Institute of Physiology, University of Regensburg, Germany.
Acta Physiol (Oxf). 2010 Jul 1;199(3):327-38. doi: 10.1111/j.1748-1716.2010.02099.x. Epub 2010 Feb 8.
Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms.
RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms.
All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl(-) affinity as determined by (86)Rb(+) uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4.
The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function.
在亨利氏升支粗段（TAL）细胞的顶端膜上，盐的重吸收主要由布美他尼敏感的 Na(+) / K(+) / 2Cl(-) 协同转运蛋白 NKCC2 介导。已经描述了 NKCC2 的三种全长剪接变体（NKCC2B、NKCC2A 和 NKCC2F）。兔、大鼠和小鼠的 NKCC2 同工型具有特定的定位和转运特性。在本研究中，我们旨在确定人 NKCC2 同工型的定位和转运特性。
通过 RT-PCR、原位杂交和 Xenopus 卵母细胞摄取研究来描述人 NKCC2 同工型。
在人肾脏中检测到所有三种经典的 NKCC2 同工型；此外，我们还发现了带有可变外显子 4 串联重复的剪接变体。与 NKCC2F 是啮齿动物中最丰富的 NKCC2 同工型相反，在人中 NKCC2A 是主要的同工型；同样，同工型特异性原位杂交显示人 NKCC2A 在 TAL 上表达水平较高。与 NKCC2B 和 NKCC2F 相比，人 NKCC2A 的 Cl(-) 亲和力最低，这是通过卵母细胞中 (86)Rb(+) 摄取研究确定的。所有 NKCC2 同工型对布美他尼的抑制作用均强于对呋塞米的抑制作用。对exon 4 变体编码的氨基酸序列进行分析表明，人与啮齿动物 NKCC2 同工型之间具有高度相似性，这表明物种间离子转运特性的差异可能与 exon 4 编码的高度保守序列之外的序列变异有关。
人 NKCC2 是一个例证，说明不同的剪接如何为转运蛋白功能的多样化奠定基础。
