STRUCTURAL BASIS OF ION CHANNEL OPENING

离子通道开口的结构基础

• 批准号: 3418489
• 负责人: ANTONIUS M VANDONGEN
• 金额: $ 14.96万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3418489
• 关键词: Xenopus; Xenopus oocyte; conformation; gene expression; molecular cloning; mutant; point mutation; potassium channel; protein sequence; protein structure function; radiotracer; recombinant DNA; site directed mutagenesis; structural biology; voltage /patch clamp; voltage gated channel

Ion channels play a crucial role in the physiology of the nervous system. By forming ion specific pores that open and close stochastically, they control membrane potential and ion concentrations both inside and outside the cell. They play an important role in excitability, neuromodulation, and neurotransmission. The long term goal is to explain the behavior of ion channels in terms of their molecular structure. This is important for understanding the molecular basis of many nervous system disorders, including epilepsy, depression, neurotoxicity, and learning and memory deficits. The behavior of ion channels is controlled by voltage, ligands or G-proteins, which determine the time the channel is open. This proposal addresses the following key questions: what is the mechanism by which ion channels open and close, how is this behavior regulated ? Drk1, a delayed rectifier K+ channel cloned from rat brain, is used as a model ion channel. The functional channel consists of four identical subunits each containing six putative transmembrane segments (S1-S6), that surround a central aqueous pore. A beta-hairpin loop region between S5 and S6 forms the pore. Membrane potential is sensed by the positively charged S4 segment, the movement of which control open/close behavior in way that is not understood. Preliminary results on heteromeric channels and subconductance levels suggest that the individual subunits play a key role in channel opening and permeation. Also, two glutamate residues flanking S5, which are unique for K+ channels, are shown to be involved in stabilizing the open state. These preliminary data, together with the structural assignment of both the voltage sensor (S4) and the pore region (S5-S6), set the stage for addressing basic questions concerning the structural basis of the open/close mechanism and its regulation by voltage. The specific aims of this project are: (i) localize regions of the channel that are critically involved in channel opening and closing, (ii) investigate how the voltage sensor is coupled to the open/close mechanism, (iii) determine what the role of the individual subunits is in voltage sensing and channel opening. Site-directed mutants (point mutations and chimaeras) of drk1 will be constructed and expressed in Xenopus oocytes. The single channel behavior of the mutants will be studied using patch clamp techniques. An important tool will be the study of heteromeric channels, obtained by tandem constructs or co-injection of different cRNAs.

离子通道在神经系统的生理学中起着至关重要的作用。 通过形成随机打开和关闭的离子特异性孔， 控制膜电位和内外离子浓度 牢房 它们在兴奋性，神经调节， 和神经传递。长期目标是解释 离子通道的分子结构。 这很重要 为了理解许多神经系统疾病的分子基础， 包括癫痫、抑郁、神经毒性、学习和记忆 赤字 离子通道的行为受电压、配体、 或G蛋白，它们决定通道开放的时间。 这 建议涉及以下关键问题： 哪些离子通道打开和关闭，这种行为是如何调节的？Drk1， 从大鼠脑中克隆的延迟整流钾通道被用作模型 离子通道 功能通道由四个相同的亚单位组成 每个含有六个推定的跨膜区段（S1-S6）， 围绕中心含水孔。 S5之间的β-发夹环区 而S6形成孔隙。 膜电位是由正的 充电S4段，其运动控制打开/关闭行为， 这种方式不被理解。 异聚体通道的初步研究结果 和亚电导水平表明，单个亚基起着关键作用， 在通道开放和渗透中的作用。 还有两个谷氨酸残基 侧翼S5对K+通道来说是独一无二的，被证明参与其中 稳定开放状态。这些初步数据，连同 - 电压传感器（S4）和孔区域两者的结构分配 （S5-S6），为解决有关 开闭机制的结构基础及其调控 电压. 这一项目的具体目标是：（一）将 这些通道在通道的打开和关闭中起着关键作用， (ii)研究电压传感器如何耦合到打开/关闭 机制，（iii）确定各个亚单位的作用是什么， 电压感测和通道打开。 定点突变体（点突变体） drk 1的突变和嵌合体）将在细胞中构建和表达 非洲爪蟾卵母细胞。 突变体的单通道行为将是 用膜片钳技术研究。 一个重要的工具将是 异聚体通道的研究，通过串联构建体或 共注射不同的cRNA。