Opioids inside Organelles

细胞器内的阿片类药物

• 批准号: 9810082
• 负责人: Henry A. Lester
• 金额: $ 20.56万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810082
• 关键词: Acids; Area; Betaine; Binding; Binding Proteins; Biological; Biosensor; Brain; Brain region; Cartoons; Cell Membrane Permeability; Cell membrane; Choline; Collaborations; Coupled; Crystallization; Cytoplasm; Dangerousness; Data; Dose; Endoplasmic Reticulum; Endosomes; Enterobacteria phage P1 Cre recombinase; Exploratory/Developmental Grant; Family; Fluorescence; Globus Pallidus; Green Fluorescent Proteins; Left; Ligands; LoxP-flanked allele; Measurement; Measures; Methods; Molecular; Molecular Conformation; Morphine; Mus; Neurons; Nicotine; Opioid; Opioid Peptide; Opioid agonist; Opioid user; Organelles; Periplasmic Binding Proteins; Pharmaceutical Preparations; Pharmacology; Plants; Property; Protein Engineering; Proteins; Publishing; Reporter; Research Personnel; Research Project Grants; Resolution; Site; Slice; Substantia nigra structure; Synaptic Vesicles; Techniques; Testing; Time; Ventral Tegmental Area; Venus Flytrap; adeno-associated viral vector; analog; base; endogenous opioids; experimental study; extracellular; midbrain central gray substance; mu opioid receptors; nanomolar; novel; opioid overdose; presynaptic; presynaptic neurons; response; synthetic opioid; temporal measurement; vesicular GABA transporter

Biological membranes are permeable to exogenous opioid drugs--both plant-derived molecules such as morphine, and synthetic molecules of which hundreds exist. Now a genetically encoded fluorescent biosensor technique allows us to measure opioids within neutral organelles such as the endoplasmic reticulum (ER). We term these molecules the intensity-based opioid-sensitive fluorescent reporter, iOpioidSnFR, family (Figure 1). Ongoing experiments, before the project begins, will extend the iOpioidSnFR family to the major classes of µ-opioid agonists. Aim 1 Further extends the iOpioidSnFRs for measurements within acidic organelles such as endosomes and synaptic vesicles. Aim 1a utilizes the present circularly permutated green fluorescent protein (cpGFP) moiety. Aim 1b develops novel circularly permuted HaloTags, which are pH-insensitive. Aim 1c, Extends the existing measurements to measure the entry of opioids into organelles, and their exit from organelles. Quantification involves both dynamics and steady- state measurements. Aim 2 tests the hypothesis that some effects of opioid drugs result after synaptic vesicles accumulate opioids via acid trapping. The synaptic vesicles would then release the opioids upon presynaptic stimulation. This mechanism would extend the patho-pharmacology of exogenous opioids to their release from many types of presynaptic neurons—even those neurons that do not release endogenous opioid peptides. Aim 2a evolves iOpioidSnFR sensitivity further, to the required nanomolar levels. Aim 2b Identifies the most sensitive method for testing presynaptic release. Aim 3 tests the hypothesis that brain regions expressing µ-opioid receptors vary in the extent and timing of organellar opioids. Aim 3a generates adeno-associated viral vectors that encode “floxed” iOpioidSnFRs. These will be expressed under the control of vesicular GABA transporter (vGAT) cre recombinase in suitable mouse lines. Aim 3b measures in brain slices from ventral tegmentum area (VTA) / substantia nigro pars reticulata (SnR), periaqueductal gray (PAG), and ventral pallidum (VP).The results will aid in the ongoing efforts to understand the cellular and molecular basis of tolerance to µ-opioid ligands.

生物膜可渗透到外源性阿片类药物 - 植物衍生的分子 例如吗啡和数百个存在的合成分子。现在是遗传编码的 荧光生物传感器技术使我们能够测量中性细胞器中的阿片类药物，例如 内质网（ER）。我们将这些分子称为基于强度的阿片类药物敏感 荧光记者，iopioidsnfr，家族（图1）。正在进行的实验，在项目之前 开始，将将iopioidsnfr家族延伸到主要的阿片类激动剂的主要类别。 AIM 1进一步扩展了二倍菌素，以进行酸性细胞器中的测量 内体和突触蔬菜。 AIM 1A利用当前的圆形排列的绿色 荧光蛋白（CPGFP）部分。 AIM 1B开发新的循环置换的挂钩，这是 对pH不敏感。 AIM 1C，扩展现有测量值以测量OIDS的进入 进入细胞器，并从细胞器中退出。定量涉及动力学和稳态 国家测量。 AIM 2检验了一个假设，即突触蔬菜后产生阿片类药物的某些作用 通过酸捕获积累阿片类药物。然后，突触蔬菜会在 突触前刺激。这种机制将扩展外源性的病理学 从多种类型的突触前神经元中释放出卵子类药物，即使是那些没有的神经元 释放内源性阿片类肽。 AIM 2A进一步发展了iopioidsnfr的灵敏度 所需的纳摩尔水平。 AIM 2B识别测试突触前的最敏感方法 发布。 AIM 3检验了以下假设：表达µ-阿片受体的大脑区域在范围内有所不同 有机化阿片类药物的时机。 AIM 3A生成与腺相关的病毒载体，该病毒向量编码为“ floxed” iopioidsnfrs。这些将在囊泡GABA转运蛋白（VGAT）的控制下表达 合适的小鼠系中的CRE重组酶。 AIM 3B脑切片中的3B测量 面积（VTA） /底块nigro pars enticulata（SNR），灰灰色（PAG）和腹侧 Pallidum（VP）。结果将有助于理解细胞和分子的持续努力 对µ-阿片类配体的耐受性的基础。