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有变化，并通过通过一系列构象变化打开响应。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，可以鉴定VGC的一部分作为电压传感器。这些信息以及三个VGC的可用晶体结构，导致了几种电压感应机械模型的建议，以及如何将这些变化转化为通道开口。然而，在电压门控发生的事件的分子和物理性质尚未解决，这是正在进行的讨论和争议的问题。该提案的长期目标是通过使用光学工具以及功能记录来研究静止分子内距离和通道时如何通过研究静止分子内距离和开放通道时的物理分子模型的长期目标。我们将使用细菌钾通道KVAP，可以在细菌培养物中大量生产，纯化和重构为脂质膜，这为分子细节提供了独特的机会来解决这些问题。而且，我们还将使用精心研究的振动钾通道，哺乳动物肌肉通道和电压敏感的磷酸酶进行体内研究。具体目的是：目标1。确定在KVAP通道中的体外和振动通道中的体内距离及其对膜电势变化的变化，重点是电压传感域；并且，目标2。将体内距离测量扩展到其他依赖电压依赖性膜蛋白，包括哺乳动物钠通道（NAV1.4）和CI-VSP（电压依赖性磷酸酶）。为了衡量距离，将特定的灯笼结合标签（LBT结合，与terbium结合并充当供体）被编码为蛋白质的不同部分，并且是另一个遗传编码的标签（Hexa-histidine Tag）或半胱氨酸（用荧光探针标记）在同一蛋白质中引入了另一部分。 Terbium在lbt中编码的附近色氨酸残基激发后发出。由于供体和受体将被放置在涉嫌参与电压门口的区域中，因此预计这些测量值将贡献实际分子距离，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。这项工作的结果将扩大我们对健康至关重要的大量电压门控蛋白的理解，并有助于制定策略改善或最终治愈涉及这种重要蛋白质家族功能障碍的某些疾病。公共卫生相关性：使用电生理学和光谱法中最先进的技术（Lanthanide能量转移），结合分子生物学，我们在这里建议在体内确定膜蛋白的关键功能元件的运动，这些元素对细胞膜中电场的变化响应。这些蛋白质（离子通道）在大多数细胞中都发现，尤其是在神经和肌肉细胞中，当它们确定并在受刺激（化学或电气）挑战时确定并调节细胞的反应性。这些蛋白质中的天然突变通常会导致神经和肌肉相关的疾病称为通道病，因此要克服或治愈通道病，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构象的变化，以了解系统的失败。