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

这个更新项目的目标仍然是确定 引起速尿的膜蛋白的性质(S)- 敏感的钠-钾-氯共转运系统(FS系统)。这层膜 运输系统广泛存在于动物细胞中，是一种重要的 红细胞体积调节中的成分和其他 细胞。它对称地调节一个电子中和耦合的通量 钠、钾和氯对其总和电化学的响应 潜力。抑制这种运输系统的机制是通过 哪些速尿和其他有效利尿剂作用于肾小管 细胞通过肾脏来诱导盐分和水分的流失。分子 钠、钾、氯共转运的基础和机制 任何细胞膜上的离子都是未知的。在这笔赠款中，我们 建议使用一个突变细胞系，多个细胞系 用突变的DNA和粘粒基因组DNA转化 从一个转化子中解救出来的克隆，所有的转化子都带有一个 突变现在已经被广泛描述并显示出 在…中产生主要的、定性的、可能是结构性的变化 FS系统；进一步纯化和鉴定蛋白质(S) 组成这一制度；并建立一种机制 系统可能会正常工作。突变蛋白的性质将会 通过分析编码基因的序列来确定 它。这一信使核糖核酸将使用基因分离，该基因已经 被克隆到粘粒载体中，作为探针。分离出的mRNA 序列用于产生蛋白质的主要氨基酸 序列，直接，并通过分析表明它的次要 三级结构也是如此。更重要的是，它提供了一个 可用来在 共转运体的结构，用于稍后分析其对 功能。函数分析可以通过表达 在培养细胞或卵母细胞中的体外诱变信息中， 并分析共转运蛋白的活性。以这种方式，关键字的映射 蛋白质上的功能区可以定义为：一个功能区 对载体的解剖最终必须融入到 其行为的动力学模型。这一项目是 致病蛋白(S)的分离与鉴定 共转运机制--一种重要的转运机制 到目前为止，这一点一直没有得到结构性定义。

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.