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包括一个独特的肝脏大型通道家族，它们正在成为新的目标 用于治疗常见但难以治愈诸如高血压和慢性疼痛之类的疾病。先前的研究 表明通过刺激结构无关的受体（例如G蛋白）激活PANX1 偶联受体，配体门控离子通道和肿瘤坏死因子受体。但是，它仍然没有 清除哪种蜂窝机制实际上打开并关闭了这种看似在下游的Panx1通道 无关刺激。此外，panx2和3是对 这些亚型的激活机制。长期目标是阐明基本机制 Pannexin门控，调节和物理信号通路。该提议的具体目标 识别物理泛素激活剂并阐明亚型特异性激活机制。 中心假设是panx1和2均通过天然发生的信号摩尔直接激活 活细胞中的Cules和PANX1专门要求翻译后修饰为其“启动” 激活。拟议研究的理由是，一旦直接激活刺激和亚型 确定了具体机制，它将使我们能够填补依赖Pannexin的信号传导中的临界空隙 通过连接上游细胞刺激和下游ATP渗透膜孔的途径 形成。为了实现总体目标，将执行以下三个特定目标：1）确定 用于活细胞的直接泛毒素激活剂； 2）阐明N末端结构域（NTD）在Pannexin中的作用 激活; 3）揭示泛毒素的亚型特异性结构特征。这些研究目的将是 通过使用基于细胞的Pannexin活性测定，电生理学，功能重新调节的组合来执行 条件和冷冻EM。本应用程序中提出的研究具有创新性，因为它引入了一本小说 pannexin（包括理解的panx2）直接通过信号分子激活的概念 在活细胞中的各种刺激下游产生。这也是创新的，因为它将提供重要的 洞悉开放通道的结构，以及为什么PANX2和3与Panx1的行为不同。这 拟议的研究很重要，因为它将为缺失的链接提供具体的分子机制 在Pannexin信号传导函数中。拟议的研究还预计将提供强大的结构 泛毒素通道的亚型特异性机制的基础。这些结果预计将具有支持 发现积极的影响不仅是因为它们提供了详细的基本机制，还因为它们会 打开一个新的门，用于筛查/设计Pannexin特异性抑制剂 - 非常需要的分子工具 对于各种目前无法保育的疾病，有很大的潜力作为新的疗法。