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

Na-K-CI和K-CI联合转运体在跨上皮细胞中的重要作用 盐分运输、细胞体积和电解质平衡。Na-K-CI 共转运蛋白是Naci跨膜重吸收过程中的一个关键因素 粗大升支中的哺乳动物肾上皮细胞 亨勒(Henle)，在那里它是利尿剂的作用部位，如 速尿。K-CI联合转运体已被提议参与Na 近端小管和TAL的CI重吸收以及K的分泌 在远端小管中，但缺乏特定的抑制剂和分子 探测器，到目前为止，还不可能确认这些作用。在 在过去的几年中，编码Na-K-CI共转运体(NKCC1)的cDNA 和NKCC2)和K-CI共转运蛋白(KCC1)已在 实验室，能够对运输过程进行分子检查。这个 这个项目的目标是了解 Na-K-CI和K-CI共转运体在哺乳动物肾脏中的功能 并了解辅助转运体对整个肾脏的意义 功能。建议的研究将在基因敲除小鼠身上进行。 NKCC2和KCC1模型，以及与兔组织和 重组NKCC和KCC蛋白在组织培养细胞中表达。 具体地说：1)肾脏Na-K-CI协同作用的结构-功能关系 Transporter(NKCC2)将被解决，并检验四个假设： 致密黄斑中的NKCC2b是小管小球反馈的中枢； NKCC2的三种剪接变体表达不同的离子亲和力和 这些差异在功能上是显著的；而NKCC2是 受直接磷酸化的调节。2)共同运输者的领地 在极化上皮中引导顶端/基底侧分选的蛋白质 将由嵌合的NKCC1/NKCC2结构在 上皮细胞系。3)KCC1的肾脏分布为 确定，联合转运体的功能意义将是 在基因敲除小鼠模型中进行了评估。

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.