Structural determinants of lipid modulation of ligand-gated ion channels

配体门控离子通道脂质调节的结构决定因素

• 批准号: 10285984
• 负责人: John T Petroff
• 金额: $ 6.64万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10285984
• 关键词: Address; Affect; Affinity; Anesthetics; Antiepileptic Agents; Binding; Binding Sites; Biochemical; Biological Models; Charge; Chemicals; Chemistry; Data; Dependence; Disease; Environment; Epilepsy; Erwinia; Fatty Acids; Foundations; Functional disorder; Gases; Goals; Head; Homologous Gene; Hydrophobicity; Ion Channel Gating; Label; Learning; Length; Ligands; Light; Lipid Binding; Lipids; Liposomes; Mass Spectrum Analysis; Measures; Mediating; Membrane; Modification; Molecular; Mutagenesis; Neurodegenerative Disorders; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Phospholipid Interaction; Phospholipids; Photoaffinity Labels; Play; Positioning Attribute; Proteins; Reagent; Research; Research Personnel; Research Project Grants; Role; Site; Specificity; Sterols; Structural Models; Structure; Synaptic Transmission; System; Tail; Techniques; Testing; Work; addiction; analog; biophysical techniques; desensitization; design; field study; ion mobility; methanethiosulfonate; mutant; neurosteroids; novel; patch clamp; receptor; reconstitution; small molecule; small molecule therapeutics; stoichiometry

PROJECY SUMMARY/ ABSTRACT Pentameric ligand-gated ion channels (pLGICs) play a primary role in synaptic transmission, and are modulated by a variety of endogenous molecules, including phospholipids, sterols, and fatty acids. pLGICs are also modulated by small molecule therapeutics (e.g. anesthetics and anti-epileptics). The structural mechanism by which phospholipids modulate pLGICs is poorly understood. Anionic phospholipids are allosteric modulators of mammalian pLGICs, and structural studies suggest that phospholipid binding sites overlaps with binding sites of small molecules such as neuroteroids. The goal of this project is to investigate the hypothesis that lipids and certain allosteric modulating drugs bind to specific sites on pLGICs, and that these drugs induce their modulatory effect through a positive, or negative, effect on lipid binding. To accomplish this goal, I will use a combination of cutting edge techniques, including native mass spectrometry (MS), covalent chemical modification, and patch- clamp recordings of giant liposomes of defined lipid composition. To apply these techniques, I will use the prototypical prokaryotic pLGIC, Erwinia ligand-gated ion channel (ELIC), as a tractable model system. ELIC is an ideal system for MS and readily permits expression and purification of mutant proteins for biochemical and reconstitution studies. Work in the Cheng lab has measured direct binding of phospholipids to ELIC by MS, and demonstrated that specific binding of anionic phospholipids reduces desensitization in ELIC. Building upon this work, this research project will address two aims. The first is to determine the specificity and sites of phospholipid binding that mediate their modulatory effects on ELIC. I hypothesize that phospholipid head group charge determines the lipid binding affinity to ELIC, but that the structure of the hydrophobic tail (e.g. length and position of unsaturations) is the critical determinant of the native modulatory effect. Phospholipid binding affinity and stoichiometry will be determined by MS. The functionally-relevant binding sites for phospholipids will be determined using mutagenesis and chemical modification with methanethiosulfonate (MTS) reagents. The second aim is to elucidate the interaction between phospholipids and allosterically modulating drugs in relation to ELIC binding and modulation. Within this aim I will determine the sites of binding of allopregnanalone (alloP) in ELIC using photo-affinity labels, and then examine the effect this labeling (or non-covalent binding in MS) has on phospholipid binding. Preliminary results indicate that alloP enhances ELIC desensitization, and I hypothesize that alloP induces its pharmacologic effect by competing for binding of sites otherwise occupied by phospholipids. Functional studies in liposomes will determine whether alloP competitively or non-competitively antagonize anionic phospholipid effect. This work will be foundational in understanding the modulation of pLGICs by relevant small molecules. The experimental framework developed within this proposal will be critical in understanding the mechanism of channel modulation by other bioactive lipids and small molecule modulators.

PROJECY摘要/摘要 五聚合配体门控离子通道（PLGICS）在突触传播中起主要作用，并被调制 通过多种内源分子，包括磷脂，固醇和脂肪酸。 PLGICS也是 由小分子疗法（例如麻醉和抗癫痫病）调节。结构机制 哪种磷脂调节PLGIC的理解很少。阴离子磷脂是变构调节剂 哺乳动物PLGIC和结构研究表明，磷脂结合位点与结合位点重叠 小分子，例如神经类动物。该项目的目的是研究脂质和 某些变构调节药物与PLGIC上的特定位点结合，并且这些药物诱导其调节 通过对脂质结合的阳性或负面影响的影响。为了实现这一目标，我将使用 尖端技术，包括天然质谱法（MS），共价化学修饰和斑块 定义脂质组成的巨型脂质体的夹具记录。要应用这些技术，我将使用 典型的原型原核PLGIC，Erwinia配体门控离子通道（ELIC）作为可拖动的模型系统。 Elic是 MS的理想系统，很容易允许表达和纯化突变蛋白的生化和 重建研究。 Cheng Lab中的工作测量了MS的磷脂与Elic的直接结合， 表明阴离子磷脂的特异性结合减少了ELIC中的脱敏。以此为基础 工作，该研究项目将解决两个目标。首先是确定磷脂的特异性和位点 结合介导其调节作用的效果。我假设磷脂头组电荷 确定脂质结合与Elic的亲和力，但疏水尾巴的结构（例如长度和位置 不饱和）是天然调节作用的关键决定因素。磷脂结合亲和力 化学计量法将由MS确定。磷脂与功能相关的结合位点将是 使用甲烷硫酸盐（MTS）试剂使用诱变和化学修饰确定。第二个 目的是阐明磷脂和相对于ELIC的变构调节药物之间的相互作用 结合和调制。在此目标中，我将确定Alopregnanalone（Allop）的结合位点 使用照片亲和力标签，然后检查该标签（或MS中非共价结合）的影响 磷脂结合。初步结果表明Allop增强了敏感性，我假设 Allop通过竞争与其他人所占用的地点的结合来诱导其药理作用 磷脂。脂质体中的功能研究将确定竞争性还是非功能性研究 拮抗阴离子磷脂效应。这项工作将是理解调制的基础 相关小分子的PLGIC。本提案中开发的实验框架将是至关重要的 在理解其他生物活性脂质和小分子调节剂的通道调节机理时。