Mercado Adriana, Vázquez Norma, Song Luyan, Cortés Rosa, Enck Alissa H, Welch Rick, Delpire Eric, Gamba Gerardo, Mount David B
Renal Division, Brigham and Women's Hospital, Boston, MA 02115, USA.
Am J Physiol Renal Physiol. 2005 Dec;289(6):F1246-61. doi: 10.1152/ajprenal.00464.2004. Epub 2005 Jul 26.
The SLC12A6 gene encoding the K(+)-Cl(-) cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH(2)-terminal isoforms of human and mouse KCC3, along with intrarenal localization and functional characterization in Xenopus laevis oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5' of two distinct first coding exons. Northern blot analysis of mouse tissues indicates that KCC3b expression is particularly robust in the kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3-specific antibody reveals that the kidney is also unique in expressing immunoreactive protein of a lower mass, suggestive evidence that the shorter KCC3b protein predominates in kidney. Immunofluorescence reveals basolateral expression of KCC3 protein along the entire length of the proximal tubule, in both the mouse and rat. Removal of the 15-residue exon 2 by alternative splicing generates the KCC3a-x2M and KCC3b-x2M isoforms; other splicing events at an alternative acceptor site within exon 1a generate the KCC3a-S isoform, which is 60 residues shorter than KCC3a. This variation in sequence of NH(2)-terminal cytoplasmic domains occurs proximal to a stretch of highly conserved residues and affects the content of putative phosphorylation sites. Kinetic characterization of KCC3a in X. laevis oocytes reveals apparent K(m)s for Rb(+) and Cl(-) of 10.7 +/- 2.5 and 7.3 +/- 1.2 mM, respectively, with an anion selectivity of Br(-) > Cl(-) > PO(4) = I(-) = SCN(-) = gluconate. All five NH(2)-terminal isoforms are activated by cell swelling (hypotonic conditions), with no activity under isotonic conditions. Although the isoforms do not differ in the osmotic set point of swelling activation, this activation is more rapid for the KCC3a-x2M and KCC3a-S proteins. In summary, there is significant NH(2)-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in the kidney. Basolateral swelling-activated K(+)-Cl(-) cotransport mediated by KCC3 likely functions in cell volume regulation during the transepithelial transport of both salt and solutes by the proximal tubule.
编码K(+)-Cl(-)协同转运蛋白KCC3的SLC12A6基因在包括肾脏在内的多种组织中表达。在此，我们报告了人和小鼠KCC3几种氨基末端异构体的分子特征，以及在非洲爪蟾卵母细胞中的肾内定位和功能特征。两种主要异构体KCC3a和KCC3b是由两个不同的第一个编码外显子5'端的转录起始产生的。对小鼠组织的Northern印迹分析表明，KCC3b在肾脏中的表达特别强烈，肾脏也表达KCC3a。使用外显子3特异性抗体对小鼠组织进行蛋白质印迹分析显示，肾脏在表达较低分子量的免疫反应性蛋白方面也很独特，这暗示了较短的KCC3b蛋白在肾脏中占主导地位的证据。免疫荧光显示，在小鼠和大鼠中，KCC3蛋白在近端小管全长的基底外侧表达。通过可变剪接去除15个残基的外显子2产生KCC3a-x2M和KCC3b-x2M异构体；外显子1a内另一个可变受体位点的其他剪接事件产生KCC3a-S异构体，其比KCC3a短60个残基。氨基末端胞质结构域序列的这种变化发生在一段高度保守的残基附近，并影响推定磷酸化位点的含量。对非洲爪蟾卵母细胞中KCC3a的动力学特征分析显示，Rb(+)和Cl(-)的表观Km分别为10.7±2.5和7.3±1.2 mM，阴离子选择性为Br(-)>Cl(-)>PO(4)=I(-)=SCN(-)=葡萄糖酸盐。所有五种氨基末端异构体都被细胞肿胀（低渗条件）激活，在等渗条件下无活性。尽管这些异构体在肿胀激活的渗透设定点上没有差异，但KCC3a-x2M和KCC3a-S蛋白的这种激活更快。总之，KCC3存在显著的氨基末端异质性，KCC3b在肾脏中表达特别强烈。由KCC3介导的基底外侧肿胀激活的K(+)-Cl(-)协同转运可能在近端小管对盐和溶质的跨上皮转运过程中的细胞体积调节中起作用。
