Elucidating structures and molecular mechanisms of Pannexin channels

阐明 Pannexin 通道的结构和分子机制

• 批准号: 10028649
• 负责人: Wei Lu
• 金额: $ 47.5万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-02 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10028649
• 关键词: Adenosine Triphosphate; Anions; Apoptotic; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Calcium; Caspase; Cells; Complex; Connexins; Consensus; Cryoelectron Microscopy; Data; Development; Disease; Dyes; Electrophysiology (science); Environment; Exocytosis; Family; Family Study; Family member; Foundations; Gap Junctions; Human; Inflammation; Ion Channel; Ischemia; Knowledge; Link; Lipids; Mechanics; Membrane; Mission; Molecular; Molecular Structure; Neoplasm Metastasis; Nervous system structure; Nociception; Oocytes; Pathologic; Permeability; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Physiological; Physiology; Play; Potassium; Process; Public Health; Reperfusion Injury; Reperfusion Therapy; Research; Role; Signal Transduction; Site; Skeleton; Skin; Solid; Structure; Synaptic Cleft; Synaptic Transmission; Taste Perception; United States National Institutes of Health; Work; base; blood pressure regulation; experimental study; extracellular; glucose uptake; neuron development; neuroprotection; novel therapeutics; particle; receptor; small molecule; targeted treatment; therapeutic development; therapeutic target; tumor

PROJECT SUMMARY Purinergic signaling plays fundamental roles in activities of the nervous system as diverse as neuroprotection, synaptic transmission, nociception, inflammation, and taste. This process is initiated by releasing adenosine triphosphate (ATP) across the membrane through the classic exocytosis or ATP-permeable channels into the synaptic cleft; the ATP then binds downstream receptors on an adjacent cell. The pannexin family is one of the key ATP-permeable channels and consists of three family members, PANX1-3. PANX1 is the best characterized functionally, and it plays crucial roles in a variety of contexts, including blood pressure regulation, glucose uptake, apoptotic cell clearance, and human oocyte development. Although PANX2 and PANX3 have been less studied than PANX1, they are important in neuronal development, ischemia-reperfusion injury, and skin/skeleton development. Thus, the PANX channels have emerged as promising therapeutic targets for a diverse range of diseases. The PANX1-3 are nonselective, large-pore ion channels, and they are predicted to share a four-transmembrane- helix (4-TM) topology with connexins, innexins, and volume-regulated anion channels. Biochemical and physiological studies provide a consensus view that PANX family members form hexameric channels but do not form gap junctions. PANX can be modulated by various factors, including mechanical scratch, extracellular potassium, intracellular calcium, phosphorylation, and caspase-dependent cleavage, but the molecular mechanisms aren’t known. PANX1 activity is modulated by a wide range of small-molecule compounds, but most of them are not specifically targeting PANX1. There is currently no well-characterized agent that modulates the activity of PANX2 and PANX3. Although PANXs are central to human physiology and are potential targets of therapeutic agents, we do not know their structures. We do not understand, in molecular detail, how the channel is activated or inhibited, or how it is modulated by small molecules binding at specific sites. In this proposed work, we will carry out in-depth structural and functional studies of the three pannexin channels to understand how these molecules work. We have determined the first cryo-EM structure of human PANX1 in the apo state at 3.7 Å and found a heptameric assembly. We have also shown that human PANX1 can be purified in a native-like lipid environment. Building on this preliminary data, we propose to continue the structural studies of these family members, combined with complementary electrophysiology experiments, proteolipsome-based dye transfer assays, binding assays, and other functional approaches, to define the molecular basis for a comprehensive gating mechanism. We will also locate the binding sites of various drugs and the molecular basis underlying their actions on PANX channels, using a combination of structural and functional approaches. These advances will provide a solid foundation for developing new drugs against PANX-linked diseases and for a deeper understanding of the function of the ATP release channel family.

项目摘要 嘌呤能信号传导在神经保护和神经保护作用的神经系统活动中起着基本作用， 突触传递，伤害感受，炎症和味道。此过程是通过释放腺苷来启动的 三磷酸（ATP）穿过经典的胞吐作用或ATP渗透通道进入膜 突触裂；然后，ATP在相邻单元格上结合下游接收器。 Pannexin家族是 关键的ATP可渗透渠道，由三个家庭成员Panx1-3组成。 panx1是最好的特征 在功能上，它在多种情况下扮演着至关重要的作用，包括调节血压，葡萄糖摄取， 凋亡细胞清除和人类卵母细胞的发育。尽管PANX2和PANX3较少研究 比PANX1，它们在神经元发育，缺血 - 重新灌注损伤和皮肤/骨骼中很重要 发展。那就出现了PANX渠道，作为承诺的潜水员范围的治疗目标 疾病。 panx1-3是非选择性的大孔离子通道，预计它们具有四跨膜 - 螺旋（4-TM）拓扑，带有连接素，innexins和体积调节的阴离子通道。生化和 生理研究提供了共识的观点，即Panx家族成员形成六聚体渠道，但不 形成间隙连接。 Panx可以通过各种因素调节，包括机械划痕，细胞外 钾，细胞内钙，磷酸化和caspase依赖性清洁，但分子 机制尚不清楚。 PANX1活性由多种小分子化合物调节，但大多数 它们不是专门针对panx1的。当前没有特定的代理可以调节 PANX2和PANX3的活性。尽管PANX是人类生理学的核心，并且是 治疗剂，我们不知道它们的结构。我们不理解分子细节的通道 被激活或抑制，或如何通过特定位点的小分子结合来调节它。 在这项拟议的工作中，我们将对三个泛素通道进行深入的结构和功能研究 了解这些分子如何工作。我们已经确定了人类panx1的第一个冷冻EM结构 Apo状态为3.7Å，发现了一个七聚体组件。我们还表明人可以纯化人Panx1 在类似于本地的脂质环境中。在此初步数据的基础上，我们建议继续进行结构研究 在这些家庭成员中，结合互补的电生理实验，基于蛋白质的基础 染料转移测定，结合测定和其他功能方法，以定义A的分子基础 全面的门控机制。我们还将找到各种药物的结合位点和分子基础 使用结构和功能方法的组合，将其在Panx通道上的行动为基础。这些 进步将为开发针对PANX连接疾病的新药提供坚实的基础，并为 对ATP发行渠道家族功能的更深入了解。