STRUCTURAL BASIS OF ION CHANNEL OPENING

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有的孔，它们 控制膜内外的膜电位和离子浓度 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。这事很重要 为了了解许多神经系统疾病的分子基础， 包括癫痫、抑郁、神经毒性以及学习和记忆 赤字。离子通道的行为受电压、配体的控制 或G蛋白，它们决定了通道开放的时间。这 提案涉及以下关键问题：该机制是什么 哪些离子通道打开和关闭，这一行为是如何调节的？Drk1， 以从大鼠脑中克隆的延迟整流钾通道为模型 离子通道。功能通道由四个相同的子单元组成 每个包含六个假定的跨膜片段(S1-S6)，其 围绕着一个中心的水孔。S5之间的一个β-发夹环区 S6形成毛孔。膜电势是由正极感应的 带电的S4段，其运动控制在 这是一种无法理解的方式。关于异相通道的初步研究结果 而亚电导水平表明，单个亚基起着关键作用 在渠道开放和渗透中的作用。另外，两种谷氨酸残留物 显示了参与其中的侧翼S5，其对于K+通道是唯一的 在稳定开放状态方面。这些初步数据，连同 电压传感器(S4)和孔区的结构指认 (S5-S6)，为解决有关以下方面的基本问题奠定基础 开/关机构的结构基础及其调节 电压。该项目具体目标是：(I)使区域本地化 与通道开启和关闭密切相关的通道， (Ii)调查电压传感器如何与打开/关闭相耦合 机制，(3)确定各个亚基在其中的作用 电压传感和通道打开。定点突变体(点 突变和嵌合体)将被构建并在 非洲爪哇卵母细胞。突变体的单通道行为将是 采用膜片钳技术进行研究。一个重要的工具将是 通过串联构建或获得的异构体通道的研究 联合注射不同的CRNAs。

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.