Abstract

Inhibitory GABA_A receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk^– cells stably expressing human recombinant GABA_A subunits (α1β1–3γ2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl^– buffer using a voltage/ion probe reader. The influence of different βsubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at α1β1γ2, α1β2γ2, and α1β3γ2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC₅₀ values of 2.4 ± 0.2, 2.5 ± 0.2, and 1.3 ± 0.1 μM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC₅₀ values of 8.7 ± 0.9, 9.2 ± 0.5, and 11.7 ± 1.2, and 43.7 ± 6.4, 24.8 ± 1.6, and 26.1 ± 2.4 μM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate, pentobarbital, and steroids, 5α-pregnan-3α-ol-20-one and 5β-pregnan-3α-ol-20-one, were unaffected by the β isoform present in the receptor complex. However, several compounds displayed β2/3 subunit selectivity, notably loreclezole, R(–)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at β2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at α1β1γ2. Diflunisal was the most efficacious compound, eliciting greater potentiation than loreclezole (90 ± 14% and 109 ± 14% at β3 and β2, respectively, compared with 62 ± 6% and 56 ± 3%), whereas niflumic acid exhibited the lowest efficacy. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity. This study identifies and characterizes a variety of allosteric modulators for which βsubunits are an important determinant of efficacy and potency.