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Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes

Research output: Contribution to journalJournal articleResearchpeer-review

Standard

Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes. / Falk-Petersen, Christina B; Søgaard, Rikke; Madsen, Kenneth L; Klein, Anders B; Frølund, Bente; Wellendorph, Petrine.

In: Basic & Clinical Pharmacology & Toxicology, Vol. 121, No. 2, 08.2017, p. 119-129.

Research output: Contribution to journalJournal articleResearchpeer-review

Harvard

Falk-Petersen, CB, Søgaard, R, Madsen, KL, Klein, AB, Frølund, B & Wellendorph, P 2017, 'Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes' Basic & Clinical Pharmacology & Toxicology, vol. 121, no. 2, pp. 119-129. https://doi.org/10.1111/bcpt.12778

APA

Falk-Petersen, C. B., Søgaard, R., Madsen, K. L., Klein, A. B., Frølund, B., & Wellendorph, P. (2017). Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes. Basic & Clinical Pharmacology & Toxicology, 121(2), 119-129. https://doi.org/10.1111/bcpt.12778

Vancouver

Falk-Petersen CB, Søgaard R, Madsen KL, Klein AB, Frølund B, Wellendorph P. Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes. Basic & Clinical Pharmacology & Toxicology. 2017 Aug;121(2):119-129. https://doi.org/10.1111/bcpt.12778

Author

Falk-Petersen, Christina B ; Søgaard, Rikke ; Madsen, Kenneth L ; Klein, Anders B ; Frølund, Bente ; Wellendorph, Petrine. / Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes. In: Basic & Clinical Pharmacology & Toxicology. 2017 ; Vol. 121, No. 2. pp. 119-129.

Bibtex

@article{bf741b40fa0b43b7a46836bca67effdd,
title = "Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes",
abstract = "δ-Containing GABAA receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ-containing GABAA receptors. Inconveniently, the recombinant expression of especially α4 β1/3 δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96-well format cell-based assay for extrasynaptic α4 β1/3 δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABAA δ-subunit. Upon co-transfection of α4 and β1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 β1/3 δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α4 β1/3 δ and binary α4 β1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn(2+) had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF, were non-selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set-up, based on transient expression of human α4 and β1/3 subunits into a δ-subunit stable HEK293 Flp-In™ cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABAA receptors.",
author = "Falk-Petersen, {Christina B} and Rikke S{\o}gaard and Madsen, {Kenneth L} and Klein, {Anders B} and Bente Fr{\o}lund and Petrine Wellendorph",
note = "{\circledC} 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).",
year = "2017",
month = "8",
doi = "10.1111/bcpt.12778",
language = "English",
volume = "121",
pages = "119--129",
journal = "Basic & Clinical Pharmacology & Toxicology",
issn = "1742-7835",
publisher = "Wiley-Blackwell",
number = "2",

}

RIS

TY - JOUR

T1 - Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes

AU - Falk-Petersen, Christina B

AU - Søgaard, Rikke

AU - Madsen, Kenneth L

AU - Klein, Anders B

AU - Frølund, Bente

AU - Wellendorph, Petrine

N1 - © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

PY - 2017/8

Y1 - 2017/8

N2 - δ-Containing GABAA receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ-containing GABAA receptors. Inconveniently, the recombinant expression of especially α4 β1/3 δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96-well format cell-based assay for extrasynaptic α4 β1/3 δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABAA δ-subunit. Upon co-transfection of α4 and β1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 β1/3 δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α4 β1/3 δ and binary α4 β1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn(2+) had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF, were non-selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set-up, based on transient expression of human α4 and β1/3 subunits into a δ-subunit stable HEK293 Flp-In™ cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABAA receptors.

AB - δ-Containing GABAA receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ-containing GABAA receptors. Inconveniently, the recombinant expression of especially α4 β1/3 δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96-well format cell-based assay for extrasynaptic α4 β1/3 δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABAA δ-subunit. Upon co-transfection of α4 and β1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 β1/3 δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α4 β1/3 δ and binary α4 β1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn(2+) had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF, were non-selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set-up, based on transient expression of human α4 and β1/3 subunits into a δ-subunit stable HEK293 Flp-In™ cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABAA receptors.

U2 - 10.1111/bcpt.12778

DO - 10.1111/bcpt.12778

M3 - Journal article

VL - 121

SP - 119

EP - 129

JO - Basic & Clinical Pharmacology & Toxicology

T2 - Basic & Clinical Pharmacology & Toxicology

JF - Basic & Clinical Pharmacology & Toxicology

SN - 1742-7835

IS - 2

ER -

ID: 180916856