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

这项资助的长期目标是在分子水平上了解 水平噻嗪类利尿剂的结构、功能和调节- 存在于哺乳动物肾脏中的敏感Na+：Cl-协同转运蛋白。 的 这项拨款建议的具体目标是：（i） 大鼠肾脏噻嗪敏感性Na+：Cl-协同转运蛋白的研究 从编码噻嗪敏感性 Na+：Cl-协同转运蛋白，我们最近从尿中克隆 膀胱的冬比目鱼，Pseudopleuronectes americanus;（ii）， 测定大鼠肾组织中的核苷酸和氨基酸序列， 噻嗪敏感的协同转运蛋白，开发拓扑模型， 共转运蛋白，并比较核苷酸和氨基酸序列的 Na+：Cl-协同转运蛋白与其他已测序蛋白，特别是其他 协同转运蛋白;（iii）评估功能特征 非洲爪蟾卵母细胞中表达的协同转运蛋白， 将这些特征与比目鱼膀胱的特征进行比较 （iv）制备针对所述转运蛋白的多克隆抗体; 共转运蛋白，其将用于评估 协同转运蛋白;（v）确定大鼠的分布 肾脏和非肾脏Na+：Cl-转运蛋白mRNA和蛋白 通过北方和西方分析，单个肾小管的PCR， （vi）评估噻嗪类的同种型是否- 在大鼠肾脏中存在一种敏感的协同转运蛋白， 小管和集合管]，如果可以鉴定同种型， 以确定这些异构体的结构并评估它们的 功能特性 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 分子探针，以评估]的功能和调节，这 在分子水平上是一种重要的肾离子转运蛋白。

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.