[H-3] RY-80 - A HIGH-AFFINITY, SELECTIVE LIGAND FOR GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS CONTAINING ALPHA-5 SUBUNITS

The radiochemical synthesis and pharmacological properties are describ ed of[H-3]RY 80 tylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1, 4]benzodiazepine-3-carboxylate, [ethyl-H-3]). This compound is one of a series of 8-substituted imidazobenzodiazepines that exhibits both hi gh affinity and selectivity for gamma-aminobutyric acid (GABA)(A) rece ptors containing alpha-5 subunits. Saturable, high-affinity (K-d simil ar to 0.7 nM) binding of [H-3]RY 80 was observed in hippocampal membra nes. The maximum number (B-max) of [H-3]RY 80 binding sites was simila r to 18% of that obtained with [H-3]flunitrazepam, a radioligand that labels all ''diazepam-sensitive'' GABA(A) receptors. This value is con sistent with previous estimates (10-20%) of the proportion of rat hipp ocampal GABA(A) receptors containing alpha-5 subunits determined by im munoprecipitation with selective antibodies and competition experiment s using an alpha-5-selective ligand. In recombinant GABA(A) receptors composed of alpha-5 beta-3 gamma-2 subunits, the K-d of [H-3]RY 80 (si milar to 0.5 nM) was consistent with the value obtained in hippocampus , whereas the B-max value was not significantly different from that ob tained with [H-3]flunitrazepam. The potencies of several benzodiazepin e site ligands to inhibit [H-3]RY 80 binding to hippocampal membranes were in agreement with the values obtained in recombinant (alpha-5 bet a-3 gamma-2) GABA(A) receptors. [H-3]RY 80 was used both in a ''GABA s hift'' assay to correctly predict the in vivo actions of a novel, alph a-5-selective ligand and to characterize a population of GABA(A) recep tors containing alpha-5 subunits in neonatal rat cortex. These finding s demonstrate that [H-3]RY 80 can be used as a radioligand to examine the properties of GABA(A) receptors containing alpha-5 subunits.