title: Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye.
abstract: 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.
authors.affiliation: Australian School of Advanced Medicine, Macquarie University, Level 1, 2 Technology Place, Sydney, NSW, 2109, Australia.
authors.forename: Alisa
authors.initials: A
authors.lastname: Knapman
authors.forename: Mark
authors.initials: M
authors.lastname: Connor
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000701:Analgesics, Opioid / Q000008:administration & dosage; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D008564:Membrane Potentials; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D015533:Transcriptional Activation
chemical_list: D000701:Analgesics, Opioid; D017450:Receptors, Opioid, mu; D000242:Cyclic AMP; D000262:Adenylyl Cyclases