Control of ENaC ubiquitination.

Shujie Shi, Gustavo Frindt, Sarah Christine M Whelan, Lawrence G Palmer
{"title":"Control of ENaC ubiquitination.","authors":"Shujie Shi, Gustavo Frindt, Sarah Christine M Whelan, Lawrence G Palmer","doi":"10.1152/ajprenal.00037.2024","DOIUrl":null,"url":null,"abstract":"<p><p>Ubiquitination influences the expression of the epithelial Na<sup>+</sup> 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<sup>+</sup> could stimulate ubiquitination. Administration of amiloride to block Na<sup>+</sup> 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<sup>+</sup> produced by monensin in FRT cells or acute Na<sup>+</sup> repletion in rats increased ubiquitination and decreased overall ENaC expression.<b>NEW & NOTEWORTHY</b> We have explored the mechanisms underlying the ubiquitination of the γ subunit of epithelial Na<sup>+</sup> 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<sup>+</sup> can stimulate ENaC ubiquitination.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":" ","pages":"F265-F276"},"PeriodicalIF":0.0000,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11444504/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"American journal of physiology. Renal physiology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1152/ajprenal.00037.2024","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/6/13 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0

Abstract

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.NEW & NOTEWORTHY 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.

控制 ENaC 泛素化。
泛素化影响上皮Na+通道(ENaC)的表达。我们在原生啮齿动物肾脏和异源表达γENaC通道的费舍尔大鼠甲状腺(FRT)细胞中评估了成熟的、已裂解的γENaC选择性泛素化的机制。在这两种模型中,单一裂解和完全裂解的γENaC都被强烈泛素化,这意味着释放抑制肽的第二次裂解对这一过程并不重要。为了了解影响泛素化的因素是否是蛋白质在顶端膜中或顶端膜附近的位置,而不是裂解本身,我们研究了γENaC 的突变体,在这些突变体中,裂解位点被取消。这些亚基只有在与α和βENaC共同表达时才会被泛素化,从而促进通过高尔基体的运输。为了检验到达顶端表面是否必要,我们进行了原位表面生物素化,并测量了大鼠肾脏顶端膜中ENaC泛素化的情况。在整个肾脏和表面部分,裂解的γENaC泛素化相似,这意味着肾尖和肾尖下通道都可能被改变。在 FRT 细胞中,用 Dyngo-4a 抑制凝集素介导的内吞作用会增加细胞表面泛素化的γENaC 总量。最后,我们测试了细胞内 Na+ 的增加会刺激泛素化的观点。在 FRT 细胞或大鼠肾脏中施用阿米洛利来阻断 Na+ 通过通道的进入并不影响 γENaC 的泛素化。然而,FRT 细胞中的莫能菌素或大鼠急性 Na+ 补给所产生的推测的细胞 Na+ 大量增加会增加泛素化并降低 ENaC 的整体表达。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信