Guanidinium Toxins as Tools for Ion Channel Study

描述(申请人提供)：正常的神经元功能依赖于电压门控钠离子通道(NAV)的严格调控表达和离散定位，NAV是控制离子跨细胞膜移动的大型蛋白质复合体。为了更好地了解NAV在轴突可塑性和信号传导中的作用，以及它们的失调与特定人类病理之间的关系，人们开始开发高精度的方法来研究它们在生命系统中的作用。然而，在活的神经细胞中对NAV的实时研究受到限制，因为缺乏可用的方法来调节单个NAV亚型的功能并‘标记’它们的细胞分布。我们正在开发用于NAV研究的小分子探针，这些探针基于自然产生的鸟粪毒素--岩藻毒素、刚鱼毒素和哲氏毒素AB。这些试剂起到了分子‘软木塞’的作用，堵住了离子电导孔的胞外口。从头化学合成使这些毒素的修饰形式成为可能，我们将结合蛋白质诱变和电生理学来深入了解毒素结合部位的三维结构。这些信息是推进我们所构建的NAV同源模型所必需的，并将使显示选择性抑制个别NAV亚型的毒素衍生物的合理设计成为可能。我们对毒素结合的结构研究告诉我们 开发新的荧光成像和基于亲和力的工具来研究与NAV功能相关的动态事件。我们的目的是了解NAV表达的调节如何影响神经细胞的输入-输出反应(即细胞可塑性)毒素结合物将在最初的实验中用于测量通道合成和周转率，并最终定量分析这些动力学数据作为神经细胞刺激和神经细胞损伤的函数的变化程度。小分子试剂提供的时间控制和此类探针的微创性质为标记内源性NAV通道提供了显著的优势。因此，用于NAV成像研究的毒素衍生物的可用性应该会为这些通道蛋白在电生成中的动态作用提供前所未有的洞察。

DESCRIPTION (provided by applicant): 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 axonal plasticity and signal conduction, and the relationship between their disregulation and specific human pathologies motivates the development of high precision methods 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 distribution. We are developing small molecule probes for NaV studies based on naturally occurring guanidinium toxins - saxitoxin, gonyautoxin, and zetekitoxin AB. 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 NaV homology model that we have constructed, and will empower the rational design of toxin derivatives that show selective inhibition of individual NaV isoforms. Our structural investigations of toxin binding are informing the development of new fluorescent imaging and affinity-based tools for investigating dynamic events associated with NaV function. We are motivated to understand how modulation of NaV expression influences the input-output responsiveness of neuronal cells (i.e., cellular plasticity) Toxin conjugates will be employed in initial experiments to measure channel synthesis and turnover rates, and ultimately to analyze quantitatively the extent to which these kinetic data vary as a function of nerve cell stimulation and nerve cell injury. The temporal control afforded by small molecule agents and the minimally invasive nature of such probes offer significant advantages over biological methods for labeling endogenous NaV channels. As such, the availability of toxin derivatives for NaV imaging studies should offer unprecedented insight into the dynamic role of these channel proteins in electrogenesis.