Structure and Function of Pannexins: Activation Mechanism

Pannexins 的结构和功能：激活机制

基本信息

批准号：
9540128
负责人：
Toshimitsu Kawate
金额：
$ 4.31万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9540128
关键词：
Agonist Amino Acid Sequence Apoptosis Apoptotic Arthritis Binding Carbenoxolone Cells Chimeric Proteins Connexins Crystallization Crystallography Cysteine Cytochromes Data Distant Drug Design Electrophysiology (science)Epilepsy Event Excitatory Postsynaptic Potentials Family Foundations Functional disorder Gap Junctions Gated Ion Channel Goals Human body Immune Immune response Impairment In Vitro Inflammatory Inflammatory Response Invertebrates Investigation Ion Channel Ion Channel Gating Knockout Mice Lead Learning Mechanical Stress Mediating Membrane Memory Loss Methods Molecular Molecular Conformation Monoclonal Antibodies Movement Muramidase Mutagenesis N-Methyl-D-Aspartate Receptors N-terminal Neuroglia Neurons Pharmaceutical Preparations Pharmacology Physiological Play Positioning Attribute Rana Recruitment Activity Regulation Research Resolution Role Signal Transduction Solid Stimulus Stroke Structure Synapses T-Cell Activation Tail Techniques Testing base chronic pain cytokine design experimental study extracellular flexibility in vivo inhibitor/antagonist innovation insight nervous system disorder neurotransmission novel object recognition patch clamp postsynaptic protein E public health relevance reconstitution response screening sensor three dimensional structure tool voltage

项目摘要

DESCRIPTION (provided by applicant): Pannexins (Panx1-3) comprise a unique family of membrane channels that mediate a variety of immune responses including apoptotic cell clearance, cytokine secretion, and T-cell activation. Pannexins also play crucial roles in modulating synaptic activity and plasticity; for instance, Panx1 knockout mice display diminished excitatory postsynaptic potentials, resulting in impaired object recognition and spatial learning. Also, Panx1 promotes aberrant postsynaptic activity after repetitive NMDA receptor stimulation, suggesting that Panx1 plays important roles in epileptic seizures. These studies highlight the vital roles of pannexins throughout the body, however, little is understood about what activates pannexin channels, how they open and close, and what cellular events accompany these changes. Our long-term goal is to uncover the mechanisms underlying pannexin gating, regulation, and downstream signaling using structural and functional approaches. The specific aims of this proposal are approached through the following lines of investigation: 1) Solve the first crystal structure of a pannexin. We have identified a pannexin species that expresses at a high level, assembles into a stable and monodisperse oligomer in solution, and forms crystals that diffract to ~9Å. We will continue optimizing crystallization conditions and determine the firt crystal structure of a pannexin. We expect the atomic resolution structure of a pannexin will serve as a solid foundation for determining what constitutes the channel pore and how the opening and closing are controlled. 2) Dissect the mechanism underlying Panx1 channel gating. Our preliminary experiments suggest that Panx1--the most studied subtype thus far--most likely harbors an intrinsic voltage sensor. We also discovered that carbenoxolone, the most commonly used nonspecific antagonist of Panx1, allosterically inhibits the voltage-gated Panx1 channel activity. Taking advantage of this drug and electrophysiology, we will identify the key residues that govern voltage gating, defining their position relative to the membrane field, and probing how they move in response to voltage. Our proposed research is innovative not only because it will uncover the first atomic resolution structure of a pannexin, but also because it wil provide the first detailed molecular mechanism of this unique class of a voltage-gated membrane channel. Moreover, this is the first detailed investigation for the mechanism of action of any known pannexin inhibitors. We expect that our studies will open new avenues for developing long-awaited Panx1 specific agonists or antagonists through structure-based drug design or in vitro screening based on the conformational changes associated with Panx1 channel opening. Once such molecular tools become available, we hope to clarify why pannexin misexpression results in widespread dysfunctions in vivo, and in the long term, potential new strategies for treating devastating conditions such as chronic pain and epilepsy.

描述（由适用提供）：Pannexins（Panx1-3）包括一个独特的膜通道家族，这些家族介导了各种免疫调查，包括凋亡细胞清除，细胞因子分泌和T细胞激活。 Pannexins在调节突触活动和可塑性中也起着至关重要的作用。例如，panx1敲除小鼠显示出兴奋性的突触后电位减少，从而导致对象识别和空间学习受损。此外，PANX1在重复性NMDA受体刺激后促进异常的突触后活性，这表明PANX1在癫痫发作中起重要作用。这些研究强调了泛毒素在整个人体中的至关重要作用，但是，关于激活泛毒素通道，如何打开和关闭以及哪些细胞事件涉及这些变化的是什么几乎没有理解。我们的长期目标是使用结构和功能方法揭示Pannexin门控，调节和下游信号的基础机制。该提案的具体目的是通过以下投资来实现的：1）解决泛素的第一个晶体结构。我们已经确定了一种以高水平表达，在溶液中聚集成稳定和单分散的寡聚物，形成衍射至〜9Å的晶体。我们将继续优化结晶条件，并确定泛素的FIRT晶体结构。我们预计，泛氧蛋白的原子分辨率结构将成为确定构成渠道孔的构成以及如何控制开口和关闭的坚实基础。 2）解剖PANX1通道门控的机制。我们的初步实验表明，PANX1（这是研究型最大的亚型），很可能是固有的电压传感器。我们还发现，最常用的非特异性拮抗剂碳氧氧母在panx1的拮抗剂上构质抑制了电压门控panx1通道活性。利用该药物和电生理学，我们将确定控制电压门控的关键保留，定义其相对于膜场的位置，并探测它们如何响应电压移动。我们提出的研究具有创新性，这不仅是因为它将揭示泛蛋白的第一个原子分辨率结构，而且还因为它将提供这种独特的电压门控膜通道的第一个详细分子机制。此外，这是任何已知的泛素抑制剂作用机理的首次详细研究。我们预计我们的研究将通过基于结构的药物设计或基于与Panx1通道开放相关的构象变化来开发期待已久的PANX1激动剂或拮抗剂的新途径。一旦这种分子工具可用，我们希望阐明为什么泛氧化物pannexin misexpression会导致体内宽度障碍，并且从长远来看，潜在的新策略来治疗诸如慢性疼痛和癫痫病等毁灭性状况。