Biochemical aspects of tolerance to chronic benzodiazepine exposure

Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. In this study we establish Sf9 cells expressing recombinant α1β2γ2 GABA_A receptors (GABAR) as a model to study mechanisms of tolerance to chronic BZ exposure. We show that in Sf9 cells expressing GABAR, there is an uncoupling of the BZ and GABA binding sites after chronic BZ exposure. This uncoupling is not associated with any significant change in either affinity or density of the GABA or BZ binding sites. Also, this uncoupling is rapidly reversed by a brief exposure to the BZ antagonist, flumazenil. Treatment of Sf9 cells with various phosphorylation drugs revealed that although phosphorylation by protein kinase C does not play a role, phosphorylation by protein kinase A (PKA) has an effect on uncoupling and recoupling. Studies showed that dephosphorylation by PKA resulted in an uncoupling of the BZ and GABA sites and phosphorylation resulted in coupling or recoupling. Mutation of the only PKA phosphorylation site among the subunits used, α1β2S410Aγ2, proved however, that direct phosphorylation of the GABAR was not involved in either uncoupling after chronic BZ exposure or reversal of uncoupling after exposure to flumazenil. Studies using osmotic-shock to lyse open intracellular compartments suggested that GABAR became internalized after chronic BZ exposure. Uncoupling was replicated in untreated Sf9 cells with binding assays done in an acidic pH, suggesting that GABAR became internalized into intracellular acidic environment where normal BZ binding can occur but potentiation by GABA was hindered. Immunofluorescence studies support the internalization hypothesis; fluorescein-labeled receptors were shown to be internalized after chronic BZ exposure and to return to the surface after brief exposure to flumazenil. Immunofluorescence studies also linked internalization to phosphorylation since chronic exposure of GABAR to the PKA inhibitor H-89 resulted in an internalization of the receptor. Together, the results from this study suggest an internalization of the GABAR receptor after chronic BZ exposure into an intracellular, acidic compartment where normal BZ binding occurs but not potentiation by GABA. This internalization somehow involves phosphorylation, although not direct phosphorylation of the GABAR.