Guanidinium Toxins as Tools for Ion Channel Study

描述（由申请人提供）：适当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运动的大蛋白复合物。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，推动了在生命系统中研究nav的高精度方法的发展。然而，由于缺乏调节单个NaV亚型功能和“标记”其细胞分布的可用方法，对活神经元细胞中NaV的实时研究受到限制。我们正在开发用于NaV研究的小分子探针，该探针基于天然存在的胍毒素——蛤蚌毒素、gonyautoxin和zetekitoxin AB。这些药物的功能就像分子“软木塞”一样，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位点的三维结构。我们需要这些信息来推进我们已经构建的NaV同源性模型，并将授权合理设计毒素衍生物，以显示对单个NaV亚型的选择性抑制。我们对毒素结合的结构研究提供了信息

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.