ISOLATION AND CHARACTERIZATION OF A MAMMALIAN K+ CHANNEL

哺乳动物 K 通道的分离和表征

• 批准号: 3286897
• 负责人: J. Jay Gargus
• 金额: $ 14.14万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286897
• 关键词: Xenopus; action potentials; cell membrane; complementary DNA; egg /ovum; gene expression; genetic library; genetic manipulation; genetic transcription; hybrid cells; laboratory mouse; lipid bilayer membrane; membrane potentials; membrane proteins; membrane reconstitution /synthesis; messenger RNA; molecular cloning; mutant; nucleic acid sequence; point mutation; potassium channel; protein biosynthesis; protein sequence; protein structure function; recombinant DNA; restriction mapping; structural genes; tissue /cell culture; transfection; transposon /insertion element; voltage /patch clamp

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.

该项目的目的是更新保留依据，以隔离和定义 负责血浆中K+通道活性的膜蛋白 哺乳动物细胞的膜。 K+通道在动物中广泛发现 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播以及反应 多种细胞类型的各种神经激动剂。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P.I的分离和特征的细胞系的使用。 它具有影响单个特定的K+通道的突变，并且 质膜的扩散K+途径。它建议扩展 通过该通道的功能表征 斑块电极记录和双层的电生理技术 重组。此外，它提出了一种允许隔离的方法 介导这种转运活性的通道蛋白。这依赖 最近开发了重组DNA技术的创新。 体细胞杂交和DNA介导的基因转移实验 已经证明该突变为主导，可选和 可转移。宇宙基因组库是由突变体形成的 DNA和突变通道基因已定位到特定体积 在此库中，通过SIB选择的迭代过程。每个 隔离程序的步骤，基因赋予的改变 通过测量K+通道的功能测试 通过确定K+，细胞在亚阈值低K+培养基中生存 通量，并带有单个通道记录。这个孤立的基因将服务 作为探针，将允许隔离其信息的cDNA，并从 它的序列是突变体的氨基酸序列的测定 和家长运输蛋白。这个项目是在 负责蛋白质的隔离和表征 重要的运输机制，到目前为止，它已经避开了分子 定义。由于双层重建和补丁电极记录 演示K渠道的改变，这是 拟议的工作将是对双层，卵巢和 由分离的和 具体突变的基因，更完全定义结构和 这些重要的生理机制的功能，并希望， 进一步扩展其在生理学中角色的表征 过程。