The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel
The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel
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DOI：
10.1074/jbc.m707989200
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发表时间：
2007-12-28
影响因子：
4.8
通讯作者：
Frizzell, Raymond A.
中科院分区：
文献类型：
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作者：
Butterworth, Michael B.;Edinger, Robert S.;Frizzell, Raymond A.
上皮钠通道 (ENaC) 被极化皮质集合管 (CCD) 上皮细胞顶膜上的 E3 连接酶 Nedd4-2 泛素化。这导致 ENaC 内吞作用和可能的降解。由于已知 ENaC 在 CCD 细胞的顶膜上循环，因此去泛素化酶 (DUB) 可能通过促进 ENaC 循环而不是降解来参与调节 ENaC 表面密度。使用化学探针方法标记活性 DUB，我们鉴定出泛素 C 末端水解酶 (UCH) 亚型 L3 是 CCD 细胞内体区室中的主要 DUB。通过选择性敲低来阻断 UCH-L3 活性或减少其表达会增加 ENaC 泛素化，并导致其从 CCD 细胞顶膜上去除。从功能上讲，这导致跨 CCD 上皮细胞的跨上皮 Na+ 电流迅速减少。表面生物素化表明当 UCH-L3 受到抑制时，ENaC 从顶端表面丢失。全细胞或顶端表面免疫沉淀证明 ENaC 泛素化随着 UCH-L3 抑制而增加。这构成了 UCH 在上皮细胞和离子通道调节中的新功能，并证明了通过再循环对位于顶端的 ENaC 进行动态调节，这是由这种 DUB 促进的。
The epithelial sodium channel ( ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na+ currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immuno-precipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.