Voltage-Gating in Bacterial Ion Channels

细菌离子通道中的电压门控

• 批准号: 6878466
• 负责人: ANA M CORREA
• 金额: $ 26.99万
• 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/6878466
• 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，其通过打开和关闭（门控）事件使离子在细胞的内部和外部环境之间流动，充当非常快速的信号实体。VGC最具特色和最有趣的方面是它们的功能受电压调制。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，已经有可能确定VGC的部分，将作为电压传感器，这导致了几个机制模型的建议，电压传感事件是如何转化为通道开放。然而，在电压门控期间发生的事件的分子和物理性质尚未解决。这是这项建议的长期目标，贡献一个物理分子模型的VGC门如何通过研究分子内距离在休息，而通道是开放的，使用光学工具沿着与功能记录。最近克隆的细菌钠通道，NaChBac，它可以大量生产，纯化和重组成脂质膜，提供了一个独特的机会，以解决这些问题在很大的分子细节。具体目标是：1）使用基于镧系元素的共振能量转移（LRET），在由脂蛋白体中的电压变化诱导的不同构象状态下的重构蛋白质中搜索经历与电压传感器相关的距离变化以及传感器和门区域之间的距离变化的区域和残基; 2）使用与目的1中相同的技术测量纯化和重构的串联蛋白中的距离，所述串联蛋白带有单个供体受体对;和3）使用电生理学和位点定向荧光的电压感测和门控的功能分析及其与目标1和2中研究的结构和结构变化的相关性。为了测量距离，在蛋白质的不同部分引入半胱氨酸，并在同一蛋白质的另一部分引入特殊序列，即结合镧系元素的EF-手基序。然后使用荧光探针标记半胱氨酸基团，并在用光激发镧系元素时发出荧光。由于将组放置在怀疑参与电压门控的区域中，因此这些测量预计将有助于获得电压门控期间发生的真实的分子距离和分子重排信息。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的VGC，这些VGC对健康至关重要，并有助于我们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。