Elucidating structures and molecular mechanisms of Pannexin channels

项目摘要 嘌呤能信号传导在神经系统活动中起着重要作用， 突触传递、伤害感受、炎症和味觉。这一过程是由释放腺苷 三磷酸（ATP）通过经典的胞吐作用或ATP渗透通道穿过膜进入细胞。 然后ATP结合相邻细胞上的下游受体。泛连接蛋白家族是一种 关键的ATP渗透通道，并由三个家庭成员，PANX 1 -3。PANX 1是最好的表征 在功能上，它在各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， 凋亡细胞清除和人卵母细胞发育。虽然PANX 2和PANX 3的研究较少， 与PANX 1相比，它们在神经元发育、缺血-再灌注损伤和皮肤/骨骼中是重要的。 发展因此，PANX通道已经成为多种肿瘤的有希望的治疗靶点。 疾病 PANX 1 -3是非选择性的大孔离子通道，预计它们共享一个四跨膜通道。 螺旋（4-TM）拓扑结构与连接蛋白，连接蛋白，和体积调节阴离子通道。生化和 生理学研究提供了一种共识，即PANX家族成员形成六聚体通道，但不形成六聚体通道。 形成缝隙连接。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，磷酸化和半胱天冬酶依赖的切割，但分子 机制尚不清楚。PANX 1活性受多种小分子化合物调节，但大多数 它们中的一种不是专门针对PANX 1的。目前还没有充分表征的药物来调节 PANX 2和PANX 3的活性。虽然PANX是人类生理学的核心，并且是 治疗药物，我们不知道它们的结构。我们不了解，在分子细节上， 被激活或抑制，或者它是如何通过在特定位点结合的小分子来调节的。 在本研究中，我们将对这三个泛连接蛋白通道进行深入的结构和功能研究 来了解这些分子是如何工作的。我们已经确定了人类PANX 1的第一个冷冻电镜结构， APO在3.7 ℃下稳定，发现了七聚体组装。我们还表明，人PANX 1可以被纯化， 在类似天然的脂质环境中。根据这些初步数据，我们建议继续进行结构研究 这些家庭成员，结合互补的电生理学实验，蛋白脂质体为基础的 染料转移测定、结合测定和其他功能方法，以确定 综合浇口机制我们还将定位各种药物的结合位点和分子基础 使用结构和功能方法的组合，支持他们对PANX通道的作用。这些 这些进展将为开发抗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.