MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF THE BUMETANIDE-SENSITIVE NA-K-CL COTRANSPORTER
MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF THE BUMETANIDE-SENSITIVE NA-K-CL COTRANSPORTER
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DOI：
10.1073/pnas.91.6.2201
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发表时间：
1994-03-15
影响因子：
11.1
通讯作者：
FORBUSH, B
中科院分区：
文献类型：
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作者：
XU, JC;LYTLE, C;FORBUSH, B
通过介导 Na、K 和 Cl 离子跨过大多数动物细胞质膜的耦合运动，布美他尼敏感的 Na-K-Cl 协同转运蛋白 (NKCC) 在离子平衡和细胞体积的调节中发挥着至关重要的作用。转运蛋白是分泌和吸收上皮细胞盐载体转运过程中的核心元件。通过用天然鲨鱼协同转运蛋白的单克隆抗体筛选，从鲨鱼直肠腺文库中分离出编码 Na-K-Cl 协同转运蛋白的 cDNA。从 cDNA 序列预测的 1191 个残基的蛋白质具有 12 个假定的跨膜结构域，两侧是大的细胞质 N 和 C 末端。分离肽中的调节性磷酸受体残基在预测序列中被鉴定为 Thr-189 和 Thr-1114。 Northern 印迹分析在直肠腺和大多数其他鲨鱼组织中发现了 7.4 kb 的 mRNA； 5.2 kb mRNA 仅限于鲨鱼肾。在大部分编码区都发现了与来自秀丽隐杆线虫的未表征基因以及与比目鱼膀胱的噻嗪类敏感的 Na-Cl 协同转运蛋白的同源性；在秀丽隐杆线虫 cDNA 和蓝细菌的未表征基因中发现了较短的同源性。人类 HEK-293 细胞已被鲨鱼 cDNA 稳定转染，并显示出表达 Na K-Cl 共转运活性以及鲨鱼蛋白的布美他尼敏感性。表达的转运蛋白在宿主细胞中功能静止，可以通过消耗细胞中的氯来激活。
By mediating the coupled movement of Na, K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2-kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.