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

肽毒素与人钠通道电压传感器域之间相互作用的生物物理分析

• 批准号: 10799056
• 负责人: Sebastien F Poget
• 金额: $ 9.95万
• 依托单位: COLLEGE OF STATEN ISLAND
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799056
• 关键词: Affect; Affinity; Amino Acids; Animals; Arrhythmia; Bacteria; Basic Science; Binding; Biological Assay; Biophysics; Cardiac; Cell membrane; Cells; Chemicals; Communication; Complex; Development; Disease; Drug Targeting; Electrophysiology (science); Epilepsy; Funding; Human; Human body; Ion Channel; Knowledge; MALDI-TOF Mass Spectrometry; Measures; Medicine; Membrane; Migraine; Muscle Cells; Mutation; Neoplasm Metastasis; Neuromuscular Diseases; Neurons; Pain; Pain management; Peptides; Peripheral Nervous System; Pharmacotherapy; Protein Isoforms; Recombinants; Regulation; Reporting; Resolution; Signal Transduction; Sodium Channel; Spider Venoms; Subgroup; System; Targeted Toxins; Therapeutic; Toxin; Vendor; Venoms; Work; biophysical analysis; biophysical techniques; cancer prevention; disease-causing mutation; drug development; experimental study; instrument; interest; mass spectrometer; mimetics; reconstitution; sensor; sodium channel proteins; sodium ion; sudden cardiac death; tool; transmission process; voltage

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.

电压门控钠通道调节钠离子通过细胞膜的快速和特定流动。 它们对于人体的功能非常重要，例如调节心跳和 神经细胞中的电信号传导。由钠通道突变引起的疾病包括致命的 心律失常、癫痫、神经肌肉疾病和严重偏头痛。此外，钠通道 也是治疗疼痛和预防癌症转移的有希望的目标。钠 通道是大量天然毒素的目标，其中许多是动物无法吸收的高度选择性肽毒素。 用于防御或制服猎物。这些毒素是生物活性化合物的宝库，具有 作为基础研究工具以及治疗药物开发的潜在应用 钠通道相关疾病。门控修饰毒素是这些毒素的一个亚组，它改变了 通过与通道的电压传感器域（VSD）结合来激活通道的电压。当前的 对门控修饰毒素作用方式的了解主要基于功能和突变研究。 一些毒素通道复合物的直接结构研究也已被报道，但分辨率在 毒素结合的区域通常较差。在这个项目中，从两个人体内分离出钠通道 VSD 钠通道亚型将用作毒素分离的目标，然后毒素将在功能上和 具有结构特征。此外，新的和/或已知的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，来自两个人类钠通道（心脏通道 NaV1.5 和 参与疼痛传递的周围神经系统的 NaV1.7）将在细菌中表达， 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重建 研究。重组 VSD 将用于从多种动物的粗毒液中分离出相互作用的毒素 已知含有门控修饰毒素的物种。此类毒素的初步鉴定将通过 MALDI-TOF 质谱分析。目前用于此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。购买替换质量的资金 因此需要光谱仪，因为该仪器在毒素表征的第一步中至关重要。 在此毒素鉴定之后，将通过氨基酸测序进一步表征感兴趣的毒素， 化学或重组合成以及NMR结构分析。 VSD 和 VSD 之间交互的细节 通过测量不同残基的突变方式，可以阐明已知或新的相互作用毒素。 通过电生理学和直接测量，毒素和 VSD 会影响结合亲和力和通道调节。 结合测定。这些实验的结果将提供可以利用的有用的结构信息 开发针对离子通道的药物来治疗心律失常和疼痛等疾病。