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+.
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.
Department of Physiology, University of Sydney, New South Wales 2006, Australia.
A
A
Dinudom
K F
KF
Harvey
P
P
Komwatana
C N
CN
Jolliffe
J A
JA
Young
S
S
Kumar
D I
DI
Cook
D000584:Amiloride / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D053503:Epithelial Sodium Channels; D005982:Glutathione Transferase / Q000378:metabolism; D008334:Mandible / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018076:DNA, Complementary; D053503:Epithelial Sodium Channels; D010455:Peptides; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D010566:Virulence Factors, Bordetella; D000584:Amiloride; D012964:Sodium; D005982:Glutathione Transferase