title: [3H]RY 80: A high-affinity, selective ligand for gamma-aminobutyric acidA receptors containing alpha-5 subunits.
abstract: The radiochemical synthesis and pharmacological properties are described of [3H]RY 80 (ethyl-8-acetylene-5, 6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1, 4]benzodiazepine-3-carboxylate, [ethyl-3H]). This compound is one of a series of 8-substituted imidazobenzodiazepines that exhibits both high affinity and selectivity for gamma-aminobutyric acid (GABA)A receptors containing alpha-5 subunits. Saturable, high-affinity (Kd approximately 0.7 nM) binding of [3H]RY 80 was observed in hippocampal membranes. The maximum number (Bmax) of [3H]RY 80 binding sites was approximately 18% of that obtained with [3H]flunitrazepam, a radioligand that labels all "diazepam-sensitive" GABAA receptors. This value is consistent with previous estimates (10-20%) of the proportion of rat hippocampal GABAA receptors containing alpha-5 subunits determined by immunoprecipitation with selective antibodies and competition experiments using an alpha-5-selective ligand. In recombinant GABAA receptors composed of alpha-5 beta-3 gamma-2 subunits, the Kd of [3H]RY 80 (approximately 0.5 nM) was consistent with the value obtained in hippocampus, whereas the Bmax value was not significantly different from that obtained with [3H]flunitrazepam. The potencies of several benzodiazepine site ligands to inhibit [3H]RY 80 binding to hippocampal membranes were in agreement with the values obtained in recombinant (alpha-5 beta-3 gamma-2) GABAA receptors. [3H]RY 80 was used both in a "GABA shift" assay to correctly predict the in vivo actions of a novel, alpha-5-selective ligand and to characterize a population of GABAA receptors containing alpha-5 subunits in neonatal rat cortex. These findings demonstrate that [3H]RY 80 can be used as a radioligand to examine the properties of GABAA receptors containing alpha-5 subunits.
authors.affiliation: Laboratory of Neuroscience, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0008, USA. dpopa@helix.nih.gov
authors.forename: P
authors.initials: P
authors.lastname: Skolnick
authors.forename: R J
authors.initials: RJ
authors.lastname: Hu
authors.forename: C M
authors.initials: CM
authors.lastname: Cook
authors.forename: S D
authors.initials: SD
authors.lastname: Hurt
authors.forename: J D
authors.initials: JD
authors.lastname: Trometer
authors.forename: R
authors.initials: R
authors.lastname: Liu
authors.forename: Q
authors.initials: Q
authors.lastname: Huang
authors.forename: J M
authors.initials: JM
authors.lastname: Cook
mesh_terms: D000480:Alkynes; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D001569:Benzodiazepines / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D005260:Female; D005445:Flunitrazepam / Q000378:metabolism; D007093:Imidazoles / Q000378:metabolism*; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000480:Alkynes; D000927:Anticonvulsants; D007093:Imidazoles; C109021:RY 80; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam