ISOLATION & CHARACTERIZATION OF A MAMMALIAN K+ CHANNEL

这个项目的目标是更新仍然是隔离和定义一个 负责血浆中K+通道活性的膜蛋白 哺乳动物细胞的膜。K+通道广泛存在于动物体内 细胞，是细胞兴奋性的核心参与者， 动作电位在神经和肌肉中的传递和反应 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 提出了使用分离的细胞系并通过P.I. 具有影响单个特定K+通道的突变， 质膜中的扩散K+途径。它建议延长 该通道的功能表征通过 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。这依赖 最近在重组DNA技术上的创新。 体细胞杂交和DNA介导的基因转移实验 已经证明突变是显性的，可选择的， 可转让的从突变体中构建了粘粒基因组文库 DNA和突变通道基因已经被定位到特定的体积 在这个文库中通过同胞选择的迭代过程。在每个 在分离程序的步骤中， K+通道的功能是通过测量的能力， 细胞在阈下低K+培养基中存活，通过测定K+ 流量和单通道记录。这个分离的基因 作为探针，这将允许分离其信息的cDNA， 其序列，突变体氨基酸序列的测定 和亲本转运蛋白。这个项目是一个必要的步骤， 分离和表征负责此的蛋白质 迄今为止，分子生物学还没有发现重要的转运机制， 定义.由于双层重构和贴片电极记录 展示K通道的改变，这是 这项拟议的工作将是研究双层，卵细胞， 从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， 这些重要的生理机制的功能，希望， 进一步扩展了它们在生理学中的作用的特征， 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.