NA+/K+/CL- AND K+/CL- COTRANSPORTERS IN MAMMALIAN KIDNEY

Na-K-Cl和K-Cl共转运体在跨上皮细胞转运中起重要作用。 盐运输和细胞体积和电解质平衡。Na-K-Cl 共转运蛋白是跨膜重吸收NaCl过程中的关键要素。 哺乳动物的肾上皮在环的厚的上升肢中， Henle（TAL），它是利尿剂的作用部位， 呋塞米。K-CI共转运蛋白被认为参与Na +-Na 近端小管和TAL以及K分泌中的CI重吸收 在远端小管，但缺乏特异性抑制剂和分子 探测器，直到现在还无法确认这些角色。内 最近几年，编码Na-K-Cl共转运蛋白（NKCC 1）的cDNA 和NKCC 2）和K-CI共转运蛋白（KCC 1）已在本发明中被克隆。 实验室，使分子检查的运输过程。的 这个项目的目标是了解的机制， 哺乳动物肾脏中Na-K-Cl和K-Cl共转运蛋白的功能， 并了解共转运蛋白对整体肾脏 功能拟用基因敲除小鼠进行研究 NKCC 2和KCC 1模型，以及兔组织，以及 重组NKCC和KCC蛋白在组织培养细胞中表达。 具体而言：1）肾Na-K-Cl共- 转运蛋白（NKCC 2）将被解决，检查四个假设： 致密斑中的NKCC 2b是肾小管肾小球反馈的中心; NKCC 2的三种剪接变体传递不同的离子亲和力， 这些差异在功能上是重要的; NKCC 2是 由直接磷酸化调节。2)协同转运蛋白的结构域 一种指导极化上皮细胞顶端/基底侧分选的蛋白质 将通过嵌合NKCC 1/NKCC 2构建体在一个细胞中的表达来确定。 上皮细胞系3)KCC 1的肾脏分布将是 确定，以及协同转运蛋白的功能意义将是 在敲除小鼠模型中评估。

The Na-K-CI and K-CI co-transporters play vital roles in transepithelial salt transport and cellular volume and electrolyte balance. The Na-K-CI co-transporter is a key element in the process of NaCI reabsorption across the mammalian renal epithelium in the thick ascending limb of the loop of Henle (TAL), where it is the site of action of diuretics such as furosemide. The K-CI co-transporter has been proposed to be involved in Na CI reabsorption in the proximal tubule and TAL as well as in K secretion in the distal tubule, but lacking specific inhibitors and molecular probes, it has until now been impossible to confirm these roles. Within the last several years, cDNAs encoding the Na-K-CI co-transporter (NKCC1 and NKCC2) and K-CI co-transporter (KCC1) have been cloned in this laboratory, enabling molecular examination of the transport process. The goal of this project is to understand the mechanisms that underly the function of the Na-K-CI and K-CI co-transporters in the mammalian kidney, and to understand the significance of the co-transporters to overall renal function. The proposed studies will be carried out with knockout mouse models of NKCC2 and KCC1, as well as with rabbit tissues, and with recombinant NKCC and KCC protein expressed in tissue culture cells. Specifically: 1) Structure-function relationships in the renal Na-K-CI co- transporter (NKCC2) will be addressed, examining four hypotheses: that NKCC2b in the macula densa is central to tubuloglomerular feedback; that the three splice variants of NKCC2 convey different ion affinities and that these differences are functionally significant; and that NKCC2 is regulated by direct phosphorylation. 2) The domain of the co-transporter protein that directs apical/basolateral sorting in polarized epithelia will be determined by expression of chimeric NKCC1/NKCC2 constructs in an epithelial cell line. 3) The renal distribution of KCC1 will be determined, and the functional significance of the co-transporter will be assessed in a knockout mouse model.