Elucidating structures and molecular mechanisms of Pannexin channels

项目总结 嘌呤能信号在神经系统的各种活动中起着基本的作用，包括神经保护， 突触传递、伤害性感受、炎症和味觉。这个过程是通过释放腺苷来启动的。 三磷酸(ATP)穿过细胞膜，通过经典的胞吐作用或ATP通透通道进入 突触分裂；然后，三磷酸腺苷与相邻细胞上的下游受体结合。Pannexin家族是 关键的ATP通透通道，由三个家族成员组成，Panx1-3。Panx1是最具特点的 在功能上，它在各种环境中发挥着关键作用，包括调节血压、葡萄糖摄取、 细胞凋亡清除和人类卵母细胞发育。尽管对PANX2和PANX3的研究较少 与Panx1相比，它们在神经元发育、缺血再灌注损伤和皮肤/骨骼中都具有重要作用。 发展。因此，PANX通道已成为治疗多种疾病的有希望的靶点。 疾病。 Panx1-3是非选择性的大孔离子通道，它们被预测共享一个四跨膜通道- 具有连接蛋白、内联蛋白和容量调节的阴离子通道的螺旋(4-TM)拓扑结构。生化和 生理研究提供了一个一致的观点，即PANX家族成员形成六聚体通道，但不是 形成缝隙连接。PANX可受多种因素的调节，包括机械划痕、细胞外 钾、细胞内钙、磷酸化和caspase依赖的切割，但分子 机制尚不清楚。Panx1的活性受到一系列小分子化合物的调节，但大多数 其中一些并不是专门针对Panx1的。目前还没有一种具有良好特性的试剂来调节 PANX2和PANX3的活性。尽管PANX是人类生理的中枢，也是 治疗药物，我们不知道它们的结构。我们不知道，在分子细节上，通道是如何 是被激活还是被抑制，或者它是如何被结合在特定位置的小分子调制的。 在这项拟议的工作中，我们将对三个pannin通道进行深入的结构和功能研究 以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-EM结构 Apo状态为3.7？，并发现了一个七聚体组装。我们还证明了人类Panx1可以被提纯 在一个天然的类脂环境中。基于这些初步数据，我们建议继续进行结构研究 在这些家族成员中，结合互补的电生理实验，蛋白质脂体为基础 染料转移分析、结合分析和其他功能方法，以确定 全面的浇口机构。我们还将定位各种药物的结合部位和分子基础 他们在PANX渠道上的行动，使用结构和功能方法的组合。这些 进展将为开发治疗PANX相关疾病的新药和 对ATP释放通道家族的功能有更深入的了解。

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.