Diazepam-insensitive GABAA receptors in rat cerebellum and thalamus

Three major populations of GABAA receptor binding sites are present in cerebellar membranes: diazepam-sensitive [3H]Ro15-4513 binding sites, diazepam-insensitive [3H]Ro15-4513 binding sites and high-affinity [3H]muscimol binding sites. All three populations contain a beta subunit as shown by immunoprecipitation with antibodies that recognize all beta subunits. The beta 3 subtype of beta subunit is contained in all three populations, but only a similar low fraction (< 20%) in each. Thus, the majority contain beta subunits other than beta 3 (beta 2 and beta 1) and beta 3 subunits are not selectively associated with nor lacking in any of the three binding populations. Antibodies to the gamma 2 subunit precipitated similar fractions of [3H]Ro15-4513, [3H]flunitrazepam and [3H]muscimol binding sites, showing that gamma 2 subunits are present in high-affinity muscimol binding isoforms, as well as a significant fraction of the diazepam-insensitive [3H]Ro15-4513 binding sites. Under conditions that identify the 56 kDa alpha 6 subunit on SDS-PAGE as the diazepam insensitive site of [3H]Ro15-4513 binding in cerebellum, no polypeptide showing diazepam-insensitive binding of [3H]Ro15-4513 could be photoaffinity-labeled in rat thalamus. These results suggest that alpha 4 subunits in the thalamus participate primarily in subunit combinations which bind muscimol but not any benzodiazepine site ligands.