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

该项目的目标是分离和确定一种膜蛋白。 负责哺乳动物质膜上K+通道的活动 手机。K+通道广泛存在于动物细胞中，是细胞的中枢 参与细胞兴奋性，允许动作的传递 神经和肌肉中的电位以及多种细胞类型对 多种神经激素激动剂。K+的分子基础和作用机制 通道活动未知。这项拨款建议使用一种细胞系 分离并以具有影响单个基因突变的PI为特征 质膜上特异的K+扩散途径。它提议 通过扩展这一途径的功能特征 膜片电极记录和双层膜的电生理技术 重建。此外，还提出了一种允许隔离的方法 介导这一转运活动的通道蛋白。这依赖于 最近在重组DNA技术方面取得了创新。体细胞 使用该突变体形成的杂交后代显示该突变是显性的 可选。因此，DNA介导的基因转移技术，一 一种在过去多次成功应用的技术 为了分离其他显性可选择基因，建议分离 该突变基因影响通道功能。在隔离的每一步 程序，遗传赋予的K+通道功能的改变 将通过测量细胞在体内存活的能力进行测试 用于分离突变体的亚阈值低K+培养基。这 分离的基因将作为探针，允许分离其 并根据ITS序列测定了其氨基酸序列 突变体和亲本运输蛋白。这个项目是必要的 分离和鉴定与蛋白质有关的步骤 这一重要的运输机制到目前为止还不清楚分子 定义。由于膜上K+通道的双层重构 突变体的囊泡已经被证明是可行的，这是这一长期目标 提出的工作路线将是对纯通道双层的研究 从分离的和特定突变的基因体外合成的蛋白质， 更全面地定义这些重要的结构和功能 生理机制。

The goal of this project is to isolate and define a membrane protein responsible for K+ channel activity in the plasma membrane of a mammalian cell. K+ channels are found widely in animal cells and are central participants in cellular excitability, allowing transmission of the action potential in nerve and muscle and the response of many cell types to a variety of neurohormonal agonists. The molecular basis and mechanism of K+ channel activity is unknown. This grant proposes the use of a cell line isolated and characterized by the PI that has a mutation affecting a single specific diffusional K+ pathway in the plasma membrane. It proposes to extend the functional characterization of this pathway through the electrophysiological techniques of patch electrode recording and bilayer reconstitution. In addition, it proposes a method to allow the isolation of the channel protein mediating this transport activity. This relies upon recently developed innovations in recombinant DNA technology. Somatic cell hybrids formed using the mutant reveal the mutation to be dominant and selectable. Therefore the technique of DNA-mediated gene transfer, a technique which has been successfully applied a number of times in the past to the isolation of other dominant selectable genes, is proposed to isolate this mutant gene affecting channel function. In each step of the isolation procedure, the genetically conferred altered function of the K+ channel will be tested by measuring the ability of the cells to survive in the subthreshold low K+ medium from which the mutant was isolated. This isolated gene will serve as a probe which will allow the isolation of its mRNA and, from its sequence, the determination of the amino acid sequence of the mutant and parent transport proteins. This project is a necessary step in the isolation and characterization of the protein responsible for this important transport mechanism which has thus far eluded molecular definition. Since bilayer reconstitution of K+ channels from membrane vesicles of the mutant has proven feasible, a long-term objective of this proposed line of work would be the study in bilayers of pure channel proteins synthesized in vitro from isolated and specifically mutated genes, more completely defining the structure and function of these important physiological mechanisms.