Structure and Function of Pannexins: Activation Mechanism

摘要 Pannexins由一个独特的七聚体大孔通道家族组成，正在成为新的靶点 用于治疗常见但难以治愈的疾病，如高血压和慢性疼痛。以前的研究 表明Panx 1通过刺激结构上不相关的受体如G蛋白- 偶联受体、配体门控离子通道和肿瘤坏死因子受体。然而，它仍然没有- 明确什么样的细胞机制（S）实际上打开和关闭Panx 1通道下游的这种看似 无关的刺激。此外，Panx 2和3的研究严重不足，基本上一无所知。 这些亚型的激活机制。长期目标是阐明潜在的机制 泛连接蛋白门控、调节和生理信号通路。本提案的具体目标 目的是鉴定生理性泛连接蛋白激活剂并阐明亚型特异性激活机制。 核心假设是Panx 1和2都是由天然存在的信号分子直接激活的。 Panx 1特别需要翻译后修饰才能“启动”其表达， activation.这项研究的基本原理是，一旦直接激活刺激和亚型- 确定了具体的机制，这将使我们能够填补泛连接蛋白依赖性信号传导的关键空白， 通过连接上游细胞刺激和下游ATP渗透膜孔的途径 阵为实现总体目标，将实现以下三个具体目标：1）确定 活细胞的直接泛连接蛋白激活剂; 2）阐明N-末端结构域（NTD）在泛连接蛋白中的作用 3）揭示pannexins的亚型特异性结构特征。这些研究目标将是 通过使用基于细胞的泛连接蛋白活性测定、电生理学、功能性重组 替代和冷冻EM。本申请中提出的研究是创新的，因为它引入了一种新的 pannexins--包括未充分研究的Panx 2--直接被信号分子激活的概念 在活细胞中的各种刺激的下游产生。它也是创新的，因为它将提供重要的 深入了解开放通道的结构，以及为什么Panx 2和3的行为与Panx 1不同。的 拟议中的研究意义重大，因为它将为缺失的一环提供具体的分子机制 在泛连接蛋白信号传导功能中的作用。拟议的研究还有望提供一个强大的结构性 泛连接蛋白通道亚型特异性机制的基础。这些结果预计将有亲- 发现积极的影响不仅是因为它们提供了详细的基本机制，还因为它们将 为筛选/设计pannexin特异性通道打开了一扇新的大门-急需的分子工具， 具有作为多种目前无法治愈的疾病的新疗法的巨大潜力。

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.