Elucidating structures and molecular mechanisms of Pannexin channels

阐明 Pannexin 通道的结构和分子机制

• 批准号: 10437844
• 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/10437844
• 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.

项目概要 嘌呤能信号在神经系统的各种活动中发挥着重要作用，如神经保护、 突触传递、伤害感受、炎症和味觉。这个过程是通过释放腺苷来启动的 三磷酸 (ATP) 通过经典的胞吐作用或 ATP 可渗透通道跨膜进入 突触间隙；然后 ATP 与相邻细胞上的下游受体结合。 pannexin 家族是其中之一 关键的 ATP 渗透通道，由三个家族成员 PANX1-3 组成。 PANX1 是特征最好的 在功能上，它在多种情况下发挥着至关重要的作用，包括血压调节、葡萄糖摄取、 凋亡细胞清除和人类卵母细胞发育。尽管 PANX2 和 PANX3 研究较少 比 PANX1 更重要的是神经元发育、缺血再灌注损伤和皮肤/骨骼 发展。因此，PANX 通道已成为多种有前景的治疗靶点。 疾病。 PANX1-3 是非选择性大孔离子通道，预计它们共享一个四跨膜 具有连接蛋白、内连接蛋白和容量调节阴离子通道的螺旋 (4-TM) 拓扑结构。生化和 生理学研究提供了一个共识，即 PANX 家族成员形成六聚体通道，但不形成六聚体通道。 形成间隙连接。 PANX 可以受到多种因素的调节，包括机械划痕、细胞外 钾、细胞内钙、磷酸化和半胱天冬酶依赖性切割，但分子 机制尚不清楚。 PANX1 活性受到多种小分子化合物的调节，但大多数 其中不专门针对 PANX1。目前还没有特征明确的药物可以调节 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。我们不明白，在分子细节上，通道是如何 被激活或抑制，或者如何通过在特定位点结合的小分子进行调节。 在这项拟议的工作中，我们将对三个pannexin通道进行深入的结构和功能研究 了解这些分子是如何工作的。我们已经确定了人类 PANX1 的第一个冷冻电镜结构 3.7 Å 处的 apo 态并发现了七聚体组装体。我们还表明，人类 PANX1 可以被纯化 在类似天然的脂质环境中。基于这些初步数据，我们建议继续进行结构研究 这些家庭成员，结合补充电生理学实验，基于蛋白脂质体 染料转移测定、结合测定和其他功能方法，以确定分子基础 全面的门控机制。我们还将定位各种药物的结合位点和分子基础 使用结构性和功能性方法相结合，为他们在 PANX 渠道上的行动提供基础。这些 进展将为开发针对 PANX 相关疾病的新药和 更深入地了解 ATP 释放通道家族的功能。