Structural determinants of lipid modulation of ligand-gated ion channels

PROJECY摘要/摘要 五聚体配基门控离子通道(PLGIC)在突触传递中起主要作用，并受到调制 由各种内源分子，包括磷脂、甾醇和脂肪酸。PLGIC也是 受小分子治疗药物(如麻醉药和抗癫痫药)的调节。结构机制是通过 哪些磷脂对pLGICs的调节作用知之甚少。阴离子磷脂是一种变构调节剂 哺乳动物的pLGICs，结构研究表明磷脂结合部位与磷脂结合部位重叠 小分子，如神经类固醇。这个项目的目标是研究一种假设，即血脂和 某些变构调节药物与pLGICs上的特定位点结合，并诱导它们的调节 通过对脂类结合的正面或负面影响来发挥作用。为了实现这一目标，我将结合使用 尖端技术，包括天然质谱学(MS)、共价化学修饰和贴片-- 规定脂类成分的巨型脂质体的钳制记录。要应用这些技术，我将使用 原型原核生物pLGIC，欧文氏菌配体门控离子通道(ELIC)，作为一个易于处理的模型系统。伊利克是 一种用于MS的理想系统，并易于表达和纯化用于生化和 重建研究。程实验室的工作已经通过MS测量了磷脂与ELIC的直接结合，并且 证明了阴离子磷脂的特异性结合减少了ELIC中的脱敏。以此为基础 在工作中，这项研究项目将解决两个目标。首先是确定磷脂的特异性和位置。 介导它们对ELIC的调节作用的结合。我假设磷脂头基团电荷 决定与ELIC的脂类结合亲和力，但疏水尾巴的结构(如长度和位置 不饱和度)是自然调制效应的关键决定因素。磷脂结合亲和力和 化学计量比将由MS确定，磷脂的功能相关结合部位将是 用甲硫磺酸盐(MTS)试剂进行诱变和化学修饰测定。第二 目的是阐明磷脂和变构调节药物之间的相互作用与ELIC的关系。 捆绑和调制。在这一目标下，我将确定别孕酮在ELIC中的结合部位 使用光亲和标记，然后检查这种标记(或MS中的非共价结合)对 磷脂结合。初步结果表明，allP增强了ELIC的脱敏作用，I假设 AllP通过竞争结合其他由 磷脂。脂质体的功能研究将确定allP是竞争性的还是非竞争性的 拮抗阴离子磷脂效应。这项工作将是理解调制的基础 PLGIC通过相关的小分子。在这项提案中制定的实验框架将是至关重要的 了解其他生物活性脂类和小分子调节剂调节通道的机制。

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.