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

这个项目的目标仍然是确定更新 负责速尿的膜蛋白的性质- 敏感的Na-K-Cl共转运系统（fs系统）。 该膜 运输系统广泛存在于动物细胞中，是一个中心的 红细胞体积调节和其他 细胞 它对称地介导一个电中性耦合通量， Na、K和Cl响应于它们的电化学总和 潜力 抑制该转运系统是通过 速尿和其他强效利尿剂作用于肾小管 细胞诱导肾脏的盐和水流失。 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。 在这份协议中，我们 我建议使用一个突变的细胞系， 用突变体DNA转化，和粘粒基因组DNA 克隆人从一个机器人中获救，所有这些机器人都携带着一个 这种突变现在已经被广泛地描述， 产生一个主导的，质量上的，可能是结构上的改变， FS系统;进一步纯化和表征蛋白质 并建立一个机制， 系统可能会运行。 突变蛋白质的性质 通过分析mRNA编码序列来确定 了 这种mRNA将被分离使用的基因，这已经 克隆到粘粒载体中作为探针。 分离的mRNA 一个序列用来产生蛋白质的主要氨基酸 序列，直接，并通过分析，表明其次要 和三级结构。 更重要的是，它提供了 具体的改变可以通过什么方式产生， 协同转运体的结构，以便以后分析其对 功能 函数分析可以通过表示 在培养细胞或卵母细胞中进行体外诱变信使， 并测定协同转运蛋白活性。 以这种方式， 蛋白质上的功能区域可以定义为： 最终必须融合到载体的解剖结构中， 动力学模型。 这个项目是必要的一步， 分离和表征负责 共转运机制是一种重要的转运机制 迄今为止还没有结构定义。

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.