Molecular pharmacology

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

PMID 28428226


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 GABAA receptor (GABAAR) 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]. GABAAR-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABAAR-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, 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, and β3 homopentamers were 6 ± 1, 40 ± 11, and >18 mM, respectively), 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 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 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.

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