A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels.
The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(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 DiBAC(4)(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 DiBAC(4)(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 DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.
Baxter
Deborah F
DF
Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA.
Kirk
Martin
M
Garcia
Amy F
AF
Raimondi
Alejandra
A
Holmqvist
Mats H
MH
Flint
Kimberly K
KK
Bojanic
Dejan
D
Distefano
Peter S
PS
Curtis
Rory
R
Xie
Yu
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001331:Automation; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D002412:Cations; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D007473:Ion Channels; D007700:Kinetics; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D011189:Potassium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002412:Cations; D005456:Fluorescent Dyes; D007473:Ion Channels; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D011188:Potassium