基于荧光的高通量检测方法，用于使用膜电位敏感染料检测μ-阿片受体激活。
Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye.
作者信息
Knapman Alisa, Connor Mark
机构信息
Australian School of Advanced Medicine, Macquarie University, Level 1, 2 Technology Place, Sydney, NSW, 2109, Australia.
出版信息
Methods Mol Biol. 2015;1230:177-85. doi: 10.1007/978-1-4939-1708-2_14.
The development of new and improved opioid analgesics requires high-throughput screening (HTS) methods to identify potential therapeutics from large libraries of lead compounds. Here we describe two simple, real-time fluorescence-based assays of μ-opioid receptor activation that may be scaled up for HTS. In AtT-20 cells expressing the μ-opioid receptor (MOPr), opioids activate endogenous G protein gated inwardly rectifying K channels (GIRK channels), leading to membrane hyperpolarization. In Chinese hamster ovary cells expressing MOPr, adenylyl cyclase activation via forskolin results in membrane hyperpolarization, which is inhibited by opioids. Changes in membrane potential can be measured using a proprietary membrane potential-sensitive dye. In contrast to many HTS methods currently available, these assays reflect naturalistic coupling of the receptor to effector molecules.
新型和改良阿片类镇痛药的研发需要高通量筛选（HTS）方法，以便从大量先导化合物库中识别潜在的治疗药物。在此，我们描述了两种简单的、基于实时荧光的μ-阿片受体激活检测方法，这些方法可扩大规模用于高通量筛选。在表达μ-阿片受体（MOPr）的AtT-20细胞中，阿片类药物激活内源性G蛋白门控内向整流钾通道（GIRK通道），导致膜超极化。在表达MOPr的中国仓鼠卵巢细胞中，通过福斯高林激活腺苷酸环化酶会导致膜超极化，而阿片类药物会抑制这种超极化。膜电位的变化可以使用一种专有的膜电位敏感染料来测量。与目前可用的许多高通量筛选方法不同，这些检测方法反映了受体与效应分子的自然偶联。
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