Loreclezole modulates [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding via a distinct site on the...

The allosteric modulation of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) and [3H]flunitrazepam binding was utilized to evaluate the actions of loreclezole at the GABAA receptor complex in the rat brain. Loreclezole was observed to allosterically inhibit the binding of [35S]TBPS in a dose-dependent manner with micromolar potency (IC50 = 1 microM). Loreclezole was found to have an additive effect on neuroactive steroid modulation of [35S]TBPS binding, but merely potentiated the effect of Ro5-4864 (4"-chlorodiazepam) modulation of [35S]TBPS binding. These observations suggest that loreclezole modulates [35S]TBPS binding through a site independent of the neuroactive steroid and Ro5-4864 sites on the GABAA receptor complex. The enhancement of [3H]flunitrazepam binding to the benzodiazepine receptor by loreclezole as well as the effect of loreclezole on CL218872/[3H]flunitrazepam dose-response curves suggest that loreclezole does not act through the benzodiazepine site on the GABAA receptor complex, nor does it selectively modulate benzodiazepine receptor subtypes. The potency of loreclezole as and inhibitor of [35S]TBPS binding in rat brain was regionally dependent and GABA-sensitive. Loreclezole modulation of [35S]TBPS binding showed greater potency and GABA sensitivity in the cerebellum and thalamus when compared to other brain regions such as the cortex, hippocampus and striatum. This finding is consistent with previous reports of the selectivity of loreclezole for GABAA receptor complex's containing beta 2 and beta 3 subunits. These beta subunit isoforms predominate in the cerebellum and thalamus. Collectively the evidence suggests that loreclezole modulates [35S]TBPS and [3H]flunitrazepam binding through a site distinct from benzodiazepine, neuroactive steroid, Ro5-4864 and GABA sites on the GABAA receptor complex.