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.

生物膜可渗透外源性阿片类药物（两种植物来源的分子） 例如吗啡，以及存在数百种的合成分子。现在是基因编码的 荧光生物传感器技术使我们能够测量中性细胞器内的阿片类药物，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分子 荧光报告基因，iOpioidSnFR，家族（图 1）。项目实施前正在进行的实验 开始，将把 iOpioidSnFR 系列扩展到 µ-阿片类激动剂的主要类别。 目标 1 进一步扩展 iOpioidSnFR 用于酸性细胞器内的测量，例如 内体和突触小泡。目标 1a 利用当前循环排列的绿色 荧光蛋白（cpGFP）部分。 Aim 1b 开发了新颖的循环排列 HaloTags， 对 pH 值不敏感。目标 1c，扩展现有测量以测量阿片类药物的进入 进入细胞器，以及从细胞器排出。量化涉及动态和稳态 状态测量。 目标 2 检验阿片类药物的某些作用是在突触小泡后产生的假设 通过酸捕获积累阿片类药物。然后突触小泡会释放阿片类药物 突触前刺激。该机制将扩展外源性药物的病理药理学 阿片类药物从多种类型的突触前神经元中释放，甚至是那些不释放的神经元 释放内源性阿片肽。目标 2a 进一步提高 iOpioidSnFR 敏感性 所需的纳摩尔水平。目标 2b 确定测试突触前的最灵敏方法 发布。 目标 3 检验以下假设：表达 µ-阿片受体的大脑区域在程度和范围上存在差异。 细胞器阿片类药物的时机。 Aim 3a 生成编码“floxed”的腺相关病毒载体 i阿片类药物 SnFR。这些将在囊泡 GABA 转运蛋白 (vGAT) 的控制下表达 cre 重组酶在合适的小鼠品系中。 Aim 3b 测量腹侧被盖脑切片 区域 (VTA) / 黑质网状部 (SnR)、导水管周围灰质 (PAG) 和腹侧 苍白球 (VP)。该结果将有助于持续努力了解细胞和分子 µ-阿片类配体耐受的基础。