Voltage-Gating in Bacterial Ion Channels

细菌离子通道中的电压门控

• 批准号: 7036500
• 负责人: ANA M CORREA
• 金额: $ 1.85万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036500
• 关键词: Bacillus; bacterial proteins; cell cell interaction; conformation; cysteine; electric field; electronic recording system; electrophysiology; fluorescent dye /probe; membrane activity; membrane model; protein localization; protein purification; protein reconstitution; protein structure function; sodium channel; spectrometry; voltage gated channel

DESCRIPTION (provided by applicant): Voltage-gated ion channels (VGC) are proteins found in the membranes of practically all cells, that through opening and closing (gating) events let ions flow through between the internal and external milieu of the cells acting as very fast signaling entities. The most characteristic and intriguing aspect of VGC is that their function is modulated by voltage. That means that the protein senses changes in the electrical field and responds by opening, possibly through a sequence of conformational changes. With the advent of high resolution electrical recording techniques combined with the molecular cloning and engineering of ion channel proteins, it has been possible to identify parts of VGC that would serve as voltage-sensors, which has led to proposal of several mechanistic models on how the voltage-sensing event is translated into channel opening. Yet, the molecular and physical natures of the events that take place during voltage-gating are not resolved. It is the long-term goal of this proposal to contribute a physical molecular model of how VGC gate by studying intramolecular distances at rest and while channels are open, using optical tools along with functional recordings. The recent cloning of a bacterial sodium channel, NaChBac, which can be produced in large quantities, purified and reconstituted into lipid membranes, provides a unique opportunity to address these questions in great molecular detail. The specific aims are: 1) Search for regions and residues that undergo distances changes associated with the voltage sensor and between the sensor and the gate region using lanthanide-based resonance energy transfer (LRET) in the reconstituted protein in different conformational states induced by voltage changes in proteoliposomes; 2) Measurement of distances in tandem proteins, purified and reconstituted, bearing a single donor acceptor pair using the same technique as in aim 1; and 3) Functional analysis of voltage sensing and gating using electrophysiology and site directed fluorescence and its correlation to structure and structural changes studied in aims 1 and 2. To measure distances, cysteines are introduced in different parts of the protein and a special sequence, an EF-hand motif that binds lanthanides, is introduced in another part of the same protein. Fluorescent probes are then used to label the cysteine group and are prompted to emit upon excitation of the lanthanide with light. Because groups will be placed in areas suspected to participate in voltage gating, these measurements are expected to contribute real molecular distances and information on molecular rearrangements occurring during voltage gating. VGC are particularly important in nerve and muscle cells because they determine cell excitability and participate in cell-to-cell communication. The results from this work should help in our understanding of a large number of VGC that are crucial in health and in drawing strategies to 1'ameliorate or perhaps eventually cure some illnesses that involve the dysfunction of this important family of channels.

描述（由申请人提供）：电压门控离子通道（VGC）是在几乎所有细胞的膜中发现的蛋白质，通过打开和关闭（门控）事件让离子在细胞的内外环境之间流动，作为非常快速的信号实体。VGC最具特色和吸引人的地方是它们的功能是由电压调制的。这意味着蛋白质感知到电场的变化，并通过打开反应，可能是通过一系列的构象变化。随着高分辨率电记录技术的出现，结合离子通道蛋白的分子克隆和工程技术，已经有可能确定VGC中作为电压传感器的部分，这导致了几个关于电压感应事件如何转化为通道打开的机制模型的提出。然而，在电压门控过程中发生的事件的分子和物理性质尚未解决。本提案的长期目标是利用光学工具和功能记录，通过研究静止和通道打开时的分子内距离，提供VGC门的物理分子模型。最近克隆的细菌钠通道NaChBac，可以大量生产，纯化和重组成脂质膜，提供了一个独特的机会来解决这些问题，在很大的分子细节。具体目标是：1)利用蛋白脂质体电压变化诱导的不同构象状态重构蛋白中镧系共振能量转移（LRET），寻找与电压传感器相关的、传感器与栅极区域之间距离变化的区域和残基；2)串联蛋白的距离测量，纯化和重组，携带单个供体受体对，使用与目标1相同的技术；3)利用电生理学和位点定向荧光分析电压传感和门控的功能及其与目标1和目标2中研究的结构和结构变化的相关性。为了测量距离，将半胱氨酸引入蛋白质的不同部分，并在同一蛋白质的另一部分引入一个特殊序列，即结合镧系元素的EF-hand基序。然后用荧光探针标记半胱氨酸基团，并在光激发镧系元素时提示发射。由于组将被放置在可能参与电压门控的区域，因此这些测量有望提供电压门控期间发生的分子重排的真实分子距离和信息。VGC在神经和肌肉细胞中特别重要，因为它们决定细胞的兴奋性并参与细胞间的通讯。这项工作的结果应该有助于我们了解大量对健康至关重要的VGC，并制定策略来改善或最终治愈涉及这一重要通道家族功能障碍的一些疾病。