Fluorometric Imaging Plate Reader (FLIPR®) membrane potential dye (FMP-Red-Dye) is a proprietary tool for basic discovery and high throughput drug screening for G-protein coupled receptors and ion channels. We optimized and validated this potentiometric probe to assay functional modulators of heterologous expressed GABAA receptor (GABAAR) isoforms (synaptic α1β3γ2, extrasynaptic α4β3δ, and β3 homopentomers). High-resolution mass spectrometry identified FMP-Red-dye as DisSBAC1(3). GABAAR expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane (Em) potentials compared to GABAAR-null cells. The channel blockers picrotoxin, fipronil, tetramethylenedisulfotetramine (TETS), and the competitive antagonist bicuculline reduced fluorescence near the levels in GABAAR-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABAAR-mediated outward Cl- current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABAAR isoforms consistent with results from voltage-clamp experiments (EC50 values for α4β3δ, α1β3γ2, β3 homopentamers were 6 ± 1 nM, 40 ± 11 nM, >18 mM), whereas GABAAR-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABAAR expressing cells in the absence of GABA and demonstrated positive allosteric modulation (PAM) in the presence of GABA, whereas benzodiazepines only exhibited PAM activity. Of 20 NAS tested, allopregnanolone, (3α,5α,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile (XJ-42), pregnanolone, 5β-pregnan-3α,21-diol-20-one, and ganaxolone showed the highest potency. The FMP-Red-Dye-based assay described here provides a sensitive and quantitative method of assessing the activity of GABAAR agonists, antagonists and PAMs on diverse GABAAR isoforms. The assay has a wide range of applications, including screening for antiseizure agents and identifying channel blockers of interest to insecticide discovery or biosecurity.
- GABAA, GABAC
- Ion channel regulation
- Mass Spectroscopy
- Imaging with fluorescent indicators (e.g. Ca2+ imaging)
- The American Society for Pharmacology and Experimental Therapeutics