Guanidinium Toxins as Molecular Probes for NaV Study

项目总结 神经元的兴奋性依赖于严格调控的表达和离散的亚细胞 电压门控钠离子通道(NAV)的定位。这些大的膜蛋白 复合体控制钠离子在细胞膜上的移动，并负责 启动和传播动作电位。希望更好地了解NAV的作用 电信号传导的亚型及其与通道失调的关系 特定的人类病理(例如，心律失常、癫痫、骨骼肌疾病、神经病理性 Pain推动了高精度试剂的发展，以促进NAV研究。 NAV生理学的研究目前由于缺乏可用的方法而受到限制 调节单个通道亚型的功能并标记细胞内的变化 分布、膜表达水平和结构修饰(即蛋白质翻译后 修饰)在活细胞中，并对外部提示作出反应。 我们正在开发用于NAV研究的小分子探针，该探针基于自然生成的双胍 毒素，其中以毒素(STX)为原型。这些代理的功能是 用分子‘软木塞’堵住离子导孔的胞外口，是一种理想的方法 我们希望访问的工具化合物类型的功能。从头开始的化学合成有 使我们能够对STX的修饰形式进行工程设计，我们将与蛋白质结合使用 诱变和电生理学研究NAV活性对动作电位动力学的影响。 了解NAV的表达是如何被外部因素--胶质细胞--调节和改变的 炎症介质、pH、神经损伤-以及这些变化如何调节动作电位 这是我们研究的长期目标。为了解决这些问题，我们将开发和验证 三类工具化合物。这些试剂将使1)急性时空抑制 单个NAV亚型；2)膜NAV的选择性荧光标记；以及3)亲和力 用于蛋白质组学分析的膜NAV的纯化。随着我们研究的成功 计划，我们将提供一套独特的高精度化学探测器，以帮助照亮 NAV的复杂生理学基础上的生物电信号。

PROJECT SUMMARY Neuronal excitability relies on the tightly regulated expression and discrete subcellular localization of voltage-gated sodium ion channels (Navs). These large membrane protein complexes control the movement of sodium ions across cell membranes and are responsible for initiating and propagating action potentials. A desire to better understand the role of Nav subtypes in electrical signal conduction and the relationship between channel dysregulation and specific human pathologies (e.g., arrhythmia, epilepsy, skeletal muscle disorders, neuropathic pain} motivates the development of high precision reagents to facilitate Nav studies. Investigations of Nav physiology are currently limited by a lack of available methods with which to modulate the function of individual channel subtypes and to mark changes in cellular distributions, membrane expression levels, and structural modifications (i.e., protein posttranslational modification) in live cells and in response to external cues. We are developing small molecule probes for Nav studies based on naturally occurring bis-guanidinium toxins, among which saxitoxin (STX) is the archetype. These agents function as molecular 'corks' to occlude the extracellular mouth of the ion conductance pore, a desirable feature for the types of tool compounds we wish to access. De novo chemical synthesis has enabled the engineering of modified forms of STX, which we will use in combination with protein mutagenesis and electrophysiology to investigate Nav activity on action potential dynamics. Understanding how Nav expression is regulated and altered by extrinsic factors-glial cells, inflammatory mediators, pH, nerve injury-and how such changes modulate action potentials is a long-term goal of our research. To address these questions, we will develop and validate three classes of tool compounds. These reagents will enable 1) acute, spatiotemporal inhibition of individual Nav subtypes; 2) selective fluorescent labeling of membrane Navs; and 3) affinity purification of membrane Navs for proteomics analysis. With the success of our research program, we will deliver a unique set of high precision chemical probes to help illuminate the complex physiology of Navs that under1ies bioelectrical signaling.