CHARACTERIZATION OF U-97775 AS A GABA(A) RECEPTOR-LIGAND OF DUAL FUNCTIONALITY IN CLONED RAT GABA(A) RECEPTOR SUBTYPES

1 U-97775 (tert-butyl arbonyl]-imidazo[1,5-a])quinoxaline-3-carboxylat e) is a novel GABA(A) receptor ligand of dual functionality and was ch aracterized for its interactions with cloned rat GABA(A) receptors exp ressed in human embryonic kidney cells. 2 The drug produced a bell-sha ped dose-response profile in the alpha 1 beta 2 gamma 2 receptor subty pe as monitored with GABA-induced Cl- currents in the whole cell patch -clamp technique. At low concentrations (<0.5 mu M), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 mu M G ABA response (control). An agonist interaction of U-97775 with the ben zodiazepine site is suggested, because Ro 15-1788 (an antagonist at th e benzodiazepine site) abolished the current increase and [H-3]-flunit razepam binding was inhibited by U-97775 with a K-i of 1.2 nM. 3 The e nhancement of GABA currents progressively disappeared as the U-97775 c oncentration was raised above 1 mu M, and the current amplitude was re duced to 40% below the control at 10 mu M U-97775. The current inhibit ion by U-97775 (10 mu M) was not affected by Ro 15-1788. It appears th at U-97775 interacts with a second site on GABA receptors, distinct fr om the benzodiazepine site, to reverse its agonistic activity on the b enzodiazepine site and also to inhibit GABA currents. 4 U-97775 at low concentrations reduced and at high concentrations enhanced [S-35]-TBP S binding. Ro 15-1788 selectively blocked the effect of U-97775 at low concentrations. Analysis of the binding data in the presence of Ro 15 -1788 yielded a single low affinity site with an estimated K-d of 407 nM. 5 In other alpha beta gamma receptor subtypes, U-97775 at low conc entrations enhanced Cl- currents in the alpha 3 beta 2 gamma 2, but no t in the alpha 6 beta 2 gamma 2 subtype. On the other hand, U-97775 at high concentrations reduced Cl- currents in all the receptor subtypes we examined, including those of two subunits, alpha 1 beta 2, beta 2 gamma 2 and alpha 1 gamma 2 subtypes. 6 Therapeutically, U-97775 could be unique among benzodiazepine ligands because of its ability to limi t its own agonistic activity such that, at high doses the appearance o f agonistic activity would be delayed until occupancy of its second si te wanes. This property should make the total agonistic activity of U- 97775 relatively constant over a wide range of drug doses, and may min imize its liability to abuse.