Structural determinants of lipid modulation of ligand-gated ion channels

项目总结/摘要 五聚体配体门控离子通道（pLGICs）在突触传递中起主要作用，并且被调节 通过多种内源性分子，包括磷脂、甾醇和脂肪酸。pLGIC还 通过小分子治疗剂（例如麻醉剂和抗癫痫剂）调节。结构机制， 对哪些磷脂调节pLGIC知之甚少。阴离子磷脂是磷脂酰胆碱的变构调节剂。 哺乳动物pLGIC，结构研究表明，磷脂结合位点重叠的结合位点， 小分子如神经类固醇。该项目的目标是调查脂质和 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作用 通过对脂质结合的正面或负面影响来影响。为了实现这一目标，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， 确定脂质组成的巨大脂质体的钳夹记录。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），作为易处理的模型系统。ELIC是 MS的理想系统，并且容易允许表达和纯化突变蛋白用于生物化学和 重建研究。Cheng实验室的工作已经通过MS测量了磷脂与ELIC的直接结合， 表明阴离子磷脂的特异性结合减少ELIC中的脱敏。在此基础上 这项研究计划将致力于两个目标。首先是确定磷脂的特异性和位点 结合，介导其对ELIC的调节作用。我假设磷脂头部基团电荷 决定了与ELIC的脂质结合亲和力，但疏水尾的结构（例如长度和位置 是天然调节作用的关键决定因素。磷脂结合亲和力和 将通过MS确定化学计量。 使用诱变和用甲硫基磺酸盐（MTS）试剂进行化学修饰来测定。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 绑定和调制。在此目标下，我将确定ELIC中别孕烷酮（alloP）的结合位点， 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，alloP增强ELIC脱敏，我假设 alloP通过竞争与其他受体的结合而诱导其药理作用， 磷脂脂质体中的功能研究将确定alloP是否竞争性或非竞争性地 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。在这一建议中制定的实验框架将是至关重要的 了解其他生物活性脂质和小分子调节剂对通道的调节机制。

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.