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: 10471051
• 负责人: John T Petroff
• 金额: $ 0.25万
• 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/10471051
• 关键词: Address; Affect; Affinity; Allopregnanolone; Anesthetics; Antidepressive Agents; Antiepileptic Agents; Binding; Binding Sites; Biochemical; Biological Models; Charge; Chemicals; Chemistry; Data; Dependence; Disease; Environment; Epilepsy; Erwinia; Fatty Acids; Foundations; Functional disorder; Gases; General anesthetic drugs; 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; Phosphatidylglycerols; 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; fatty acid analog; 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.

项目概要/摘要 五聚体配体门控离子通道 (pLGIC) 在突触传递中起主要作用，并受到调节 由多种内源性分子，包括磷脂、甾醇和脂肪酸。 pLGIC 还 由小分子疗法（例如麻醉剂和抗癫痫药）调节。其结构机理为 对于哪些磷脂调节 pLGIC 知之甚少。阴离子磷脂是变构调节剂 哺乳动物 pLGIC，结构研究表明磷脂结合位点与 pLGIC 的结合位点重叠 小分子，例如神经类固醇。该项目的目标是研究脂质和 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 通过对脂质结合产生积极或消极的影响。为了实现这个目标，我将结合使用 尖端技术，包括天然质谱 (MS)、共价化学修饰和补丁技术 确定脂质成分的巨型脂质体的钳记录。为了应用这些技术，我将使用 原型原核 pLGIC，欧文氏菌配体门控离子通道 (ELIC)，作为易于处理的模型系统。 ELIC 是 一个理想的 MS 系统，可以轻松表达和纯化突变蛋白，用于生化和 重组研究。 Cheng 实验室的工作通过 MS 测量了磷脂与 ELIC 的直接结合，并且 证明阴离子磷脂的特异性结合减少了 ELIC 中的脱敏。在此基础上 工作中，该研究项目将实现两个目标。首先是确定磷脂的特异性和位点 介导其对 ELIC 的调节作用的结合。我假设磷脂头基带电荷 决定了脂质与 ELIC 的结合亲和力，但疏水尾部的结构（例如长度和位置） 不饱和度）是天然调节效应的关键决定因素。磷脂结合亲和力和 化学计量将通过MS确定。磷脂的功能相关结合位点是 使用甲硫代磺酸盐 (MTS) 试剂进行诱变和化学修饰来确定。第二个 目的是阐明磷脂和与 ELIC 相关的变构调节药物之间的相互作用 结合和调制。在此目标下，我将确定 ELIC 中异孕酮 (alloP) 的结合位点 使用光亲和标记，然后检查该标记（或 MS 中的非共价结合）对 磷脂结合。初步结果表明 alloP 增强 ELIC 脱敏作用，我假设 alloP 通过竞争结合位点来诱导其药理作用，否则该位点被 磷脂。脂质体的功能研究将决定 alloP 是竞争性的还是非竞争性的 拮抗阴离子磷脂作用。这项工作将为理解调制奠定基础 pLGIC 由相关小分子组成。该提案中开发的实验框架至关重要 了解其他生物活性脂质和小分子调节剂的通道调节机制。