Y) in their carboxyl-terminal end. Mutations in this PP X Y domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions
with the ubiquitin ligase N eural precursor cell- e xpressed d evelopmentally d own- r egulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading
to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC
channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132,
increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules
by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal
function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important
regulator of ENaC function.