Structure and Function of Pannexins: Activation Mechanism

摘要 PAnnexins包括一个独特的七聚体大孔通道家族，正在成为新的靶点 用于治疗高血压和慢性疼痛等常见但难以治愈的疾病。以前的研究 表明Panx1是通过刺激结构无关的受体激活的，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- 清楚是什么细胞机制(S)实际上打开和关闭了Panx1通道下游的这种看似 无关的刺激。此外，对Panx2和Panx3的研究严重不足，基本上对其一无所知 这些亚型的激活机制。其长期目标是阐明潜在的机制。 Pannexin门控、调节和生理信号通路。这项提案的具体目标 鉴定生理上的pAnnexin激活剂，并阐明亚型特异性的激活机制。 中心假设是Panx1和Panx2都是由自然产生的信号分子直接激活的- 而且Panx1特别需要翻译后的修饰才能为其 激活。这项拟议研究的基本原理是，一旦直接激活刺激和亚型- 具体的机制被确定，它将使我们能够填补pannin依赖的信号转导的关键空白 连接上游细胞刺激和下游ATP通透膜孔的途径 队形。为实现总体目标，将实现以下三个具体目标：1)确定 活细胞直接膜联蛋白激活剂；2)阐明N末端结构域(NTD)在膜联蛋白中的作用 3)揭示PAnnexins亚型特有的结构特征。这些研究目标将是 通过使用基于细胞的膜联蛋白活性分析、电生理学、功能重组- 制度，和冷冻-EM。本申请中提出的研究具有创新性，因为它引入了一种新的 Pannexins--包括未被研究的Panx2--被信号分子直接激活的概念 在活细胞中的各种刺激的下游产生的。它也是创新的，因为它将提供重要的 洞察开放通道的结构，以及为什么Panx2和Panx3的行为与Panx1不同。这个 拟议的研究具有重要意义，因为它将为缺失的链提供具体的分子机制 在pAnnexin信号转导功能中。预计拟议的研究还将提供强有力的结构性 PAnnexin通道亚型特异性机制的基础。预计这些结果将有利于- 发现积极影响不仅是因为它们提供了详细的基本机制，而且还因为它们将 为筛选/设计pAnnexin特异性抑制剂打开一扇新的大门--这是一种非常需要的分子工具 具有巨大的潜力，可作为治疗各种目前无法治愈的疾病的新疗法。

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.