Nephrology Research Group, L'Hôtel-Dieu de Québec Research Center, Department of Medicine, Faculty of Medicine, Laval University , Quebec City, Quebec , Canada.
Cardiometabolic Research Group, Department of Kinesiology, Faculty of Medicine, University of Montreal , Montreal, Quebec , Canada.
Am J Physiol Cell Physiol. 2019 Jul 1;317(1):C20-C30. doi: 10.1152/ajpcell.00041.2019. Epub 2019 Mar 27.
Na-K-Cl cotransporter type 2 (NKCC2) is confined to the apical membrane of the thick ascending limb of Henle, where it reabsorbs a substantial fraction of the ultrafiltered NaCl load. It is expressed along this nephron segment as three main splice variants (called NKCC2A, NKCC2B, and NKCC2F) that differ in residue composition along their second transmembrane domain and first intracellular cytosolic connecting segment (CS2). NKCC2 is known to be activated by cell shrinkage and intracellular [Cl] reduction. Although the with no lysine (WNK) kinases could play a role in this response, the mechanisms involved are ill defined, and the possibility of variant-specific responses has not been tested thus far. In this study, we have used the oocyte expression system to gain further insight in these regards. We have found for the first time that cell shrinkage could stimulate NKCC2A- and NKCC2B-mediated ion transport by increasing carrier abundance at the cell surface and that this response was achieved (at least in part) by the enzymatic function of a WNK kinase. Interestingly, we have also found that the activity and cell surface abundance of NKCC2F were less affected by cell shrinkage compared with the other variants and that ion transport by certain variants could be stimulated through WNK kinase expression in the absence of carrier redistribution. Taken together, these results suggest that the WNK kinase-dependent pathway can affect both the trafficking as well as intrinsic activity of NKCC2 and that CS2 plays an important role in carrier regulation.
钠-钾-2 氯共转运蛋白 2（NKCC2）局限于亨利氏升支粗段的顶端膜，在那里它重吸收了相当一部分超滤液中的 NaCl 负荷。它在这个肾单位节段表达为三种主要的剪接变体（称为 NKCC2A、NKCC2B 和 NKCC2F），它们在第二跨膜域和第一细胞内胞质连接段（CS2）的残基组成上存在差异。NKCC2 已知通过细胞收缩和细胞内[Cl]减少而被激活。尽管无赖氨酸（WNK）激酶可能在这种反应中发挥作用，但涉及的机制尚未明确，变体特异性反应的可能性迄今尚未得到检验。在这项研究中，我们使用卵母细胞表达系统进一步深入研究了这些方面。我们首次发现，细胞收缩可以通过增加细胞表面载体的丰度来刺激 NKCC2A 和 NKCC2B 介导的离子转运，并且这种反应至少部分是通过 WNK 激酶的酶功能实现的。有趣的是，我们还发现，与其他变体相比，NKCC2F 的活性和细胞表面丰度受细胞收缩的影响较小，并且在没有载体重新分布的情况下，通过 WNK 激酶表达可以刺激某些变体的离子转运。总之，这些结果表明，WNK 激酶依赖性途径可以影响 NKCC2 的运输和内在活性，并且 CS2 在载体调节中发挥重要作用。
