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，通过打开和闭合（门控）事件，使离子在细胞的内部和外部环境之间流动起来，这些细胞的内部和外部环境充当非常快速的信号传导实体。 VGC最有特征和有趣的方面是它们的功能是由电压调节的。这意味着蛋白质在电场中的感觉变化，并通过打开，可能是通过一系列构象变化的响应。随着高分辨率电记录技术的出现，结合了离子通道蛋白的分子克隆和工程，可以识别VGC的一部分，这些部分将用作电压传感器，这导致了有关如何将电压感应事件转换为通道开放的多种机械模型。然而，在电压门控发生的事件的分子和物理性质尚未解决。该提案的长期目标是通过使用光学工具以及功能性记录来研究静止分子内距离和通道开放时如何通过研究静止分子距离和通道开放的物理分子模型。最近将细菌钠通道的克隆nachbac可以大量生产，纯化和重构为脂质膜，提供了一个独特的机会，可以大量的分子细节解决这些问题。具体目的是：1）搜索与电压传感器相关的距离变化以及传感器和栅极区域之间的区域和残基，并在不同构型蛋白质中使用基于灯笼的谐振能量转移（LRET）在不同构型蛋白质中受到蛋白质脂质体的电压变化诱导的不同构象状态； 2）使用与AIM 1相同的技术携带单个供体受体对的串联蛋白中的距离，并具有单个供体受体对； 3）使用电生理学和位点的荧光对电压传感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中引入了半胱氨酸，并且在蛋白质的不同部分中引入了一种特殊的EF序列，一种EF手段，一种结合Lanthanides的EF序列，在同一蛋白质的另一部分中引入了lanthanides。然后使用荧光探针标记半胱氨酸基团，并在光线激发时发出发射。因为将组放置在涉嫌参与电压门口的区域，因此预计这些测量值将有助于实际分子距离，并了解电压门控过程中发生的分子重排的信息。 VGC在神经和肌肉细胞中尤为重要，因为它们决定了细胞兴奋性并参与细胞间通信。这项工作的结果应有助于我们理解大量在健康中至关重要的VGC，并在绘制1'Mealiestion或可能最终治愈涉及这种重要渠道功能障碍的疾病中。