THIAZIDE-SENSITIVE NA+/CL- TRANSPORTER STRUCTURE-FUNCTN

这笔赠款的长期目标是在分子水平上理解 使噻嗪类利尿剂的结构、功能和调节水平- 哺乳动物肾脏中存在敏感的Na：CL共转运蛋白。这个 这项拨款建议的具体目标是：(I)克隆 大鼠肾脏对噻嗪敏感的Na：CL-共转运蛋白的研究 由编码噻嗪敏感基因的cDNA产生的核苷酸探针 NA：我们最近从尿液中克隆的CL-辅转运蛋白 (Ii)至(Ii)至 测定大鼠肾脏的核苷酸和氨基酸序列 开发了一种拓扑模型 共转运蛋白，并比较其核苷酸和氨基酸序列。 Na：CL-共转运蛋白与其他已测序蛋白质，特别是其他 共转运蛋白；(Iii)评估其功能特性 共转运蛋白在非洲爪哇卵母细胞中的表达 将这些特征与比目鱼膀胱的特征进行比较 转运蛋白；(Iv)制备多克隆抗体 共转运体，将被用来评估表达 共转运蛋白；(V)确定大鼠的分布 肾和非肾组织中Na：CL-辅转运蛋白的表达 组织经Northern和Western分析，单肾小管聚合酶链式反应， 和免疫荧光；(Vi)评估噻嗪的异构体- 在大鼠肾脏中存在敏感的共转运蛋白[特别是。在远端 小管和集合管]，如果能鉴定出异构体， 确定这些异构体的结构并评估它们的 功能特征。这些拟议研究的结果将 提供我们可以开始建模的基本信息[和 要评估的分子探针]它的功能和调节 重要的肾脏离子转运蛋白在分子水平上。

The long-term objective of this grant is to understand at a molecular level the structure, function, and regulation of the thiazide diuretic- sensitive Na+:Cl- cotransporter present in the mammalian kidney. the specific objectives of this grant proposal are: (i) to clone the thiazide-sensitive Na+:Cl- cotransporter from rat kidney using a nucleotide probe generated from the cDNA encoding the thiazide-sensitive Na+:Cl- cotransporter that we have recently cloned from the urinary bladder of the winter flounder, Pseudopleuronectes americanus; (ii) to determine the nucleotide and amino acid sequences of the rat renal thiazide-sensitive cotransporter, to develop a topology model the cotransporter, and to compare the nucleotide and amino acid sequences of the Na+:Cl- cotransporter with other sequenced proteins, especially other cotransporter proteins; (iii) to assess the functional characteristics of the cotransporter as expressed in Xenopus laevis oocytes and to compare these characteristics with those of the flounder bladder transporter; (iv) to prepare polyclonal antibodies against the cotransporter which will be used to assess expression of the cotransporter protein; (v) to determine the distribution of the rat Na+:Cl- cotransporter mRNA and protein in both renal and non renal tissues by Northern and Western analyses, PCR of single renal tubules, and immunofluorescence; (vi) to assess whether isoforms of the thiazide- sensitive cotransporter exist in the rat kidney [esp. in the distal tubule and collecting duct] using PCR, and if isoforms can be identified, to determine the structure of these isoforms and to assess their functional characteristics. The results of these proposed studies will provide basic information on which we can begin to model [and the molecular probes on which to assess] the function and regulation of this important renal ion transporting protein at a molecular level.