STRUCTURAL BASIS OF ION CHANNEL OPENING

离子通道开口的结构基础

• 批准号: 2269486
• 负责人: ANTONIUS M VANDONGEN
• 金额: $ 18.89万
• 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 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2269486
• 关键词: Xenopus; Xenopus oocyte; alternatives to animals in research; 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定孔，它们 控制内部和外部的膜电位和离子浓度 细胞。 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 了解许多神经系统疾病的分子基础， 包括癫痫、抑郁症、神经毒性以及学习和记忆 赤字。 离子通道的行为由电压、配体控制 或 G 蛋白，它决定通道打开的时间。 这 提案解决了以下关键问题：其机制是什么？ 哪些离子通道打开和关闭，这种行为是如何调节的？德克1, 使用从大鼠大脑克隆的延迟整流 K+ 通道作为模型 离子通道。 功能通道由四个相同的亚基组成 每个包含六个假定的跨膜片段（S1-S6）， 围绕中央水孔。 S5 之间的 β-发夹环区域 S6形成孔。 膜电位是由正感测到的 带电 S4 段，其运动控制打开/关闭行为 方式不被理解。 异聚通道的初步结果 和亚电导水平表明各个亚基起着关键作用 通道开放和渗透的作用。 此外，还有两个谷氨酸残基 侧翼 S5，对于 K+ 通道是独特的，被证明参与其中 处于稳定的开放状态。这些初步数据，连同 电压传感器 (S4) 和孔隙区域的结构分配 （S5-S6），为解决有关 开/关机构的结构基础及其调节 电压。 该项目的具体目标是： (i) 本地化区域 关键参与通道打开和关闭的通道， (ii) 研究电压传感器如何与开/关耦合 机制，（iii）确定各个子单元的作用 电压传感和通道打开。 定点突变体（点 drk1 的突变和嵌合体）将被构建并表达于 爪蟾卵母细胞。 突变体的单通道行为将是 使用膜片钳技术进行研究。 一个重要的工具将是 通过串联构建体获得的异聚通道的研究或 不同cRNA的共注射。