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: 10515074
• 负责人: Sebastien F Poget
• 金额: $ 43.42万
• 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/10515074
• 关键词: Affect; Affinity; Amino Acids; Animals; Arrhythmia; Bacteria; Basic Science; Binding; Biological Assay; Biophysics; Cardiac; Cell membrane; Cells; Chemicals; Communication; Complement; Complex; Cryoelectron Microscopy; Development; Disease; Drug Design; Drug Targeting; Electrophysiology (science); Epilepsy; Escherichia coli; Fishes; Foundations; Human; Human body; Immobilization; Inclusion Bodies; Ion Channel; Ions; Knowledge; Lead; Mass Spectrum Analysis; Measures; Medicine; Membrane; Methods; Migraine; Muscle Cells; Mutation; NMR Spectroscopy; Nature; Neoplasm Metastasis; Neuromuscular Diseases; Neurons; Nuclear Magnetic Resonance; Pain; Pain management; Pathway interactions; Peptides; Peripheral Nervous System; Pharmacotherapy; Phospholipids; Physiological; Play; Protein Isoforms; Protocols documentation; Recombinants; Regulation; Reporting; Research; Resolution; Role; Sampling; Scorpions; Sea Anemones; Series; Signal Transduction; Site; Sodium Channel; Source; Specificity; Spider Venoms; Spiders; Structure; Surface Plasmon Resonance; System; Targeted Toxins; Techniques; Testing; Therapeutic; Toxin; Venoms; Vesicle; base; biophysical analysis; biophysical techniques; cancer prevention; disease-causing mutation; drug development; experimental study; interest; mimetics; mutant; neurotransmission; novel; patch clamp; protein aminoacid sequence; reconstitution; sensor; side effect; sodium channel proteins; sodium ion; sudden cardiac death; three dimensional structure; 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. 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.

电压门控钠通道调节钠离子通过细胞膜的快速且特定的流动。 它们对人体的功能非常重要，如心跳的调节， 神经细胞中的电信号由钠通道突变引起的疾病的例子包括致命的 心律失常、癫痫、神经肌肉紊乱和严重偏头痛。此外，钠离子通道 也是治疗疼痛和潜在预防癌症转移的有前景的靶点。钠 通道是由大量的天然毒素，其中许多是高度选择性的肽毒素，动物， 用于防御或制服猎物。这些毒素代表了生物活性化合物的宝库， 作为基础研究工具的潜在应用，以及在开发药物治疗 钠离子通道相关疾病一些针对钠通道的蜘蛛、蝎子和海葵毒素 通过结合到通道的电压传感器域（VSD）来改变通道的激活电压。 这类毒素被称为门控修饰毒素，由于其潜在的毒性， 以精细和受控的方式高度特异性地控制通道活动。目前对该模式的认识 门控修饰毒素的作用主要基于功能和突变研究。一些直接的结构 还报道了毒素-通道复合物的研究，但是在毒素-通道复合物的区域中的分辨率不高。 由于VSD的动力学性质和钠通道的大尺寸（~2000 氨基酸残基）。在这个项目中，从两个人钠通道亚型中分离出钠通道VSD， 将用作毒素分离的靶，然后将对毒素进行功能和结构表征。 此外，新的和/或已知的毒素和这些VSD之间的相互作用的结构细节将被 通过不同的生物物理学技术来阐明。 为了进行这些实验，使用来自两个人钠通道（心脏钠通道NaV1.5 和参与疼痛传递的外周神经系统的NaV1.7）将被重组 在细菌中表达并在适合于毒素下拉实验的膜模拟系统中重构 和生物物理相互作用研究。重组VSD将用于从细胞中钓出相互作用的毒素。 已知含有门控修饰毒素的几种动物的粗毒液。这些毒素将 进行质谱分析以确定它们的肽序列。然后，它们将在化学上 或生物合成产生用于进一步的NMR结构分析。VSD与 已知的或新的相互作用毒素将通过测量不同残基的突变如何被阐明。 毒素和VSD影响结合亲和力和通道调节，如通过电生理学和直接电生理学测量的。 结合测定。这些实验的结果将提供有用的结构信息，可以利用 在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方面。