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

电子中性型Na+-Cl-共转运体[包括布美他尼敏感的 Na+：K+：2Cl-和对噻嗪敏感的Na+：CL-共转运蛋白]包括 新发现的独特的蛋白质家族，参与 上皮盐吸收和分泌过程，以细胞体积表示 在细胞对有丝分裂因子的早期反应中的调节，以及在 血管内皮细胞和平滑肌细胞对血管内皮细胞的反应 血管活性物质。在哺乳动物的肾脏中，布美他尼敏感， Na+：K+：2Cl-共转运蛋白[BSC]在根尖细胞膜上含量最高 亨勒氏环[TAL]的粗大上行支，在那里它调节着Nac1 吸收，一个对尿液稀释和浓缩至关重要的过程。我们的 最近从大鼠外髓中克隆的BSC[BSC-R1]现在已经 为研究其结构、功能和调控提供了依据 在分子水平上研究这种重要的转运蛋白。离子和利尿剂 BSC-R1的抑制剂动力学及其活性调节因素 将在卵母细胞、分离的TAL小管和 稳定表达的细胞。共转运蛋白基因的定位 大鼠肾脏中的产物将通过Northern分析进行原位评估 单管杂交和聚合酶链式反应。产生多克隆抗体 针对共转运蛋白将被用来检测共转运蛋白 用Western分析和免疫细胞化学方法对大鼠肾脏进行免疫组织化学分析。角色 协同转运蛋白功能中的磷酸化-去磷酸化 学习。相关基因产物将在大鼠和小鼠中被鉴定 通过低严格性的cDNA文库筛选和聚合酶链式反应对肾脏进行筛选。这个 这些相关cDNA的功能特征将在#年确定 X.laevis卵母细胞。定点突变和嵌合体构建将 与同位素通量研究和电流研究结合使用 开始确定影响Na+的分子区域的测量， BSC-R1的K+和Cl-结合/转位及磷酸化 蛋白。这些研究的结果将为 了解Na+：K+：2Cl-共转运蛋白的功能及其在健康中的调节 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.