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A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels

Millennium Pharmaceuticals, Inc., Cambridge, MA

Martin Kirk

Molecular Devices Corporation, Sunnyvale, CA, Celtor Biosystems, Santa Clara, CA

Amy F. Garcia

Millennium Pharmaceuticals, Inc., Cambridge, MA

Alejandra Raimondi

Millennium Pharmaceuticals, Inc., Cambridge, MA

Mats H. Holmqvist

Millennium Pharmaceuticals, Inc., Cambridge, MA

Kimberly K. Flint

Millennium Pharmaceuticals, Inc., Cambridge, MA

Dejan Bojanic

Millennium Pharmaceuticals, Inc., Cambridge, MA

Peter S. Distefano

Millennium Pharmaceuticals, Inc., Cambridge, MA, Elixir Pharmaceuticals, Inc., Cambridge, MA

Rory Curtis

Millennium Pharmaceuticals, Inc., Cambridge, MA, Elixir Pharmaceuticals, Inc., Cambridge, MA

Yu Xie

Millennium Pharmaceuticals, Inc., Cambridge, MA

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC₄(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC₄(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K⁺ channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC₄(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC₄(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K_ATP¹ and hERG.² Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.

Journal of Biomolecular Screening, Vol. 7, No. 1, 79-85 (2002)
DOI: 10.1177/108705710200700110


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