pH-Insensitive FRET Voltage Dyes

Abstract

Many high-throughput ion channel assays require the use of voltage-sensitive dyes to detect channel activity in the presence of test compounds. Dye systems employing Förster resonance energy transfer (FRET) between 2 membrane-bound dyes are advantageous in combining high sensitivity, relatively fast response, and ratiometric output. The most widely used FRET voltage dye system employs a coumarin fluorescence donor whose excitation spectrum is pH dependent. The authors have validated a new class of voltage-sensitive FRET donors based on a pyrene moiety. These dyes are significantly brighter than CC2-DMPE and are not pH sensitive in the physiological range. With the new dye system, the authors demonstrate a new high-throughput assay for the acid-sensing ion channel (ASIC) family. They also introduce a novel method for absolute calibration of voltagesensitive dyes, simultaneously determining the resting membrane potential of a cell. (Journal of Biomolecular Screening 2007:656-667)

Measuring Resting Membrane Potential Using the Fluorescent Voltage Reporters DiBAC4(3) and CC2-DMPE

“…As the inner leaflet becomes more negative (hyperpolarized), the anionic DiBAC 4 (3) leaves the cell and its signal decreases. Conversely, as the inner leaflet becomes more positive, more DiBAC 4 (3) enters, and the DiBAC 4 (3) signal gets more intense (brighter) (Maher et al 2007). By monitoring the brightness of these two fluorophores, you can, with the appropriate corrections applied to the images, determine relative V mem by analysis of the signals coming from the two fluorophores.…”

Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5

“…Although both dye systems employ negatively charged oxonols that can translocate across the plasma membrane in response to depolarization, the MD Blue dye uses an extracellular quencher to reduce fluorescence when the oxonol is at the outer leaf of the membrane, whereas the VSP relies on FRET with a coumarin dye that is restricted to the outer leaf. 17 Whether these differences underlie the (lack of) sensitivity of the respective dye systems to inhibition by compounds 2 and 3 is not clear. However, we limited our use of the membrane potential dye delay-to-onset profile until we had confirmed that hits from this approach were active in electrophysiology assays (Fig.…”

“…[14][15][16] Two membrane potential dyes are widely used for HTS: the FLIPR Membrane Potential dye (Molecular Devices, Sunnyvale, CA) that uses a fluorescence indicator in combination with a quencher and voltage sensor probes (VSPs) that rely on fluorescence-resonance energy transfer (FRET) between a voltage-sensing oxonol acceptor and a fluorescent membrane-bound coumarin dye. 17 In the current study, we compared the performance of these membrane potential dyes, along with a sodium-sensing dye, in assay development and found that these dye systems could only detect certain subsets of Na V 1.7 inhibitors using standard data analysis methods. In this article, we describe alternate approaches to analyzing these data to identify compounds with Na V 1.7 selective activity.…”

Citations

“…Finding a suitable pH-insensitive replacement for hydroxycoumarin is not trivial: in addition to being less vulnerable to acidic conditions, potential FRET donors must also persist on the outer face of the cell membrane, resist aggregation, and exhibit suitable excitation and emission wavelengths. In a recent report in the Journal of Biomolecular Screening , Maher et al. report that certain pyrenetrisulfonate dyes can meet these criteria, serving as FRET donors that are almost as effective as the standard coumarin-linked phospholipid donor.…”