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。门控调节剂毒素是这些毒素的一个子群，它改变了 通过绑定到通道的电压传感器域(VSD)来激活通道的电压。当前 对门控修饰剂毒素作用模式的了解主要基于功能和突变研究。 一些毒素通道复合体的直接结构研究也已被报道，但在 毒素结合的地区通常很差。在本项目中，分离的钠通道VSD来自两个人 钠通道异构体将被用作毒素分离的靶标，然后毒素将被功能和 具有结构特征的。此外，新毒素和/或已知毒素之间相互作用的结构细节 这些VSD将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(心脏通道NaV1.5和 参与疼痛传递的外周神经系统的Nav1.7)将在细菌和 重组在适合毒素下拉实验和生物物理相互作用的膜模拟系统中 学习。重组VSD将用于从几种动物的粗毒中分离相互作用的毒素 已知含有门控调节剂毒素的物种。对这种毒素的初步鉴定将通过以下方式实现 MALDI-TOF质谱仪。目前用于这一目的的仪器不再可靠地工作，并且 无法修复，因为它不受供应商的支持。用于购买替换质量的资金 因此需要光谱仪，因为该仪器在毒素鉴定的第一步中是必不可少的。 在这种毒素鉴定之后，感兴趣的毒素将通过氨基酸测序进一步表征， 化学或重组合成及核磁共振结构分析。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. The gating-modifier toxins are a subgroup of these toxins that change the voltage of activation of the channels by binding to the voltage sensor domains (VSDs) of the channel. 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. 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 channel NaV1.5 and NaV1.7 of the peripheral nervous system that is involved in pain transmission) will be 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 isolate interacting toxins from the crude venoms of several animal species that are known to contain gating-modifier toxins. Initial identification of such toxins will be achieved by MALDI-TOF mass spectrometry. The instrument currently used for this purpose no longer works reliably and cannot be fixed since it is out of support from the vendor. Funding for the purchase of a replacement mass spectrometer is therefore requested, since this instrument is essential in the first step of toxin characterization. Following this toxin identification, toxins of interest will be further characterized by amino acid sequencing, chemical or recombinant synthesis and 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.