Functional expression of the Na-K-2Cl cotransporter NKCC2 in mammalian cells fails to confirm the dominant-negative effect of the AF splice variant.
Functional expression of the Na-K-2Cl cotransporter NKCC2 in mammalian cells fails to confirm the dominant-negative effect of the AF splice variant.
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DOI：
10.1074/jbc.m109.060004
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发表时间：
2009-12-18
期刊：
影响因子：
--
通讯作者：
Flatman PW
中科院分区：
文献类型：
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作者：
Hannemann A;Christie JK;Flatman PW
肾脏布美他尼敏感性Na-K-2Cl协同转运蛋白（NKCC 2）是哺乳动物粗升肢顶膜中的主要盐转运途径。它是差异剪接的，三种主要变体（A、B和F）的定位和转运特征不同。关于其调控的大多数知识来自非洲爪蟾卵母细胞的实验，因为NKCC 2被证明难以在哺乳动物系统中功能性表达。在这里，我们报告的雪貂肾（fNKCC 2A，-B，和-F）的主要剪接变体的未标记和未修饰的版本在人胚肾（HEK）293细胞的克隆和功能表达。本研究中使用的许多NKCC 2抗体检测到高分子量形式的转染蛋白，可能是NKCC 2二聚体，但不是单体。有趣的是，单体强烈检测磷酸特异性抗体针对磷酸肽的监管N端。布美他尼敏感的86 Rb摄取在转染的HEK-293细胞中显著更高，并且可以通过在摄取测量之前在含有低氯化物浓度的培养基中孵育细胞来刺激。fNKCC 2对氯离子浓度降低的敏感性低于NKCC 1。使用稳定表达fNKCC 2A的HEK-293细胞，我们还表明，变体NKCC 2AF的共表达对在非洲爪蟾卵母细胞中观察到的NKCC 2A活性没有显性负效应，也没有被运输到细胞表面。此外，fNKCC 2AF在其N末端调控区既不复杂糖基化，也不像其他变体那样磷酸化。
The renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb. It is differentially spliced and the three major variants (A, B, and F) differ in their localization and transport characteristics. Most knowledge about its regulation comes from experiments in Xenopus oocytes as NKCC2 proved difficult to functionally express in a mammalian system. Here we report the cloning and functional expression of untagged and unmodified versions of the major splice variants from ferret kidney (fNKCC2A, -B, and -F) in human embryonic kidney (HEK) 293 cells. Many NKCC2 antibodies used in this study detected high molecular weight forms of the transfected proteins, probably NKCC2 dimers, but not the monomers. Interestingly, monomers were strongly detected by phosphospecific antibodies directed against phosphopeptides in the regulatory N terminus. Bumetanide-sensitive 86Rb uptake was significantly higher in transfected HEK-293 cells and could be stimulated by incubating cells in a medium containing a low chloride concentration prior the uptake measurements. fNKCC2 was less sensitive to the reduction in chloride concentration than NKCC1. Using HEK-293 cells stably expressing fNKCC2A we also show that co-expression of variant NKCC2AF does not have the dominant-negative effect on NKCC2A activity that was seen in Xenopus oocytes, nor is it trafficked to the cell surface. In addition, fNKCC2AF is neither complex glycosylated nor phosphorylated in its N terminus regulatory region like other variants.