Voltage-Gating in Bacterial Ion Channels

细菌离子通道中的电压门控

• 批准号: 6732321
• 负责人: ANA M CORREA
• 金额: $ 26.82万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6732321
• 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(申请人提供)：电压门控离子通道(VGC)是存在于几乎所有细胞膜中的蛋白质，通过开启和关闭(门控)事件，离子作为非常快的信号实体在细胞的内部和外部环境之间流动。VGC的最大特点和最吸引人的方面是它们的功能受电压的调节。这意味着蛋白质感知电场的变化，并通过打开做出反应，可能是通过一系列构象变化。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，已经有可能识别VGC中作为电压传感器的部分，这导致了关于电压敏感事件如何转化为通道开放的几个机制模型的提出。然而，在电压门控期间发生的事件的分子和物理性质还没有解决。这项提议的长期目标是通过研究静止和通道打开时的分子内距离，利用光学工具和功能记录，为VGC如何门提供一个物理分子模型。最近克隆的细菌钠通道NaChBac可以大量生产、纯化并重组成脂膜，为从分子上详细解决这些问题提供了一个独特的机会。具体的目的是：1)在重组蛋白质中使用基于镧系元素的共振能量转移(LRET)来搜索经历与电压传感器相关的距离变化的区域和残基，以及在传感器和门区域之间的距离变化；2)使用与目标1相同的技术来测量纯化和重组的串联蛋白质中的距离，所述串联蛋白质带有与目标1相同的供体受体对；3)利用电生理学和定点荧光对电压传感和门控的功能进行分析及其与结构和结构变化的相关性在AIMS 1和2中进行了研究。为了测量距离，在蛋白质的不同部分引入了半胱氨酸，在同一蛋白质的另一部分引入了一个特殊的序列-EF-Hand基序，该基序与稀土元素结合。然后用荧光探针标记半胱氨酸基，并在镧系元素用光激发时被提示发射。由于小组将被放置在被怀疑参与电压门控的区域，这些测量有望提供真实的分子距离和关于在电压门控期间发生的分子重排的信息。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理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一些涉及这一重要通道家族功能障碍的疾病。