ISOLATION AND CHARACTERIZATION OF A MAMMALIAN K+ CHANNEL

该项目的目标是更新，以隔离和定义 与血浆K+通道活性有关的膜蛋白 哺乳动物细胞的膜。K+通道广泛存在于动物体内 细胞是细胞兴奋性的中心参与者，允许 动作电位在神经和肌肉中的传递及其反应 多种细胞类型对多种神经激素激动剂的作用。分子 K+通道活性的基础和机制尚不清楚。这笔赠款 建议使用一种分离的细胞系，其特征是P.I. 具有影响单个特定K+通道的突变，并且 质膜上的K+扩散途径。它建议延长 此渠道的功能特征通过 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 基于最近发展起来的重组DNA技术的创新。 体细胞杂交和DNA介导的基因转移实验 已经证明了这种突变是显性的、可选择的和 可转让的。从突变体中形成了粘粒基因组文库 DNA和突变的通道基因已被定位到特定的体积 在该文库中通过迭代过程进行同胞选择。在每一个中 分离程序的步骤，基因赋予的改变 K+通道的功能是通过测量 细胞在亚阈值低K+培养液中存活，通过测定K+ 磁通，并带有单声道记录。这个分离的基因将服务于 作为一种探针，它将允许分离其消息的cdna，并从 ITS序列，突变株氨基酸序列的测定 和亲本转运蛋白。这一项目是 与此相关的蛋白质的分离和鉴定 重要的运输机制，到目前为止还没有弄清楚分子 定义。由于双层重建和贴片电极记录 演示K通道的改变，这是 这项拟议的工作将是在双层、卵母细胞和 分离的和合成的通道蛋白的转染细胞 特别是突变的基因，更完整地定义了结构和 这些重要的生理机制的作用，希望， 进一步扩展了它们在生理上的作用的特征 流程。

The goal of this project is renewal remains 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 P.I. that has a mutation affecting a single specific K+ channel and diffusional K+ pathway in the plasma membrane. It proposes to extend the functional characterization of this channel 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 hybridization and DNA-mediated gene transfer experiments have demonstrated the mutation to be dominant, selectable, and transferable. A cosmid genomic library has been formed from the mutant DNA and the mutant channel gene has been localized to a specific volume in this library through the iterative process of sib selection. In each step of the isolation procedure, the genetically conferred altered function of the K+ channel is tested by measuring the ability of the cells to survive in the subthreshold low K+ medium, by determining K+ fluxes, and with single channel recording. This isolated gene will serve as a probe which will allow the isolation of its message's cDNA 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 and patch electrode records demonstrate the alteration of the K channel, an ultimate objective of this proposed line of work would be the study in bilayers, oocuytes, and transfected cells of channel proteins synthesized from isolated and specifically mutated genes, more completely defining the structure and function of these important physiological mechanisms and, it is hoped, further extending a characterization of their roles in physiological processes.