Voltage-Gating in Bacterial Ion Channels

细菌离子通道中的电压门控

• 批准号: 7581479
• 负责人: ANA M CORREA
• 金额: $ 35.68万
• 依托单位: UNIVERSITY OF CHICAGO
• 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 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7581479
• 关键词: Address; Area; Arts; Avidin; Binding; Biotin; Cell Communication; Cell membrane; Cells; Characteristics; Charge; Chemicals; Chimera organism; Cysteine; Data; Dependence; Disease; Electrophysiology (science); Elements; Energy Transfer; Engineering; Environment; Event; Fluorescent Probes; Functional disorder; Gated Ion Channel; Goals; Health; Histidine; In Vitro; Ion Channel; Ion Channel Gating; Ion Channel Protein; Ions; Label; Lanthanoid Series Elements; Lead; Mammalian Cell; Measurable; Measurement; Measures; Membrane; Membrane Lipids; Membrane Potentials; Membrane Proteins; Modeling; Molecular; Molecular Biology; Molecular Cloning; Molecular Conformation; Molecular Models; Movement; Muscle; Muscle Cells; Mutation; Nature; Nerve; Neurologic; Noise; Oocytes; Optics; Phosphoric Monoester Hydrolases; Positioning Attribute; Potassium Channel; Process; Protein Family; Proteins; Relative (related person); Resolution; Rest; Rotation; Shaker potassium channel; Signal Transduction; Sodium Channel; Source; Spectrum Analysis; Stimulus; Structure; System; Techniques; Terbium; Testing; Toxin; Translating; Translations; Tryptophan; Width; Work; base; electric field; in vivo; molecular modeling; molecular rearrangement; physical state; public health relevance; reconstitution; research study; response; sensor; tool; voltage

DESCRIPTION (provided by applicant): Voltage-gated ion channels (VGC) are proteins found in the membranes of practically all cells and 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 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 serve as voltage-sensors. This information along with the available solved crystal structures of three VGC, has led to the proposal of several mechanistic models of voltage-sensing and how these changes are translated into channel opening. Yet, the molecular and physical natures of the events that take place during voltage-gating are not resolved and are the matter of ongoing discussion and controversy. It is the long-term goal of this proposal to contribute a physical molecular model of how VGC gate by studying intra-molecular distances at rest and while channels are open, using optical tools along with functional recordings. We will use the bacterial potassium channel, KVAP, which can be produced in large quantities in bacterial culture, purified and reconstituted into lipid membranes, which provides a unique opportunity to address these questions in molecular detail. And, we will also use the well-studied Shaker potassium channel, a mammalian muscle channel and a voltage-sensitive phosphatase for in vivo studies. The specific aims are: Aim 1. To determine in vitro in KVAP channels and in vivo in Shaker channels intra-molecular distances and their changes in response to membrane potential changes focusing on the voltage sensing domain; and, Aim 2. To extend in vivo distance measurements to other voltage-dependent membrane proteins, including a mammalian sodium channel (NaV1.4) and the Ci-VSP, a voltage-dependent phosphatase. To measure distances, a specific Lanthanide Binding Tag (LBT, that binds terbium and acts as a donor) is encoded into different parts of the protein and either another genetically encoded tag (a hexa-histidine tag) or a cysteine (to be labeled with a fluorescent probe) are introduced in another part of the same protein to act as acceptor. The terbium emits upon excitation of a nearby tryptophan residue encoded in the LBT. Because the donor and acceptor 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 will broaden our understanding of a large number of voltage-gated proteins that are crucial in health and shall help to draw strategies to ameliorate or perhaps eventually cure some illnesses that involve the dysfunction of this important family of proteins. PUBLIC HEALTH RELEVANCE: Using state-of-the-art techniques in electrophysiology and spectroscopy (lanthanide energy transfer), combined with molecular biology, we propose here to determine in vivo the movement of crucial functional elements of membrane proteins that respond to changes in the electric field across the cell membrane. These proteins (ion channels) are found in most cells but especially in nerve and muscle cells where they determine and modulate the cells' responsiveness when challenged by a stimulus (chemical or electrical). Natural mutations in these proteins often lead to neurological and muscle related diseases known as channelopathies, therefore to overcome or cure channelopathies there is a need to understand at a molecular level how these proteins sense the environment and what changes in conformation occur during this process to understand how the system fails.

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，并且通过打开和关闭（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，并通过一系列构象变化来响应开放。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的部分作为电压传感器。这些信息沿着三种VGC的可用解析晶体结构，导致提出了电压感测的几种机制模型以及这些变化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，并且是正在进行的讨论和争议的问题。这是该提案的长期目标，贡献一个物理分子模型的VGC门如何通过研究分子内的距离在休息，而通道是开放的，使用光学工具沿着与功能记录。我们将使用细菌钾通道，KVAP，它可以在细菌培养中大量产生，纯化和重组成脂质膜，这提供了一个独特的机会来解决这些问题的分子细节。而且，我们还将使用研究充分的Shaker钾通道，哺乳动物肌肉通道和电压敏感性磷酸酶进行体内研究。具体目标是：目标1。在体外确定KVAP通道和在体内确定Shaker通道的分子内距离及其响应于膜电位变化的变化，重点是电压传感域;以及，目的2。将体内距离测量扩展到其他电压依赖性膜蛋白，包括哺乳动物钠通道（NaV1.4）和Ci-VSP，电压依赖性磷酸酶。为了测量距离，将特定的镧系元素结合标签（LBT，其结合铽并充当供体）编码到蛋白质的不同部分中，并且将另一个遗传编码标签（六组氨酸标签）或半胱氨酸（待用荧光探针标记）引入相同蛋白质的另一部分中以充当受体。铽在激发LBT中编码的附近色氨酸残基时发射。因为供体和受体将被放置在怀疑参与电压门控的区域中，所以这些测量预计将贡献关于在电压门控期间发生的分子重排的真实的分子距离和信息。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。这项工作的结果将拓宽我们对大量电压门控蛋白的理解，这些蛋白对健康至关重要，并将有助于制定策略来改善或最终治愈一些涉及这一重要蛋白质家族功能障碍的疾病。公共卫生相关性：使用国家的最先进的技术在电生理学和光谱学（镧系能量转移），结合分子生物学，我们建议在这里确定在体内的运动的关键功能元件的膜蛋白，响应在整个细胞膜上的电场的变化。这些蛋白质（离子通道）存在于大多数细胞中，但特别是在神经和肌肉细胞中，当受到刺激（化学或电）时，它们决定和调节细胞的反应性。这些蛋白质中的自然突变通常会导致被称为通道病的神经和肌肉相关疾病，因此为了克服或治愈通道病，需要在分子水平上了解这些蛋白质如何感知环境以及在此过程中发生了什么构象变化，以了解系统如何失败。