Gagnon Edith, Forbush Biff, Flemmer Andreas W, Giménez Ignacio, Caron Luc, Isenring Paul
Groupe de Recherche en Néphrologie, Department of Medicine, Faculty of Medicine, Laval University, Laval, Quebec, Canada G1R 2J6.
Am J Physiol Renal Physiol. 2002 Nov;283(5):F1046-55. doi: 10.1152/ajprenal.00107.2002.
The Na-K-Cl cotransporter isoform 1 (NKCC1) has been isolated from several species, including Squalus acanthias. A second kidney-specific isoform (NKCC2) has been cloned mainly from higher vertebrates. Here, we have isolated the S. acanthias NKCC2 and found that it is produced in at least four spliced variants (saNKCC2A, saNKCC2F, saNKCC2AF, and saNKCC2AFno8) of approximately 1,090 residues. Expression of these transcripts in Xenopus laevis oocytes revealed that only the A and F variants are functional and that they are more active after incubation in low-Cl or hyperosmolar media. Rates of activation after exposure to these media were exceptionally rapid, demonstrating for the first time that the NKCC2 itself represents an important site of regulation by Cl and that extracellular domains are involved. Another remarkable finding in this study was the failure to identify NKCC2B, a variant found in the kidney of higher vertebrates and expressed specifically in macula densa cells. This result, in conjunction with the fact that the shark kidney lacks a well-developed juxtaglomerular apparatus, suggests that the B exon evolved as a result of selective pressure (presumably by exon duplication) and that a restricted relationship exists between NKCC2B and macula densa.
钠-钾-氯协同转运体同工型1（NKCC1）已从包括棘鲨在内的多个物种中分离出来。第二种肾脏特异性同工型（NKCC2）主要是从高等脊椎动物中克隆出来的。在这里，我们分离出了棘鲨的NKCC2，并发现它至少以四种剪接变体（saNKCC2A、saNKCC2F、saNKCC2AF和saNKCC2AFno8）的形式产生，约有1090个残基。这些转录本在非洲爪蟾卵母细胞中的表达表明，只有A和F变体具有功能，并且在低氯或高渗培养基中孵育后它们的活性更高。暴露于这些培养基后的激活速率异常迅速，首次证明NKCC2本身是氯离子调节的重要位点，并且细胞外结构域也参与其中。这项研究中的另一个显著发现是未能鉴定出NKCC2B，这是一种在高等脊椎动物肾脏中发现并特异性表达于致密斑细胞的变体。这一结果，再加上鲨鱼肾脏缺乏发育良好的球旁器这一事实，表明B外显子是选择性压力（可能是通过外显子重复）的结果而进化而来的，并且NKCC2B与致密斑之间存在有限的关系。
