, Volume 375, Issue 3, pp 177-187
Date: 22 Mar 2007

Allosteric uncoupling and up-regulation of benzodiazepine and GABA recognition sites following chronic diazepam treatment of HEK 293 cells stably transfected with α1β2γ2S subunits of GABAA receptors

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Benzodiazepines are drugs known to produce tolerance and dependence and also to be abused and co-abused. The aim of this study was to further explore the mechanisms that underlie adaptive changes in GABAA receptors following prolonged exposure to these drugs. Human embryonic kidney (HEK 293) cells stably expressing recombinant α1β2γ2s GABAA receptors were exposed for 72 h to a high concentration of diazepam (50 μM) in the absence or presence of other drugs. Radioligand binding studies were used to determine the parameters of [3H]flunitrazepam and [3H]muscimol binding sites and allosteric interactions between these sites. Prolonged treatment with diazepam increased the maximum number (B max) of [3H]flunitrazepam and [3H]muscimol binding sites in the membranes, and of [3H]muscimol binding sites on the surface of HEK 293 cells. There was no change in the affinity (K d) of binding sites. The diazepam-induced increase in the B max value of [3H]flunitrazepam binding sites was reduced by two GABAA receptor antagonists, gabazine (1 and 10 μM) and picrotoxin (100 μM). In addition, it was reduced by cycloheximide (5 μg/ml), a protein synthesis inhibitor, and actinomycin D (7.5 μg/ml), an RNA synthesis inhibitor. Flumazenil (5 μM), the antagonist of benzodiazepine binding sites, also up-regulated [3H]flunitrazepam recognition sites. Simultaneous treatment with diazepam and flumazenil failed to produce an additive up-regulation. GABA (1 nM - 1 mM)-induced potentiation of [3H]flunitrazepam binding to membranes obtained from diazepam (50 μM)-pretreated cells was markedly reduced, suggesting functional uncoupling between GABA and benzodiazepine binding sites. The results suggest that diazepam up-regulated benzodiazepine binding sites on stably expressed GABAA receptors by stimulating their synthesis at both the transcriptional and translational levels. A comparable increase of [3H]muscimol binding sites expressed on the surface of intact HEK 293 cells suggests that internalisation of surface receptors presumably can not explain the uncoupling.