Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States.
Department of Physiology and Biophysics, Weill-Cornell Medicine, New York, New York, United States.
Am J Physiol Renal Physiol. 2024 Aug 1;327(2):F265-F276. doi: 10.1152/ajprenal.00037.2024. Epub 2024 Jun 13.
Ubiquitination influences the expression of the epithelial Na channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and Fisher rat thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaCs were strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see whether location of the protein in or near the apical membrane rather than cleavage per se influences ubiquitination, we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when coexpressed with α- and βENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed in situ surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total and ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na could stimulate ubiquitination. Administration of amiloride to block Na entry through the channels did not affect ubiquitination of γENaC in either FRT cells or the rat kidney. However, presumed large increases in cellular Na produced by monensin in FRT cells or acute Na repletion in rats increased ubiquitination and decreased overall ENaC expression. We have explored the mechanisms underlying the ubiquitination of the γ subunit of epithelial Na channel (ENaC), a process believed to control channel internalization and degradation. We previously reported that the mature, cleaved form of the subunit is selectively ubiquitinated. Here we show that this specificity arises not from the cleavage state of the protein but from its location in the cell. We also show that under some conditions, increased intracellular Na can stimulate ENaC ubiquitination.
泛素化影响上皮钠通道（ENaC）的表达。我们评估了在天然啮齿动物肾脏和表达通道异源的 Fisher 大鼠甲状腺（FRT）细胞中，成熟的、切割形式的γENaC 选择性泛素化的机制。在这两种模型中，单切割和完全切割的γENaC 都被强烈泛素化，这意味着释放抑制肽的第二次切割对于该过程不是必需的。为了观察蛋白质在质膜内部或附近的位置而不是切割本身是否会影响泛素化，我们研究了γENaC 的突变体，其中切割位点被消除。这些亚基只有在与α和βENaC 共同表达时才被泛素化，从而促进了通过高尔基体的运输。为了测试到达质膜表面是否是必需的，我们进行了原位表面生物素化，并测量了大鼠肾脏质膜中 ENaC 的泛素化。在整个肾脏和表面部分中，切割的γENaC 的泛素化是相似的，这意味着顶端和亚顶端通道都可以被修饰。在 FRT 细胞中，用 Dyngo-4a 抑制网格蛋白介导的内吞作用会增加细胞表面的总 γENaC 和泛素化 γENaC。最后，我们测试了增加细胞内 Na 可以刺激泛素化的想法。用阿米洛利阻断通道进入的 Na 不会影响 FRT 细胞或大鼠肾脏中 γENaC 的泛素化。然而，在 FRT 细胞中，莫能菌素引起的细胞内 Na 大量增加或大鼠急性 Na 补充会增加泛素化并减少整体 ENaC 表达。我们已经探索了上皮钠通道（ENaC）γ 亚基泛素化的机制，该过程被认为控制通道内化和降解。我们之前报道过成熟的、切割形式的亚基是选择性泛素化的。在这里，我们表明这种特异性不是来自蛋白质的切割状态，而是来自其在细胞中的位置。我们还表明，在某些条件下，增加细胞内 Na 可以刺激 ENaC 泛素化。
