Structure and Function of Pannexins: Activation Mechanism

Pannexins 的结构和功能：激活机制

• 批准号: 10445505
• 负责人: Toshimitsu Kawate
• 金额: $ 32.87万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445505
• 关键词: Adrenergic Receptor; Biological Assay; Blood Vessels; C-terminal; Caspase; Cell Line; Cell Membrane Permeability; Cells; Cryoelectron Microscopy; Disease; Electrophysiology (science); Extracellular Domain; Family; Foundations; G-Protein-Coupled Receptors; Genetic; Goals; Human body; Hypertension; Immune response; Inflammation; Inflammatory; Ion Channel; Ion Channel Gating; Knock-out; Knowledge; Lead; Ligands; Link; Mediating; Molecular; Molecular Conformation; N-Methyl-D-Aspartate Receptors; N-terminal; Papaverine; Pharmacology; Phospholipase A2; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Process; Protein Dephosphorylation; Regulation; Renal tubule structure; Research; Resolution; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Stimulus; Structure; Synapses; Tissues; Tumor Necrosis Factor Receptor; Tyrosine Phosphorylation; base; brain tissue; cell type; chronic pain; design; inhibitor; innovation; insight; knock-down; lymphatic vasculature; nervous system disorder; neurodevelopment; neurotransmission; nexin; novel; novel therapeutics; receptor; reconstitution; reconstruction; screening; shear stress; tool

ABSTRACT Pannexins comprise a unique family of heptameric large-pore channels that are emerging as novel targets for treating common, yet hard to cure diseases such as hypertension and chronic pain. Previous studies indicate that Panx1 is activated through stimulation of structurally unrelated receptors such as G protein- coupled receptors, ligand-gated ion channels, and tumor necrosis factor receptors. However, it remains un- clear what cellular mechanism(s) actually open and close the Panx1 channel downstream of such seemingly unrelated stimuli. Furthermore, Panx2 and 3 are severely understudied and essentially nothing is known about the activation mechanisms of these subtypes. The long-term goal is to elucidate the mechanisms underlying pannexin gating, regulation, and physiological signaling pathways. The specific objectives for this proposal are to identify the physiological pannexin activators and elucidate the subtype-specific activation mechanisms. The central hypothesis is that both Panx1 and 2 are directly activated by naturally occurring signaling mole- cules in living cells and that Panx1 specifically requires posttranslational modifications to be "primed" for its activation. The rationale for the proposed research is that once the direct activation-stimuli and the subtype- specific mechanisms are identified, it will enable us to fill the critical gap in the pannexin-dependent signaling pathway by connecting the upstream cell-stimulation and the downstream ATP-permeable membrane pore formation. To attain the overall objectives, the following three specific aims will be performed:1) Identify the direct pannexin activators for living cells; 2) Elucidate the role of the N-terminal domain (NTD) in pannexin activation; and 3) Uncover the subtype-specific structural features of pannexins. These research aims will be executed by using a combination of a cell-based pannexin activity assay, electrophysiology, functional recon- stitution, and cryo-EM. The research proposed in this application is innovative because it introduces a novel concept that pannexins—including the understudied Panx2—are directly activated by signaling molecules produced downstream of various stimuli in living cells. It is also innovative because it will provide important insights into the structure of the open channel and why Panx2 and 3 behave differently from Panx1. The proposed research is significant because it will provide concrete molecular mechanisms for the missing link in the pannexin signaling function. The proposed research is also expected to provide a strong structural foundation for subtype specific mechanisms of pannexin channels. These results are expected to have pro- found positive impact not only because they provide detailed basic mechanisms, but also because they will open a new door for screening/designing pannexin-specific inhibitors—much-needed molecular tools that have great potentials to serve as novel therapeutics for a variety of currently uncurable diseases.

抽象的 Pannexins 包含一个独特的七聚体大孔通道家族，它们正在成为新的靶标 用于治疗高血压和慢性疼痛等常见但难以治愈的疾病。之前的研究 表明 Panx1 通过刺激结构上不相关的受体（例如 G 蛋白）而被激活 偶联受体、配体门控离子通道和肿瘤坏死因子受体。然而，它仍然不 明确什么细胞机制实际上打开和关闭这种看似下游的 Panx1 通道 不相关的刺激。此外，Panx2 和 3 的研究严重不足，基本上一无所知 这些亚型的激活机制。长期目标是阐明潜在机制 pannexin 门控、调节和生理信号传导途径。本提案的具体目标 旨在鉴定生理性pannexin激活剂并阐明亚型特异性激活机制。 核心假设是 Panx1 和 2 均由自然发生的信号分子直接激活 Panx1 特别需要翻译后修饰为其“做好准备” 激活。拟议研究的基本原理是，一旦直接激活刺激和亚型 确定了具体的机制，这将使我们能够填补 pannexin 依赖性信号传导的关键空白 通过连接上游细胞刺激和下游 ATP 渗透膜孔的途径 形成。为实现总体目标，将实现以下三个具体目标：1）确定 活细胞的直接pannexin激活剂； 2) 阐明pannexin N端结构域(NTD)的作用 激活; 3）揭示pannexins的亚型特异性结构特征。这些研究目标将是 通过使用基于细胞的 pannexin 活性测定、电生理学、功能重建的组合来执行 机构和冷冻电镜。本申请提出的研究具有创新性，因为它引入了一种新颖的方法 pannexins（包括正在研究的 Panx2）由信号分子直接激活的概念 产生于活细胞中各种刺激的下游。它也是创新的，因为它将提供重要的 深入了解开放通道的结构以及为什么 Panx2 和 3 的行为与 Panx1 不同。这 拟议的研究意义重大，因为它将为缺失的环节提供具体的分子机制 在pannexin信号传导功能中。拟议的研究也有望提供强有力的结构 Pannexin 通道亚型特异性机制的基础。这些结果预计将有利于 之所以能产生积极的影响，不仅是因为它们提供了详细的基本机制，还因为它们将 为筛选/设计pannexin特异性抑制剂打开了一扇新的大门——这是急需的分子工具 具有作为多种目前无法治愈的疾病的新疗法的巨大潜力。