Rapid Throughput Analysis of GABAA Receptor Subtype Modulators and Blockers Using DiSBAC1(3) Membrane Potential Red Dye

Abstract

Fluorometric imaging plate reader 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 GABA_A receptor (GABA_AR) isoforms (synaptic α1β3γ2, extrasynaptic α4β3δ, and β3 homopentomers). High-resolution mass spectrometry identified FMP-Red-Dye as 5,5'-(1-propen-1-yl-3-ylidene)bis[1,3-dimethyl-2-thio-barbituric acid]. GABA_AR-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABA_AR-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, and the competitive antagonist bicuculline reduced fluorescence near the levels in GABA_AR-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABA_AR-mediated outward Cl^- current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABA_AR isoforms consistent with results from voltage-clamp experiments (EC₅₀ values for α4β3δ, α1β3γ2, and β₃ homopentamers were 6 ± 1, 40 ± 11, and >18 mM, respectively), whereas GABA_AR-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABA_AR expressing cells in the absence of GABA and demonstrated positive allosteric modulation in the presence of GABA, whereas benzodiazepines only exhibited positive allosteric modulator (PAM) activity. Of 20 NAS tested, allopregnanolone, (3α,5α,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile, eltanolone, 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 GABA_AR agonists, antagonists, and PAMs on diverse GABA_AR 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.