Covalent labeling endogenous G-protein coupled receptors in living cells

共价标记活细胞中的内源性 G 蛋白偶联受体

基本信息

批准号：
9891997
负责人：
JOHN T WILLIAMS
金额：
$ 19.25万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9891997
关键词：
Absence of pain sensation Acute Pain Address Affinity Alexa594 Animals Antibodies Area Binding Binding Sites Brain Brain region Cells Clinical Treatment Coupled Detection Development Dissociation Electrophysiology (science)Epitopes Future G-Protein-Coupled Receptors Gastrointestinal Motility Goals Imidazole Immunohistochemistry Label Left Ligand Binding Ligands Link Location Lysine Maps Mental Depression Methods Midbrain structure Mus Naltrexone Nervous system structure Neurons Opioid Opioid Antagonist Opioid Receptor Pathway interactions Penetration Peripheral Nervous System Pharmacology Phenotype Population Preparation Property Rattus Reaction Reproducibility Respiration Rewards Series Side Site Slice Substantia nigra structure System Time Tissues Transgenic Animals Ventral Tegmental Area Viral Whole-Cell Recordings Work analog base chronic pain endogenous opioids expectation experimental study knockin animal locus ceruleus structure mu opioid receptors novel postsynaptic receptor receptor function small molecule

项目摘要

Opioid receptors are widely distributed in the central and peripheral nervous system and upon activation have manifold actions including; depression of respiration, activation of the reward pathway, disruption of normal gastrointestinal motility and analgesia. It is currently difficult to examine this widespread distribution in wild type animals. Antibodies against the opioid receptors are often very unreliable and the epitopes are most often intracellular such that detection in living tissue is not possible. The use of transgenic or knockin animals to identify receptors requires extensive characterization, can be limited by varied expression levels and has been limited to studies in mice. The goal of this proposal is to use a ligand directed approach to covalently label endogenous opioid receptors in living tissue. The guide compound, naltrexamine, is a non-selective opioid antagonist that has high affinity for all subtype opioid receptors. Naltrexamine will be coupled through a series of linkers, a reactive acyl imidazole and fluorescent ligand. Once bound to the receptor, the reactive acyl imidazole reacts with a lysine on the receptor and simultaneously releases the naltrexamine. Thus natrexamine is free to dissociate from the receptor, however the fluorescent ligand is left covalently bound to the receptor. Preliminary results indicate that this compound interacts very effectively with the mu-opioid receptor. Thus endogenous receptor are labeled and the labeling is not dependent on species. This compound and future refinements will be used areas of the CNS where opioid receptors are expressed in only a small population of neurons. This proposal will center on the VTA and Substantia Nigra in mouse and rat. Opioid receptor expressing neurons in living brain slices will be identified and whole cell recordings will be made. By targeting these cells a complete understanding of opioid action in this key area of the reward system will be possible. The results of this cell based study will address a long-standing question surrounding the mechanisms that underlie one aspect of the addictive properties of opioids.

阿片类药物受体广泛分布在中央和周围神经中系统和激活后具有多种作用，包括：抑郁症呼吸，激活奖励途径，正常的破坏胃肠道运动和镇痛。目前很难检查一下野生型动物的广泛分布。抗阿片类药物的抗体受体通常非常不可靠，并且表位最常见于细胞内因此，无法在活组织中进行检测。使用转基因或敲击动物以识别受体需要广泛的特征，可以是受各种表达水平的限制，并且仅限于小鼠的研究。这该建议的目标是使用配体的定向方法共价标记活组织中的内源性阿片受体。指南化合物Naltrecamine，是一种非选择性阿片类药物拮抗剂，对所有亚型阿片类药物具有高亲和力受体。纳曲胺将通过一系列接头耦合，一种反应性酰基咪唑和荧光配体。一旦结合到受体，反应性酰基咪唑在受体上与赖氨酸反应，并同时释放纳曲胺。因此，纳特雷敏可以自由与受体分离，但是荧光配体与受体共价结合。初步结果表明该化合物与Mu-Apoid非常有效相互作用受体。因此，内源性受体被标记，标记不依赖关于物种。这种化合物和未来的精炼将用于中枢神经系统的区域阿片受体仅在少量神经元中表达。这提案将以小鼠和大鼠为中心，并以VTA为中心和黑质Nigra。阿片类药物将鉴定出在活脑切片中表达神经元的受体并全细胞录音将进行。通过将这些单元靶向完全了解在奖励系统的这一关键领域中，阿片类药物的行动将是可能的。结果这项基于细胞的研究将解决围绕该细胞的长期问题阿片类药物成瘾性特性的一个方面的机制。