3H Flunitrazepam binding to recombinant α1β2γ2S GABAA receptors stably expressed in HEK 293 cells

The interaction of selected compounds with the binding of the benzodiazepine 3H flunitrazepam to membranes isolated from human embryonic kidney (HEK) 293 cells, stably transfected with the α1β2γ2S subtype of GABAA receptors, was studied. This subtype of GABAA receptors is the most common type of GABAA receptor found in the brain, and benzodiazepines are drugs known to enhance the effects of the inhibitory neurotransmitter gamma-amino butyric acid (GABA) by binding to the benzodiazepine binding sites which are part of the GABAA receptor complex. Scatchard analysis of binding data revealed the existence of a single type of binding site for 3H flunitrazepam. GABA and thiopental enhanced, while the antagonist of central benzodiazepine binding sites – flumazenil, benzodiazepines such as clonazepam, flunitrazepam and diazepam, and the triazolopyridazine CI 218,872 – displaced with nanomolar potency the binding of 3H flunitrazepam. A partial displacement was obtained with the antagonist of the peripheral benzodiazepine binding sites – PK 11195 – and with the neurosteroid dehydroepiandrosterone sulfate. The potency of drugs to enhance or inhibit 3H flunitrazepam binding mainly corresponded to that observed for the modulation of the binding of 3H flunitrazepam to the native type 1 benzodiazepine binding sites. This, as well as a high density of expressed binding sites, makes the cell line under study a very reliable and economical model for the testing of effects of different compounds at the GABAA receptor.