Visualizing endogenous opioid receptors in living neurons using ligand-directed chemistry
Visualizing endogenous opioid receptors in living neurons using ligand-directed chemistry
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DOI：
10.7554/elife.49319
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发表时间：
2019-10-07
期刊：
影响因子：
7.7
通讯作者：
Williams, John T.
中科院分区：
文献类型：
--
作者：
Arttamangkul, Seksiri;Plazek, Andrew;Williams, John T.
识别具有功能性阿片受体的神经元是理解阿片类药物的细胞、突触和系统作用的基础。目前的技术仅限于对固定组织的事后分析。在这里，我们开发了一种名为纳曲胺-阿昔咪唑(NAI)的荧光探针，用于标记阿片受体，该方法基于一种被称为“无迹亲和标记”的化学方法。在这种方法中，高亲和力拮抗剂纳曲沙明被用作阿昔咪唑在受体上转移反应的引导分子。这个反应产生一种与受体共价连接的荧光染料，而纳曲胺被释放并离开结合部位。这种试剂诱导的标记使大鼠和小鼠脑内对阿片敏感的神经元得以可视化，而不会失去荧光标记的受体的功能。在活体组织中定位内源性受体的能力将有助于确定阿片受体在野生型动物中枢神经系统中的分布和生理作用。
Identifying neurons that have functional opioid receptors is fundamental for the understanding of the cellular, synaptic and systems actions of opioids. Current techniques are limited to post hoc analyses of fixed tissues. Here we developed a fluorescent probe, naltrexamine-acylimidazole (NAI), to label opioid receptors based on a chemical approach termed 'traceless affinity labeling'. In this approach, a high affinity antagonist naltrexamine is used as the guide molecule for a transferring reaction of acylimidazole at the receptor. This reaction generates a fluorescent dye covalently linked to the receptor while naltrexamine is liberated and leaves the binding site. The labeling induced by this reagent allowed visualization of opioid-sensitive neurons in rat and mouse brains without loss of function of the fluorescently labeled receptors. The ability to locate endogenous receptors in living tissues will aid considerably in establishing the distribution and physiological role of opioid receptors in the CNS of wild type animals.