Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye

Alisa Knapman; Mark Connor

doi:10.1007/978-1-4939-1708-2_14

Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye

Alisa Knapman, Mark Connor

Research output: Chapter in Book/Report/Conference proceeding › Other chapter contribution › peer-review

9 Citations (Scopus)

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.

Original language	English
Title of host publication	Opioid Receptors
Subtitle of host publication	Methods and Protocols
Editors	Santi M. Spampinato
Place of Publication	Totowa, NJ
Publisher	Springer, Springer Nature
Pages	177-185
Number of pages	9
Edition	1
ISBN (Electronic)	9781493917082
ISBN (Print)	9781493917075
DOIs	https://doi.org/10.1007/978-1-4939-1708-2_14
Publication status	Published - 2015

Publication series

Name	Methods in Molecular Biology: Methods and Protocols
Publisher	Springer
Volume	1230
ISSN (Print)	1064-3745

Keywords

adenylyl cyclase
AtT-20
CHO
fluorescent assay
GIRK
high-throughput screening
membrane potential
μ-opioid receptor

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10.1007/978-1-4939-1708-2_14

Cite this

Knapman, A., & Connor, M. (2015). Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye. In S. M. Spampinato (Ed.), Opioid Receptors: Methods and Protocols (1 ed., pp. 177-185). (Methods in Molecular Biology: Methods and Protocols; Vol. 1230). Springer, Springer Nature. https://doi.org/10.1007/978-1-4939-1708-2_14

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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.",

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Knapman, A & Connor, M 2015, Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye. in SM Spampinato (ed.), Opioid Receptors: Methods and Protocols. 1 edn, Methods in Molecular Biology: Methods and Protocols, vol. 1230, Springer, Springer Nature, Totowa, NJ, pp. 177-185. https://doi.org/10.1007/978-1-4939-1708-2_14

Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye. / Knapman, Alisa; Connor, Mark.

Opioid Receptors: Methods and Protocols. ed. / Santi M. Spampinato. 1. ed. Totowa, NJ : Springer, Springer Nature, 2015. p. 177-185 (Methods in Molecular Biology: Methods and Protocols; Vol. 1230).

Research output: Chapter in Book/Report/Conference proceeding › Other chapter contribution › peer-review

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AB - 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.

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Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye

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