Guanidinium Toxins as Molecular Probes for NaV Study

项目总结 正常的神经元功能依赖于严格调控的表达和离散的定位 电压门控钠离子通道(NAV)是一种控制运动的大型蛋白质复合体 穿过细胞膜的离子。希望更好地了解NAV在电力行业中的作用 信号传导与经络失调与特定人的关系 病理学推动了用于活体研究的高精度试剂的发展 系统。然而，在活的神经细胞中对NAV的实时研究受到 缺乏有效的方法来调节个别NAV亚型的功能并 标记它们的细胞分布和膜表达水平。 我们正在开发基于自然发生的NAV研究的小分子探针 鸟粪类毒素--萨克托毒素、刚鱼毒素和哲氏毒素。这些代理的功能是 分子‘软木塞’，堵住离子电导孔的胞外口。从头开始 化学合成使这些毒素的修饰形式可用，我们将在 结合蛋白质诱变和电生理学，深入了解这三种 毒素结合部位的空间结构。这样的信息是需要的，以推动 Fidelity NAV同源模型，并将使合理设计的毒素衍生物 显示对个别NAV亚型的选择性抑制。 我们对毒素结合的结构研究正在为新的荧光材料的发展提供信息 成像和基于亲和力的工具，将用于探索相关的动态事件 具有导航功能。我们希望了解NAV膜表达的调控和 翻译后蛋白质修饰影响神经元的输入输出反应 神经损伤后的细胞。将制备毒素衍生的荧光探针，并用于 测量活细胞中膜植入NAV的空间分布和浓度。 这些研究将提供对NAV结构(即后结构)的定量分析 翻译修饰)、离子门控、膜分布和蛋白质周转率 神经细胞损伤模型中的改变。此外，我们的试验性设计将允许我们 评估研究药物、蛋白质因子和/或其他小分子对 调节NAV运输和恢复适当的神经元信号。最终，这项工作可能会 导致确定用于治疗疼痛的新的治疗靶点或先导化合物。

PROJECT SUMMARY Proper neuronal function relies on the tightly regulated expression and discrete localization of voltage-gated sodium ion channels (NaVs), large protein complexes that control the movement of ions across cell membranes. A desire to better understand the role of NaVs in electrical signal conduction and the relationship between channel disregulation and specific human pathologies motivates the development of high precision reagents for their study in living systems. Real-time investigations of NaVs in live neuronal cells, however, are limited by the lack of available methods with which to modulate the function of individual NaV subtypes and to mark their cellular distributions and membrane expression levels. We are developing small molecule probes for NaV studies based on naturally occurring guanidinium toxins – saxitoxin, gonyautoxins, and zetekitoxin. These agents function as molecular `corks' to occlude the extracellular mouth of the ion conductance pore. De novo chemical synthesis makes available modified forms of these toxins, which we will use in combination with protein mutagenesis and electrophysiology to gain insights into the three- dimensional structure of the toxin binding site. Such information is needed to advance a high fidelity NaV homology model, and will empower the rational design of toxin derivatives that display selective inhibition of individual NaV isoforms. Our structural investigations of toxin binding are informing the development of new fluorescent imaging and affinity-based tools, which will be utilized to explore dynamic events associated with NaV function. We wish to understand how modulation of NaV membrane expression and post-translational protein modifications influence the input-output responsiveness of neuronal cells following nerve injury. Toxin-derived fluorescent probes will be prepared and used to measure the spatial distributions and concentrations of membrane-inserted NaVs in live cells. These investigations will provide a quantitative analysis of how NaV structure (i.e., post- translational modification), ion gating, membrane distribution, and protein turnover rates are altered in neuronal cell injury models. In addition, our experimental design will allow us to assess the influence of investigational drugs, protein factors, and/or other small molecules on regulating NaV trafficking and restoring proper neuronal signaling. Ultimately, this work could lead to the identification of new therapeutic targets or lead compounds for pain treatment.