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

这个项目的目标是分离和定义一种膜蛋白 负责哺乳动物质膜中的K+通道活性 cell. K+通道广泛存在于动物细胞中， 参与细胞兴奋性，允许传输的行动 神经和肌肉中的潜能以及许多细胞类型对 各种神经激素激动剂。 K+的分子基础和作用机制 频道活动未知。 这项拨款建议使用细胞系 分离并表征为具有影响单个突变的PI， 质膜上特异的K+扩散途径。 它建议 将该途径的功能特征扩展到 贴片电极记录和双层膜电生理技术 重组 此外，它还提出了一种方法，允许隔离 介导这种转运活动的通道蛋白。 这取决于 最近在重组DNA技术上的创新。 体细胞 使用突变体形成的杂交体显示突变是显性的， 可选的。 因此，DNA介导的基因转移技术， 这项技术在过去已经成功地应用了很多次 为了分离其他显性选择基因，建议分离 这个突变基因影响了通道功能。 在隔离的每一步中 过程中，基因赋予的改变功能的K+通道 将通过测量细胞在体内存活的能力来测试。 在阈下低K+培养基中分离突变体。 这 分离的基因将作为探针，这将允许分离其 mRNA，并从其序列确定氨基酸序列 突变体和亲本转运蛋白的差异。 该项目是一个必要的 分离和表征负责 这一重要的运输机制，迄今为止， 定义. 由于K+通道的双层重构 囊泡的突变体已被证明是可行的，这是一个长期的目标， 建议的工作路线将是纯通道双层的研究 从分离的和特异性突变的基因体外合成的蛋白质， 更完整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.