Voltage-Gating in Bacterial Ion 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，通过打开和关闭（门控）事件让离子在细胞的内部和外部环境之间流动，作为非常快速的信号实体。VGC最具特色和吸引人的地方是它们的功能是由电压调制的。这意味着蛋白质感知到电场的变化，并通过一系列构象变化来打开。随着高分辨率电记录技术的出现，结合分子克隆和离子通道蛋白的工程，已经有可能识别VGC中作为电压传感器的部分。这些信息以及三种VGC的晶体结构，已经导致了几种电压传感机制模型的提出，以及这些变化如何转化为通道打开。然而，在电压门控过程中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。本提案的长期目标是利用光学工具和功能记录，通过研究静止和通道打开时的分子内距离，提供VGC门的物理分子模型。我们将使用细菌钾通道KVAP，它可以在细菌培养中大量产生，纯化并重组为脂质膜，这为从分子细节上解决这些问题提供了独特的机会。并且，我们还将使用经过充分研究的Shaker钾通道，哺乳动物肌肉通道和电压敏感磷酸酶进行体内研究。具体目标是：目标1。测定体外KVAP通道和体内Shaker通道分子内距离及其对膜电位变化的响应，重点关注电压传感域；和，目标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）和电压依赖性磷酸酶Ci-VSP。为了测量距离，一个特定的镧系结合标签（LBT，结合铽并作为供体）被编码到蛋白质的不同部分，另一个遗传编码标签（六组氨酸标签）或半胱氨酸（用荧光探针标记）被引入到同一蛋白质的另一部分作为受体。铽在附近编码在LBT中的色氨酸残基的激发下发出。由于供体和受体将被放置在可能参与电压门控的区域，因此这些测量有望提供电压门控期间发生的分子重排的真实分子距离和信息。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。这项工作的结果将扩大我们对大量对健康至关重要的电压门控蛋白的理解，并将有助于制定策略来改善或最终治愈涉及这一重要蛋白质家族功能障碍的一些疾病。公共卫生相关性：利用最先进的电生理学和光谱学技术（镧系元素能量转移），结合分子生物学，我们建议在体内确定膜蛋白的关键功能元件的运动，这些功能元件响应细胞膜上电场的变化。这些蛋白质（离子通道）存在于大多数细胞中，尤其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。这些蛋白质的自然突变经常导致神经和肌肉相关疾病，称为通道病，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，以及在这个过程中发生的构象变化，以了解系统是如何失败的。

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.