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首页> 外文期刊>American Journal of Physiology >Endogenous and exogenous Na-K-Cl cotransporter expression in a low K-resistant mutant MDCK cell line.
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Endogenous and exogenous Na-K-Cl cotransporter expression in a low K-resistant mutant MDCK cell line.

机译：低耐K突变MDCK细胞系中的内源性和外源性Na-K-Cl共转运蛋白表达。

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摘要

A low K-resistant mutant Madin-Darby canine kidney (MDCK) cell line, LK-C1, has been shown previously to lack functional Na-K-Cl cotransporter (NKCC) activity, indicating that it may be a useful NKCC "knockout" cell line for structure-function studies. Using immunological probes, we first characterized the defect in the endogenous NKCC protein of the LK-C1 cells and then fully restored NKCC activity in these cells by stably expressing the human secretory NKCC1 protein (hNKCC1). The endogenous NKCC protein of the LK-C1 cells was expressed at significantly lower levels than in wild-type MDCK cells and was not properly glycosylated. This latter finding indicated that the lack of functional NKCC activity in the LK-C1 cells may be due to the inability to process the protein to the plasma membrane. In contrast, exogenously expressed hNKCC1 protein was properly processed and fully functional at the plasma membrane. Significantly, the exogenous hNKCC1 protein was regulated in a manner similar to the protein innative secretory cells as it was robustly activated by cell shrinkage, calyculin A, and low-Cl incubation. Furthermore, when the LK-C1 cells formed an epithelium on permeable supports, the exogenous hNKCC1 protein was properly polarized and functional at the basolateral membrane. The low levels of endogenous NKCC protein expression, the absence of any endogenous NKCC transport activity, and the ability to form a polarized epithelium indicate that the LK-C1 cells offer an excellent expression system with which to study the molecular physiology of the cation Cl cotransporters.
机译：先前已显示出一种耐低钾的突变型Madin-Darby犬肾（MDCK）细胞系LK-C1缺乏功能性Na-K-Cl共转运蛋白（NKCC）活性，表明它可能是有用的NKCC“敲除”细胞结构功能研究。使用免疫探针，我们首先表征LK-C1细胞内源性NKCC蛋白的缺陷，然后通过稳定表达人类分泌型NKCC1蛋白（hNKCC1）来完全恢复这些细胞中的NKCC活性。 LK-C1细胞的内源性NKCC蛋白的表达水平明显低于野生型MDCK细胞中的水平，并且未被正确糖基化。后一个发现表明，LK-C1细胞中缺乏功能性NKCC活性可能是由于无法将蛋白质加工成质膜。相反，外源表达的hNKCC1蛋白经过适当处理，在质膜上具有完整功能。重要的是，外源性hNKCC1蛋白的调节方式类似于蛋白内分泌性细胞，因为它可以通过细胞收缩，钙蛋白A和低Cl孵育而被强烈激活。此外，当LK-C1细胞在可渗透支持物上形成上皮时，外源性hNKCC1蛋白被正确极化并在基底外侧膜上起作用。内源性NKCC蛋白表达水平低，不存在任何内源性NKCC转运活性以及形成极化上皮的能力表明LK-C1细胞提供了一个出色的表达系统，可用于研究阳离子C1共转运蛋白的分子生理学。

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