Structure and Function of Pannexins: Activation Mechanism

 描述(由申请人提供)：PAnnexins(Panx1-3)包括一个独特的膜通道家族，介导各种免疫反应，包括凋亡细胞清除、细胞因子分泌和T细胞激活。PAnnexins在调节突触活性和可塑性方面也发挥着关键作用；例如，Panx1基因敲除小鼠表现出兴奋性突触后电位降低，导致物体识别和空间学习受损。此外，Panx1在重复的NMDA受体刺激后促进异常的突触后活动，表明Panx1在癫痫发作中发挥重要作用。这些研究强调了膜联蛋白在全身的重要作用，然而，关于什么激活膜联蛋白通道，它们是如何打开和关闭的，以及伴随这些变化的是什么细胞事件，人们知之甚少。我们的长期目标是使用结构和功能方法来揭示pAnnexin门控、调控和下游信号转导的潜在机制。这项建议的具体目标是通过以下几个方面的调查来实现的：1)解决PAnnexin的第一个晶体结构。我们已经确定了一种高水平表达的膜联蛋白，它在溶液中组装成稳定的单分散低聚物，并形成衍射率为~9？的晶体。我们将继续优化结晶条件，确定PAnnexin的第一个晶体结构。我们预计，PAnnexin的原子分辨结构将作为确定通道孔的组成以及如何控制通道开放和关闭的坚实基础。2)剖析Panx1通道选通的机制。我们的初步实验表明，Panx1--到目前为止研究最多的亚型--很可能含有内在电压传感器。我们还发现，Panx1最常用的非特异性拮抗剂甘苯氧酮可以变构抑制电压门控Panx1通道的活动。利用这种药物和电生理学的优势，我们将识别控制电压门控的关键残基，确定它们相对于膜场的位置，并探索它们如何随电压移动。我们提出的研究具有创新性，不仅因为它将揭示第一个pAnnexin的原子分辨结构，而且还因为它将提供这类独特的电压门控膜通道的第一个详细的分子机制。此外，这是第一次对任何已知的Pannexin抑制剂的作用机制进行详细研究。我们预计，我们的研究将为开发期待已久的Panx1特异性激动剂或拮抗剂开辟新的途径，通过基于结构的药物设计或基于Panx1通道开放相关构象变化的体外筛选。一旦这些分子工具可用，我们希望澄清为什么pannin错误表达会导致体内广泛的功能障碍，并从长远来看，潜在的治疗慢性疼痛和癫痫等破坏性疾病的新策略。

 DESCRIPTION (provided by applicant): Pannexins (Panx1-3) comprise a unique family of membrane channels that mediate a variety of immune responses including apoptotic cell clearance, cytokine secretion, and T-cell activation. Pannexins also play crucial roles in modulating synaptic activity and plasticity; for instance, Panx1 knockout mice display diminished excitatory postsynaptic potentials, resulting in impaired object recognition and spatial learning. Also, Panx1 promotes aberrant postsynaptic activity after repetitive NMDA receptor stimulation, suggesting that Panx1 plays important roles in epileptic seizures. These studies highlight the vital roles of pannexins throughout the body, however, little is understood about what activates pannexin channels, how they open and close, and what cellular events accompany these changes. Our long-term goal is to uncover the mechanisms underlying pannexin gating, regulation, and downstream signaling using structural and functional approaches. The specific aims of this proposal are approached through the following lines of investigation: 1) Solve the first crystal structure of a pannexin. We have identified a pannexin species that expresses at a high level, assembles into a stable and monodisperse oligomer in solution, and forms crystals that diffract to ~9Å. We will continue optimizing crystallization conditions and determine the firt crystal structure of a pannexin. We expect the atomic resolution structure of a pannexin will serve as a solid foundation for determining what constitutes the channel pore and how the opening and closing are controlled. 2) Dissect the mechanism underlying Panx1 channel gating. Our preliminary experiments suggest that Panx1--the most studied subtype thus far--most likely harbors an intrinsic voltage sensor. We also discovered that carbenoxolone, the most commonly used nonspecific antagonist of Panx1, allosterically inhibits the voltage-gated Panx1 channel activity. Taking advantage of this drug and electrophysiology, we will identify the key residues that govern voltage gating, defining their position relative to the membrane field, and probing how they move in response to voltage. Our proposed research is innovative not only because it will uncover the first atomic resolution structure of a pannexin, but also because it wil provide the first detailed molecular mechanism of this unique class of a voltage-gated membrane channel. Moreover, this is the first detailed investigation for the mechanism of action of any known pannexin inhibitors. We expect that our studies will open new avenues for developing long-awaited Panx1 specific agonists or antagonists through structure-based drug design or in vitro screening based on the conformational changes associated with Panx1 channel opening. Once such molecular tools become available, we hope to clarify why pannexin misexpression results in widespread dysfunctions in vivo, and in the long term, potential new strategies for treating devastating conditions such as chronic pain and epilepsy.