title: Miniaturization and HTS of a FRET-based membrane potential assay for K(ir) channel inhibitors.
abstract: The K(ir) family of potassium-selective ion channels is characterized by their inward (anomalous) rectifying current-voltage relationship. K(ir) channels are widely expressed in mammalian cells and through their role in regulation of the cell membrane potential have been implicated in diverse physiological functions. To enable the identification of novel K(ir) channel inhibitors, a fluorescence resonance energy transfer (FRET)-based membrane potential assay was developed using a Chinese hamster ovary cell line stably expressing a human K(ir) channel. The FRET-based assay incorporates the use of two dyes {N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidylethanolamine (CC2-DMPE) and bis(1,3-diethylthiobarbiturate)trimethine oxonol [DiSBAC(2)(3)]} to track changes in membrane potential, thus enabling all of the advantages of ratiometric readout: reduced inaccuracies arising from well-to-well variation in cell number, dye loading, signal intensities, and plate inconsistencies. The assay was miniaturized to a 1,536-well microtiter plate format and read on a fluorometric imaging plate reader (FLIPR(Tetra), Molecular Devices, Sunnyvale, CA). The assay was automated and utilized to perform a primary high-throughput screening campaign to identify novel inhibitors of the K(ir) channel.
authors.affiliation: Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454, USA.
authors.forename: Kelli
authors.initials: K
authors.lastname: Solly
authors.forename: Jason
authors.initials: J
authors.lastname: Cassaday
authors.forename: John P
authors.initials: JP
authors.lastname: Felix
authors.forename: Maria L
authors.initials: ML
authors.lastname: Garcia
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
authors.forename: Laszlo
authors.initials: L
authors.lastname: Kiss
mesh_terms: D000818:Animals; D001463:Barbiturates; D001514:Bee Venoms / Q000494:pharmacology; D016466:CHO Cells; D003374:Coumarins; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004983:Ethanolamines; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007555:Isoxazoles; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D013858:Thiobarbiturates
chemical_list: D001463:Barbiturates; D001514:Bee Venoms; D003374:Coumarins; D004983:Ethanolamines; D005456:Fluorescent Dyes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D007555:Isoxazoles; C502247:N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine; D026902:Potassium Channel Blockers; D013858:Thiobarbiturates; C029738:tertiapin; C025284:bis(1,3-diethylthiobarbiturate)trimethineoxonol