Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin-Darby canine kidney cells
Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin-Darby canine kidney cells
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DOI：
10.1074/jbc.m110904200
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发表时间：
2002-03-22
影响因子：
4.8
通讯作者：
Rotin, D
中科院分区：
文献类型：
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作者：
Hanwell, D;Ishikawa, T;Rotin, D
位于顶端的上皮Na+通道（alphabetagamma-ENaC）在调节肾脏和其他上皮中的盐和液体转运中起关键作用，但其运输至质膜的模式及其在哺乳动物细胞中的细胞表面稳定性知之甚少。由于ENaC在天然组织/细胞中的表达非常低，我们产生了稳定表达α-γ-ENaC的上皮Madin-Darby犬肾（MDCK）细胞，其中每个亚基在细胞内C末端被差异标记，并且β-亚基在胞外域也被Myc标记（α（HA）β（Myc），（T7）γ（FLAG））。这些细胞中的ENaC表达通过用标签的抗体进行免疫印迹来验证，并且膜片钳分析已经证实标记的通道是功能性的。此外，使用电子显微镜，我们证明了顶端，但不是基底，在这些细胞中的ENaC膜定位。ENaC细胞内库的糖基化模式揭示了肽N-糖苷酶F和内切糖苷酶H的敏感性。令人惊讶的是，通过表面生物素化分析的ENaC的细胞表面池也是核心糖基化的，并且缺乏可检测的糖苷内切酶H抗性通道。在Triton X-100中从细胞中提取通道表明，ENaC的细胞内和细胞表面池在很大程度上是可溶的。此外，漂浮试验，以分析存在的ENaC在脂筏表明，细胞内和细胞表面池的这个通道是不相关的筏。我们以前已经表明，ENaC的总细胞库迅速周转（t（1 - 2）类似于1-2小时）。使用放线菌酮处理和表面生物素化，我们现在证明，ENaC的细胞表面池具有类似的短半衰期（t（1/2）类似于1小时），不像最近报道的非洲爪蟾A6细胞的长半衰期。总的来说，这些结果有助于阐明哺乳动物肾上皮细胞中ENaC运输和周转率的关键方面。
The apically located epithelial Na+ channel (alphabetagamma-ENaC) plays a key role in the regulation of salt and fluid transport in the kidney and other epithelia, yet its mode of trafficking to the plasma membrane and its cell surface stability in mammalian cells are poorly understood. Because the expression of ENaC in native tissues/cells is very low, we generated epithelial Madin-Darby canine kidney (MDCK) cells stably expressing alphabetagamma-ENaC, where each subunit is tagged differentially at the intracellular C terminus and the beta-subunit is also Myc-tagged at the ectodomain (alpha(HA)beta(Myc),(T7)gamma(FLAG)). ENaC expression in these cells was verified by immunoblotting with antibodies to the tags, and patch clamp analysis has confirmed that the tagged channel is functional. Moreover, using electron microscopy, we demonstrated apical, but not basal, membrane localization of ENaC in these cells. The glycosylation pattern of the intracellular pool of ENaC revealed peptide N-glycosidase F and endoglycosidase H sensitivity. Surprisingly, the cell surface pool of ENaC, analyzed by surface biotinylation, was also core glycosylated and lacked detectable endoglycosidase H-resistant channels. Extraction of the channel from cells in Triton X-100 demonstrated that both intracellular and cell surface pools of ENaC are largely soluble. Moreover, floatation assays to analyze the presence of ENaC in lipid rafts showed that both intracellular and cell surface pools of this channel are not associated with rafts. We have shown previously that the total cellular pool of ENaC is turned over rapidly (t(1/2) similar to 1-2 h). Using cycloheximide treatment and surface biotinylation we now demonstrate that the cell surface pool of ENaC has a similarly short half-life (t(1/2) similar to 1 h), unlike the long half-life reported recently for the Xenopus A6 cells. Collectively, these results help elucidate key aspects of ENaC trafficking and turnover rates in mammalian kidney epithelial cells.