Roles of the C termini of alpha -, beta -, and gamma -subunits of epithelial Na+ channels (ENaC) in regulating ENaC and mediating its inhibition by cytosolic Na+

• Published in: The Journal of biological chemistry Vol. 276; no. 17; pp. 13744 - 13749
• Main Authors: Dinudom, A, Harvey, K F, Komwatana, P, Jolliffe, C N, Young, J A, Kumar, S, Cook, D I
• Format: Journal Article
• Language: English
• Published: United States 27.04.2001
• Subjects: Amiloride - pharmacology; Amino Acid Motifs; Amino Acid Sequence; Animals; Cytosol - metabolism; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Epithelial Sodium Channels; Glutathione Transferase - metabolism; Mandible - cytology; Mice; Molecular Sequence Data; Patch-Clamp Techniques; Peptides - pharmacology; Phosphorylation; Plasmids - metabolism; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; Sodium - metabolism; Sodium Channels - chemistry; Sodium Channels - metabolism; Virulence Factors, Bordetella - pharmacology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M011273200

The amiloride-sensitive epithelial Na(+) channels (ENaC) in the intralobular duct cells of mouse mandibular glands are inhibited by the ubiquitin-protein ligase, Nedd4, which is activated by increased intracellular Na(+). In this study we have used whole-cell patch clamp methods in mouse mandibular duct cells to investigate the role of the C termini of the alpha-, beta-, and gamma-subunits of ENaC in mediating this inhibition. We found that peptides corresponding to the C termini of the beta- and gamma-subunits, but not the alpha-subunit, inhibited the activity of the Na(+) channels. This mechanism did not involve Nedd4 and probably resulted from the exogenous C termini interfering competitively with the protein-protein interactions that keep the channels active. In the case of the C terminus of mouse beta-ENaC, the interacting motif included betaSer(631), betaAsp(632), and betaSer(633). In the C terminus of mouse gamma-ENaC, it included gammaSer(640). Once these motifs were deleted, we were able to use the C termini of beta- and gamma-ENaC to prevent Nedd4-mediated down-regulation of Na(+) channel activity. The C terminus of the alpha-subunit, on the contrary, did not prevent Nedd4-mediated inhibition of the Na(+) channels. We conclude that mouse Nedd4 interacts with the beta- and gamma-subunits of ENaC.