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

哺乳动物肾脏中的 NA /K /CL- 和 K /CL- 协同转运蛋白

• 批准号: 6354687
• 负责人: BLISS FORBUSH
• 金额: $ 14.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6354687
• 关键词: basolateral membrane; cellular polarity; chimeric proteins; chloride ion; complementary DNA; fluorescence microscopy; gene targeting; genetically modified animals; immunofluorescence technique; ion transport; laboratory mouse; laboratory rabbit; membrane permeability; membrane transport proteins; molecular cloning; phosphorylation; polymerase chain reaction; potassium ion; protein isoforms; protein localization; protein structure function; recombinant proteins; renal tubular transport; sodium ion; toad

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.

NA-K-CI和K-CI共发器在transepithialial中起着至关重要的作用 盐传输和细胞体积和电解质平衡。 NA-K-CI 共转运蛋白是NACI重吸收过程中的关键要素 较厚的环路上升的哺乳动物肾上皮 Henle（TAL），它是利尿剂的作用所在的地方 速尿。已经提出K-CI共转运蛋白参与NA CI近端小管和TAL以及k分泌中的Ci重吸收 在远端小管中，但缺乏特异性抑制剂和分子 探针，到目前为止不可能确认这些角色。之内 最近几年，编码NA-K-CI Co-Transporter（NKCC1）的CDNA 和NKCC2）和K-CI Co-Tansporter（KCC1）已被克隆在此中 实验室，可以对运输过程进行分子检查。这 该项目的目标是了解基本的机制 Na-k-CI和K-CI共发器在哺乳动物肾脏中的功能， 并了解共同转运者对总体肾脏的意义 功能。拟议的研究将使用敲除鼠标进行 NKCC2和KCC1的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：1）肾脏Na-k-Ci共同的结构功能关系 将解决转运蛋白（NKCC2），研究四个假设： 黄斑丁萨中的NKCC2B是微管反馈的核心；那 NKCC2的三个剪接变体传达了不同的离子亲和力和 这些差异在功能上很重要。 NKCC2是 由直接磷酸化调节。 2）共同转运蛋白的域 指导偏光性上皮的顶端/基底外侧排序的蛋白质 将通过表达嵌合nkcc1/nkcc2构建体的表达来确定 上皮细胞系。 3）KCC1的肾脏分布将是 确定的，以及共转运蛋白的功能意义将是 在敲除鼠标模型中评估。