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

Abstract

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 KATP1 and hERG.2 Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.

Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine

“…Membrane potential (⌬⌿) was evaluated using 3,3-dipentyloxacarbocyanine (DiOC 5 ), a charged lipophilic dye that emits a fluorescent signal proportional to ⌬⌿. The validity of this method to assess channel activity approaches the reliability of classical patch clamp methods, as borne out by a compelling body of evidence (8)(9)(10). Overall, hBD-2 and crotamine altered ⌬⌿ in several target organisms versus untreated controls (Fig.…”

Plasma Membrane Potential Oscillations in Insulin Secreting Ins-1 832/13 Cells Do Not Require Glycolysis and Are Not Initiated by Fluctuations in Mitochondrial Bioenergetics

“…The ⌬ p indicator we have termed PMPI for plasma membrane potential indicator (19) is a proprietary constituent of the Molecular Devices FLIPR membrane potential assay kit and responds 14 times faster than bis-(1,3-dibutylbarbituric acid)-trimethine oxonol to changes in ⌬ p (24). The anionic indicator responds to a ⌬ p depolarization with an increased fluorescence as it enters the cell.…”

Membrane potential fluorescence: A rapid and highly sensitive assay for nicotinic receptor channel function

“…Among the functional assay techniques for such screening, fluorescence has attracted much attention of late (16,(18)(19)(20)(21)(22). The most widely used fluorescent probes for nicotinic receptor functional analysis have been the calcium chelating Fluo-and Fura-dyes developed by Tsien (39)(40)(41).…”

“…Functional assays for ion channels include classical electrophysiology (12,17), radioisotopic ion flux (13)(14)(15), and fluorescent assays using ion-or voltage-sensitive dyes (16,(18)(19)(20). In the current study, nicotinic receptor activation was measured by using a membrane potential-sensitive fluorescent dye (21,22) as an indicator of ionic flux in cell lines expressing various nicotinic receptor subtypes, both native (8,23,24) and transfected (25,26). This result was compared with measurements using a calcium-sensitive dye.…”