ISOLATION OF THE GENE ENCODING THE NA-K-C1 COTRANSPORTER

该项目更新的目标仍然是确定 负责呋塞米的膜蛋白的性质 敏感的Na-K-Cl共转运系统（fs系统）。 这个膜 转运系统广泛存在于动物细胞中，是细胞的中枢。 红细胞和其他细胞体积调节的组成部分 细胞。 它对称地介导电中性耦合通量 Na、K 和 Cl 响应其电化学总和 潜力。 该运输系统的抑制机制是 哪些速尿和其他强效利尿剂作用于肾小管 细胞诱导肾脏损失盐和水。 分子 Na、K、Cl共转运的基础和机制 离子穿过任何细胞膜是未知的。 在这笔赠款中我们 提议使用突变细胞系、多种细胞系 用突变体DNA和粘粒基因组DNA转化 从一个转化体中拯救出来的克隆，所有这些转化体都携带 现在已被广泛表征并显示的突变 产生主导性的、定性的、可能的结构性改变 金融服务体系；进一步纯化和表征蛋白质 组成这个系统的；并建立一个机制 系统可能会正常工作。 突变蛋白的性质将 通过分析编码mRNA的序列来确定 它。 该 mRNA 将使用该基因进行分离，该基因已经 被克隆到粘粒载体中，作为探针。 分离的 mRNA 序列用于产生蛋白质的初级氨基酸 直接序列，并通过分析表明其次要序列 以及三级结构。 更重要的是，它提供了一个 通过这种方式可以产生特定的改变 协同转运蛋白的结构，以便稍后分析它们对 功能。 泛函分析可以通过表达来实现 培养细胞或卵母细胞中的体外诱变信息， 并测定协同转运蛋白活性。 以这种方式，键的映射 蛋白质上的功能区域可以定义为： 最终必须融入载体的解剖结构 其行为的动力学模型。 该项目是一个必要的步骤 负责蛋白质的分离和表征 协同转运机制，一种重要的转运机制 迄今为止，它还没有得到结构定义。

The goal of this project in renewal remains to determine the nature of the membrane protein(s) responsible for the furosemide- sensitive Na-K-Cl cotransport system (fs system). This membrane transport system is widely found in animal cells and is a central component in the volume regulation of erythrocytes and other cells. It symmetrically mediates an electroneutral coupled flux of Na, K, and Cl in response to their summated electrochemical potential. Inhibition of this transport system is the mechanism by which furosemide and other potent diuretics act on renal tubule cells to induce salt and water loss by the kidney. The molecular basis and mechanism underlying the cotransport of Na, K, and Cl ions across any cell membrane is unknown. In this grant we propose to use a mutant cell line, a number of cell lines transformed with the mutant DNA, and a cosmid genomic DNA clone rescued from one transformant, all of which carry a mutation that now has been characterized extensively and shown to produce a dominant, qualitative, likely structural, alteration in the fs system; to purify and characterize further the protein(s) that make up this system; and to establish a mechanism by which the system might function. The nature of the mutant protein will be determined by analyzing the sequence of the mRNA encoding it. This mRNA will be isolated using the gene, which has already been cloned in a cosmid vector, as probe. The isolated mRNA sequence serves to yield the protein's primary amino acid sequence, directly, and, through analysis, suggests its secondary and tertiary structure as well. More important, it provides a means by which specific alterations can be produced in the cotransporter's structure for later analysis of their impact on function. A functional analysis can be achieved by expressing the in vitro-mutagenized message in either cultured cells or oocytes, and assaying cotransporter activity. In this manner a map of key functional regions on the protein can be defined: a functional anatomy of the carrier which must ultimately be melded into the kinetic model of its behavior. This project is a necessary step in the isolation and characterization of the protein(s) responsible for the cotransport mechanism, an important transport mechanism which has thus far eluded structural definition.