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Journal of Biomolecular Screening
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Comparative Study of Membrane Potential-Sensitive Fluorescent Probes and their Use in Ion Channel Screening Assays

Christian Wolff

Department of in vitro Pharmacology, UCB SA, Braine l'Alleud, Belgium, christian.wolff{at}ucb-group.com

Bruno Fuks

Department of in vitro Pharmacology, UCB SA, Braine l'Alleud, Belgium

Pierre Chatelain

Department of in vitro Pharmacology, UCB SA, Braine l'Alleud, Belgium

In this study, the authors compared and evaluated 4 membrane potential probes in the same cellular assay: the oxonol dye DiBAC4(3), the FLIPR membrane potential (FMP) dye (Molecular Devices), and 2 novel fluorescence resonance energy transfer (FRET) dye systems from PanVera [CC2-DMPE/DiSBAC2(3)] and Axiom [DiSBAC1(3)/DiSBAC1(5)]. The kinetic parameters of each membrane probe were investigated in RBL-2H3 cells expressing an endogenous inward rectifier potassium channel (IRK1). The FMP dye presented the highest signal over background ratio whereas the FRET dyes from PanVera gave the fastest response. The determination of IC50 values for 8 different channel modulators indicated a good correlation between the 4 membrane probe systems. The compound-dye interaction was evaluated in the presence of compounds at 10 [.proportional]M and clearly indicated no effect on the FMP or the PanVera donor dye, whereas some major interference with the oxonol probes was observed. Using a cell permeabilization assay in the presence of gramicidin, the authors concluded that the FRET dyes from PanVera and the FMP dye are unable to measure the gramicidin-induced cell membrane hyperpolarizations. The 4 dye systems were investigated under high-throughput screening (HTS) conditions, and their respective Z' parameter was deter-mined. The characteristics of each dye system and its potential use in HTS assays is discussed. (Journal of Biomolecular Screening 2003:533-543)

Key Words: fluorescence • membrane potential probes • high-throughput screening • fluorescence resonance energy transfer

Journal of Biomolecular Screening, Vol. 8, No. 5, 533-543 (2003)
DOI: 10.1177/1087057103257806
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