Yerby T R, Vibat C R, Sun D, Payne J A, O'Donnell M E
Department of Human Physiology, School of Medicine, University of California, Davis 95616, USA.
Am J Physiol. 1997 Jul;273(1 Pt 1):C188-97. doi: 10.1152/ajpcell.1997.273.1.C188.
The Na-K-Cl cotransporter is an important regulator of endothelial cell volume and may also contribute to flux of Na and Cl across the endothelium of the blood-brain barrier. To date, two Na-K-Cl cotransport isoforms have been identified, the cotransporter in secretory epithelia, NKCC1, and that in absorptive renal epithelia, NKCC2. Our previous studies showed that a monoclonal antibody to the cotransporter of human colonic T84 epithelial cells, an NKCC1 isoform, recognizes a 170-kDa glycoprotein from endothelial cells. The molecular identity of the Na-K-Cl cotransporter present in endothelial cells, however, has been unknown. In addition, although evidence has been provided that phosphorylation of the endothelial cotransporter plays a role in regulating its activity, little is known about potential sites for protein kinase interaction with the cotransporter. The present study was conducted to determine the molecular structure of the endothelial Na-K-Cl cotransporter. Using a 1.0-kilobase (kb) cDNA fragment from a conserved region of the T84 cell cotransporter, we screened a bovine aortic endothelial cell cDNA library and subsequently identified and sequenced two overlapping clones that together spanned the entire coding region. The endothelial cotransporter is a 1,201-amino acid protein with 12 putative transmembrane segments and large amino and carboxy termini, each containing several consensus sites for phosphorylation by protein kinases. Comparison of the endothelial cotransporter amino acid sequence with known NKCC1 and NKCC2 sequences revealed a 96% identity with NKCC1. Northern blot analysis using a cDNA probe from the endothelial cotransporter revealed high expression of approximately 7.5-kb transcripts in a number of bovine tissues. Finally, a prominent expression of Na-K-Cl cotransporter was found by Western blot analysis in both cultured and freshly isolated endothelial cells of bovine aorta and cerebral microvessels.
钠-钾-氯协同转运蛋白是内皮细胞体积的重要调节因子，也可能有助于钠和氯穿过血脑屏障内皮的通量。迄今为止，已鉴定出两种钠-钾-氯协同转运亚型，即分泌上皮中的协同转运蛋白NKCC1和吸收性肾上皮中的NKCC2。我们先前的研究表明，针对人结肠T84上皮细胞（一种NKCC1亚型）的协同转运蛋白的单克隆抗体可识别来自内皮细胞的170 kDa糖蛋白。然而，内皮细胞中存在的钠-钾-氯协同转运蛋白的分子身份尚不清楚。此外，尽管有证据表明内皮协同转运蛋白的磷酸化在调节其活性中起作用，但关于蛋白激酶与协同转运蛋白相互作用的潜在位点知之甚少。本研究旨在确定内皮钠-钾-氯协同转运蛋白的分子结构。我们使用来自T84细胞协同转运蛋白保守区域的1.0千碱基（kb）cDNA片段，筛选了牛主动脉内皮细胞cDNA文库，随后鉴定并测序了两个重叠克隆，它们共同跨越了整个编码区域。内皮协同转运蛋白是一种由1201个氨基酸组成的蛋白质，有12个假定的跨膜片段以及大的氨基和羧基末端，每个末端都含有几个蛋白激酶磷酸化的共有位点。将内皮协同转运蛋白的氨基酸序列与已知的NKCC1和NKCC2序列进行比较，发现与NKCC1有96%的同一性。使用来自内皮协同转运蛋白的cDNA探针进行的Northern印迹分析显示，在许多牛组织中约7.5 kb转录本有高表达。最后，通过蛋白质印迹分析在牛主动脉和脑微血管的培养内皮细胞和新鲜分离的内皮细胞中均发现了钠-钾-氯协同转运蛋白的显著表达。
