Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland, United Kingdom.
J Biol Chem. 2009 Dec 18;284(51):35348-58. doi: 10.1074/jbc.M109.060004.
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 (86)Rb 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.
哺乳动物升支粗段顶端膜上的钠-钾-2 氯协同转运蛋白（NKCC2）是一种对呋塞米敏感的协同转运蛋白，它通过可变剪接产生 3 种主要变体（A、B 和 F），这 3 种变体在定位和转运特性上存在差异。关于 NKCC2 的调节机制，大部分知识来自于非洲爪蟾卵母细胞实验，因为 NKCC2 在哺乳动物系统中很难实现功能性表达。本研究报告了从雪貂肾脏（fNKCC2A、-B 和 -F）克隆和表达未标记和未经修饰的主要剪接变体，并在人胚肾（HEK）293 细胞中进行了功能表达。本研究中使用的许多 NKCC2 抗体检测到转染蛋白的高分子量形式，可能是 NKCC2 二聚体，但不是单体。有趣的是，单体可以被针对调节 N 端磷酸肽的磷酸特异性抗体强烈检测到。在转染的 HEK-293 细胞中，（86）Rb 的摄取对呋塞米敏感，并且可以通过在摄取测量之前将细胞在含有低氯浓度的培养基中孵育来刺激摄取。fNKCC2 对氯离子浓度降低的敏感性低于 NKCC1。使用稳定表达 fNKCC2A 的 HEK-293 细胞，我们还表明，变体 NKCC2AF 的共表达对 NKCC2A 活性没有在非洲爪蟾卵母细胞中观察到的显性负效应，也不会向细胞表面转运。此外，fNKCC2AF 既没有在其 N 端调节区进行复杂的糖基化，也没有磷酸化，而其他变体则存在这两种修饰。
