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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：
8862682
负责人：
Toshimitsu Kawate
金额：
$ 30.16万
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/8862682
关键词：
Agonist Amino Acid Sequence Apoptosis Apoptotic Arthritis Binding Carbenoxolone Cells Chimeric Proteins Connexins Crystallization Crystallography Cysteine Cytochromes Data Drug Design Electrophysiology (science)Epilepsy Event Excitatory Postsynaptic Potentials Family Foundations Functional disorder Gap Junctions Gated Ion Channel Goals Human body Immune Immune response In Vitro Inflammatory Inflammatory Response Invertebrates Investigation Ion Channel Knockout Mice Lead Learning Mechanical Stress Mediating Membrane Memory Loss Methods Molecular Monoclonal Antibodies Movement Muramidase Mutagenesis N-Methyl-D-Aspartate Receptors N-terminal Neuroglia Neurons Peptide Sequence Determination Pharmaceutical Preparations Physiological Play Positioning Attribute Rana Recruitment Activity Regulation Relative (related person)Research Resolution Role Signal Transduction Solid Solutions Stimulus Stroke Structure Synapses T-Cell Activation Tail Techniques Testing base cellular transduction chronic pain cytokine design extracellular flexibility in vivo inhibitor/antagonist innovation insight nervous system disorder neurotransmission novel object recognition patch clamp postsynaptic public health relevance reconstitution research study 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.