177 Santi M. Spampinato (ed.), Opioid Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 1230, DOI 10.1007/978-1-4939-1708-2_14, © Springer Science+Business Media New York 2015 Chapter 14 Fluorescence-Based, High-Throughput Assays for μ-Opioid Receptor Activation Using a Membrane Potential-Sensitive Dye Alisa Knapman and Mark Connor 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 hyper- polarization, 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. Key words Adenylyl cyclase, AtT-20, CHO, Fluorescent assay, GIRK, High-throughput screening, Membrane potential, μ-Opioid receptor 1 Introduction The μ-opioid receptor (MOPr) is a G-protein-coupled receptor (GPCR), and is the primary target for opioid analgesics [1]. There is increasing evidence that different ligands can stabilize GPCRs including MOPr in various active conformations, leading to the preferential activation of distinct signaling pathways via Gα and Gβγ subunits [2, 3]. The development of novel opioid pharmaco- therapies is now beginning to focus on identifying MOPr ligands that can selectively activate a subset of effector pathways associated with analgesia, without activating pathways leading to adverse effects [4–7]. Drug development typically involves the screening of large libraries of lead compounds to identify those capable of binding to and signaling via a receptor. The vast array of lead compounds available requires high-throughput screening (HTS) methods to