MOLECULAR BIOLOGY OF THE NA+/K+/2CL- COTRANSPORTER

电中性Na+-Cl-协同转运蛋白[包括布美他尼敏感的 Na+：K+：2Cl-和噻嗪敏感性Na+：Cl-共转运蛋白]包含一个 新认识的和不同的蛋白质家族，参与 上皮盐吸收和分泌过程，以细胞体积计 在细胞对促有丝分裂因子的早期反应中， 血管内皮细胞和平滑肌细胞对 血管活性剂 在哺乳动物肾脏中，布美他尼敏感， Na+：K+：2Cl-协同转运蛋白[BSC]在顶膜上最丰富， 亨利氏袢[TAL]的粗升支，在此介导NaCl 1 吸收，这是尿液稀释和浓缩的重要过程。 我们 最近从大鼠外髓质克隆BSC [BSC-r1]， 为研究其结构、功能和调控提供了依据 这个重要的转运蛋白的分子水平。 离子和利尿剂 BSC-r1的抑制剂动力学和调节活性的因素 将在卵母细胞、分离的TAL小管和 稳定转染的细胞。 协同转运蛋白基因的定位 将通过原位北方分析评估大鼠肾脏中的产物 单个小管的杂交和PCR。 产生的多克隆抗体 将用于检测协同转运蛋白 用Western分析和免疫细胞化学方法检测大鼠肾脏中的蛋白质水平。 的作用 磷酸化-去磷酸化的协同转运功能将是 研究了 相关基因产物将在大鼠和小鼠中鉴定 通过低严格性筛选cDNA文库和通过PCR对肾脏进行分析。 的 这些相关cDNA的功能特征将在 X.卵母细胞 定点诱变和嵌合体构建体将 与同位素通量研究和电流结合使用 测量开始以识别影响Na+的分子区域， BSC-r1的K+和Cl-结合/易位和磷酸化 蛋白 这些研究的结果将为 了解Na+：K+：2Cl-协同转运蛋白在健康中的功能和调节 和疾病

Electroneutral Na+-Cl- cotransporters [including bumetanide-sensitive Na+:K+:2Cl- and thiazide-sensitive Na+:Cl- cotransporters] comprise a newly recognized and distinct family of proteins that participate in epithelial salt absorptive and secretory processes, in cell volume regulation, in the early response of cells to mitogenic factors, and in the response of vascular endothelial and smooth muscle cells to vasoactive agents. In the mammalian kidney the bumetanide-sensitive, Na+:K+:2Cl- cotransporter [BSC] is most abundant on apical membranes of the thick ascending limb of Henle's loop [TAL] where it mediates NaC1 absorption, a process vital to urine dilution and concentration. Our recent cloning of the BSC [BSC-r1] from rate outer medulla has now provided the basis for studying the structure, function and regulation of this important transporter at a molecular level. The ion and diuretic inhibitor kinetics, and the factors regulating activity, of the BSC-r1 cotransporter will be determined in oocytes, isolated TAL tubules and stably transfected cells. The localization of the cotransporter gene products in the rat kidney will be assessed by Northern analysis, in situ hybridization and PCR of single tubules. Polyclonal antibodies produced against the cotransporter will be used to detect cotransporter protein in rat kidney using Western analysis and immunocytochemistry. The role of phosphorylation-dephosphorylation in cotransporter function will be studied. Related gene products will be identified in rat and mouse kidney by low stringency screening of cDNA libraries and by PCR. The functional characteristics of these related cDNAs will be determined in X. laevis oocytes. Site-directed mutagenesis and chimera constructs will be used in combination with isotopic flux studies and electrical current measurements to begin to identify the molecular regions influencing Na+, K+ and Cl- binding/translocation and phosphorylation of the BSC-r1 protein. The results of these studies will provide a molecular basis for understanding Na+:K+:2Cl- cotransporter function and regulation in health and disease.