Dinudom A, Harvey K F, Komwatana P, Jolliffe C N, Young J A, Kumar S, Cook D I
Department of Physiology, University of Sydney, New South Wales 2006, Australia.
J Biol Chem. 2001 Apr 27;276(17):13744-9. doi: 10.1074/jbc.M011273200. Epub 2001 Feb 1.
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.
小鼠下颌下腺小叶内导管细胞中的氨氯地平敏感上皮钠通道（ENaC）受到泛素蛋白连接酶Nedd4的抑制，细胞内钠离子浓度升高会激活该酶。在本研究中，我们采用全细胞膜片钳技术，研究小鼠下颌下腺导管细胞中ENaC的α、β和γ亚基的C末端在介导这种抑制作用中的作用。我们发现，对应于β和γ亚基C末端的肽（而非α亚基的肽）可抑制钠通道的活性。该机制不涉及Nedd4，可能是由于外源性C末端竞争性干扰了维持通道活性的蛋白质-蛋白质相互作用。就小鼠β-ENaC的C末端而言，相互作用基序包括βSer(631)、βAsp(632)和βSer(633)。在小鼠γ-ENaC的C末端，它包括γSer(640)。一旦这些基序被删除，我们就能利用β和γ-ENaC的C末端来防止Nedd4介导的钠通道活性下调。相反，α亚基的C末端并不能防止Nedd4介导的钠通道抑制。我们得出结论，小鼠Nedd4与ENaC的β和γ亚基相互作用。
