Biophysical analysis of interactions between peptide toxins and human sodium channel voltage-sensor domains

电压门控钠离子通道调节通过细胞膜的钠离子的快速和特定的流动。 它们对人体的功能非常重要，如调节心跳和 神经细胞中的电信号。钠通道突变引起的疾病的例子包括致命的 心律失常、癫痫、神经肌肉疾病和严重偏头痛。此外，钠离子通道 在治疗疼痛和预防癌症转移方面也是很有希望的靶点。钠 通道是大量天然毒素的靶标，其中许多是高度选择性的多肽毒素，动物 用来防御或制服猎物。这些毒素代表着具有生物活性的化合物的宝库 作为基础研究工具的潜在应用以及在治疗癌症药物开发中的应用 钠通道相关疾病。一些针对钠通道的蜘蛛、蝎子和海葵毒素 通过绑定到通道的电压传感器域(VSD)来更改通道的激活电压。 这类毒素被称为门控调节剂毒素，因其潜在的毒性而引起人们的特别兴趣。 以一种微妙的、可控的方式控制通道活动，具有高度的特异性。目前对该模式的认识 门控修饰剂毒素的作用主要基于功能和突变研究。几个直接结构 对毒素通道复合体的研究也有报道，但在毒素所在区域的分辨率 由于VSD的动态性质和钠通道的大尺寸(~2000)，结合通常较差 氨基酸残基)。在本项目中，从两种人钠通道亚型中分离出钠通道VSD 将被用作毒素分离的目标，然后将对毒素进行功能和结构表征。 此外，新的和/或已知的毒素与这些VSD之间相互作用的结构细节将是 通过不同的生物物理技术来阐明。 为了进行这些实验，来自两个人钠通道(心脏钠通道NaV1.5)的VSD 和参与疼痛传递的周围神经系统的Nav1.7)将被重组 在细菌中表达，并在适合毒素下拉实验的膜模拟系统中重组 和生物物理相互作用研究。重组的VSD将被用来从 已知含有门控调节因子毒素的几种动物的粗毒。这样的毒素将是 进行质谱分析，确定它们的多肽序列。然后它们将被化学处理 或生物合成的产物，用于进一步的核磁共振结构分析。VSD和VSD之间交互的详细信息 已知或新的相互作用的毒素将通过测量不同残基的突变如何被阐明 毒素和VSD影响电生理和直接测量的结合亲和力和通道调制 有约束力的分析。这些实验的结果将提供有用的结构信息，可以加以利用 开发针对离子通道的药物，以治疗包括心律失常和疼痛在内的疾病。

Voltage-gated sodium channels regulate the rapid and specific flow of sodium ions through the cell membrane. They are of great importance for functions in the human body such as the regulation of the heartbeat and electrical signaling in nerve cells. Examples of diseases caused by mutations in sodium channels include fatal cardiac arrhythmias, epilepsy, neuromuscular disorders and severe migraines. Furthermore, sodium channels are also promising targets in the treatment of pain and potentially in the prevention of cancer metastasis. Sodium channels are targeted by a vast array of natural toxins, many of them highly selective peptide toxins that animals use for defense or to subdue their prey. These toxins represent a treasure trove of bioactive compounds with potential applications as tools for basic research as well as in the development of drugs for the treatment of sodium channel-related diseases. Some spider, scorpion and sea anemone toxins that target sodium channels change the voltage of activation of the channels by binding to the voltage sensor domains (VSDs) of the channel. These kinds of toxins are known as gating-modifier toxins and are of special interest because of their potential to control channel activity in a subtle and controlled way with high specificity. Current knowledge of the mode of action of gating-modifier toxins is mostly based on functional and mutational studies. A few direct structural studies of toxin-channel complexes have also been reported, but the resolution in the regions where the toxin binds is generally poor due to the dynamic nature of the VSDs and the large size of the sodium channels (~2000 amino acid residues). In this project, isolated sodium channel VSDs from two human sodium channel isoforms will be used as targets for toxin isolation, and the toxins will then be functionally and structurally characterized. Additionally, structural details of the interactions between new and/or known toxins and these VSDs will be elucidated through different biophysical techniques. For conducting these experiments, VSDs from two human sodium channels (the cardiac sodium channel NaV1.5 and NaV1.7 of the peripheral nervous system that is involved in pain transmission) will be recombinantly expressed in bacteria and reconstituted in a membrane mimetic system suitable for toxin pull-down experiments and biophysical interaction studies. The recombinant VSDs will be used to fish out interacting toxins from the crude venoms of several animal species that are known to contain gating-modifier toxins. Such toxins will be subjected to mass spectrometry analysis for determining their peptide sequence. They will then be chemically or biosynthetically produced for further NMR structural analysis. The details of interactions between VSDs and known or new interacting toxins will be elucidated by measuring how the mutation of different residues on the toxin and VSD affect the binding affinities and channel modulation as measured by electrophysiology and direct binding assays. The results of these experiments will provide useful structural information that can be exploited in the development of drugs targeting ion channels to treat disorders including cardiac arrhythmias and pain.