Structure and Function of Pannexins: Activation Mechanism

 描述（由申请人提供）：泛联蛋白（Panx 1 -3）包含介导多种免疫应答（包括凋亡细胞清除、细胞因子分泌和T细胞活化）的独特膜通道家族。Pannexins在调节突触活动和可塑性方面也起着至关重要的作用;例如，Panx 1敲除小鼠显示兴奋性突触后电位减少，导致物体识别和空间学习受损。此外，Panx 1促进重复NMDA受体刺激后的异常突触后活动，表明Panx 1在癫痫发作中起重要作用。这些研究强调了泛连接蛋白在整个身体中的重要作用，然而，人们对什么激活泛连接蛋白通道，它们如何打开和关闭以及伴随这些变化的细胞事件知之甚少。我们的长期目标是使用结构和功能方法揭示泛连接蛋白门控、调节和下游信号传导的潜在机制。该提议的具体目标通过以下研究路线来实现：1）解决泛连接蛋白的第一晶体结构。我们已经鉴定了一种高水平表达的泛连接蛋白种类，其在溶液中组装成稳定且单分散的寡聚体，并形成结晶，结晶至约9 μ m。我们将继续优化结晶条件并确定泛连接蛋白的第一晶体结构。我们期望泛连接蛋白的原子分辨率结构将作为确定通道孔的组成以及如何控制打开和关闭的坚实基础。2)剖析Panx 1通道门控的机制。我们的初步实验表明，Panx 1-迄今为止研究最多的亚型-最有可能拥有内在的电压传感器。我们还发现，甘珀酸，最常用的非特异性拮抗剂Panx 1，变构抑制电压门控Panx 1通道的活动。利用这种药物和电生理学，我们将识别控制电压门控的关键残基，定义它们相对于膜场的位置，并探测它们如何响应电压而移动。我们提出的研究是创新的，不仅因为它将揭示泛连接蛋白的第一个原子分辨率结构，而且因为它将提供这种独特的电压门控膜通道的第一个详细的分子机制。此外，这是第一次详细研究任何已知的泛连接蛋白抑制剂的作用机制。我们期望我们的研究将为开发期待已久的Panx 1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与Panx 1通道开放相关的构象变化的体外筛选。一旦这些分子工具变得可用，我们希望澄清为什么泛连接蛋白的错误表达会导致体内广泛的功能障碍，以及从长远来看，治疗慢性疼痛和癫痫等破坏性疾病的潜在新策略。

 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.