Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells.
Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells.
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上皮细胞中截短的 Na-K-2Cl 协同转运蛋白的误定位。
DOI：
10.1152/ajpcell.00130.2018
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Delpire,Eric
中科院分区：
文献类型：
--
作者：
Koumangoye,Rainelli;Omer,Salma;Delpire,Eric
我们最近报道了一例多系统功能衰竭的年轻患者，该患者携带编码Na-K-2Cl共转运蛋白-1 （NKCC1）的基因slc12a2的新生突变。在非上皮细胞中的异源表达研究未能证明显性负作用。在这项研究中，我们检测了突变的共转运蛋白在上皮细胞中的表达。利用在玻璃罩、通透性支架和基质上生长的Madin-Darby犬肾（MDCK）细胞，我们发现荧光标记的突变共转运蛋白在细胞质和顶膜上表达，并影响上皮的完整性。突变转运蛋白在根尖膜的表达也会导致一些野生型转运蛋白在根尖膜上的错误定位。由于Na+-K+- atp酶仍然局限在基底外侧膜上，因此这种错误靶向是NKCC1所特有的。为了评估转运体在体内的定位，我们使用CRISPR/cas9创建了一个小鼠模型，该模型再现了slc12a2外显子22上11bp的缺失。虽然小鼠没有表现出明显的表型，但我们发现结肠和唾液腺在顶极大量表达野生型NKCC1，证实了在培养上皮细胞中获得的数据。然而，必须在基底外侧膜上保留足够的共转运蛋白来参与唾液分泌，因为在突变小鼠中没有观察到唾液分泌的显著减少。
We recently reported the case of a young patient with multisystem failure carrying a de novo mutation inSLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na+-K+-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 ofSlc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.