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

Na-K-CI 和 K-CI 协同转运蛋白在跨上皮细胞中发挥重要作用 盐运输和细胞体积和电解质平衡。 Na-K-CI 协同转运蛋白是 NaCl 重吸收过程中的关键元件 哺乳动物肾上皮位于环的厚升肢 Henle (TAL)，它是利尿剂的作用部位，例如 呋塞米。 K-CI协同转运蛋白已被提议参与Na 近曲小管和 TAL 中的 CI 重吸收以及 K 分泌 位于远曲小管，但缺乏特异性抑制剂和分子 探针，到目前为止还无法确认这些角色。内 在过去的几年里，编码 Na-K-CI 协同转运蛋白（NKCC1 和 NKCC2) 和 K-CI 协同转运蛋白 (KCC1) 已在此克隆 实验室，能够对运输过程进行分子检查。的 该项目的目标是了解潜在的机制 Na-K-CI 和 K-CI 协同转运蛋白在哺乳动物肾脏中的功能， 并了解协同转运蛋白对整体肾脏的重要性 功能。拟议的研究将使用基因敲除小鼠进行 NKCC2 和 KCC1 模型以及兔组织和 重组NKCC和KCC蛋白在组织培养细胞中表达。 具体来说： 1) 肾脏 Na-K-CI 中的结构-功能关系 将讨论转运蛋白（NKCC2），检查四个假设： 致密斑中的 NKCC2b 对肾小管肾小球反馈至关重要。那个 NKCC2 的三种剪接变体传递不同的离子亲和力 这些差异在功能上具有重要意义； NKCC2 是 直接磷酸化调节。 2) 协同转运蛋白的域 指导极化上皮细胞顶端/基底外侧排序的蛋白质 将通过嵌合 NKCC1/NKCC2 构建体的表达来确定 上皮细胞系。 3) KCC1的肾脏分布为 确定，并且协同转运蛋白的功能意义将是 在敲除小鼠模型中进行评估。

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.