Guanidinium Toxins as Molecular Probes for NaV Study

项目摘要 适当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（NaVs），控制运动的大型蛋白质复合物 离子穿过细胞膜。希望更好地了解NaV在电气中的作用 信号传导及通道失调与特定人体的关系 病理学激发了高精度试剂的发展，用于他们在生活中的研究 系统.然而，在活的神经元细胞中实时研究NaV受到以下限制： 缺乏可用于调节单个NaV亚型功能的方法， 标记它们的细胞分布和膜表达水平。 我们正在开发用于NaV研究的小分子探针， 胍毒素-石房蛤毒素、gonyautoxins和zetekitoxin。这些代理的功能是 分子“软木塞”堵塞离子传导孔的细胞外口。从头 化学合成使这些毒素的修饰形式可用，我们将使用它来 结合蛋白质诱变和电生理学，以深入了解这三个- 毒素结合位点的三维结构。需要这些信息来推动一个高水平的 保真度NaV同源性模型，并将赋予毒素衍生物的合理设计， 显示对单个NaV同种型的选择性抑制。 我们对毒素结合的结构研究为新的荧光标记的开发提供了信息。 成像和基于亲和性的工具，将用于探索相关的动态事件 Nav功能。我们希望了解NaV膜表达的调节和 翻译后蛋白质修饰影响神经元的输入-输出反应性 神经损伤后的细胞。将制备毒素衍生的荧光探针并用于 测量活细胞中插入膜的NaV的空间分布和浓度。 这些研究将提供一个定量分析如何NaV结构（即，后 翻译修饰），离子门控，膜分布和蛋白质周转率。 在神经元细胞损伤模型中改变。此外，我们的实验设计将使我们能够 评估研究药物、蛋白质因子和/或其他小分子对 调节NaV运输并恢复适当的神经元信号传导。最终，这项工作可以 导致识别新的治疗靶点或用于疼痛治疗的先导化合物。

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.