GABAA and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABAA receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk- cells stably transfected with GABAA receptors (bovine alpha 1, beta 1, and gamma 2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [3H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the KD and Bmax of [3H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t1/2 of approximately 30 min. The EC50 for clonazepam treatment was approximately 0.3 microM, and cotreatment with the benzodiazepine antagonist Ro 15-1788 (5.6 microM) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents post-translational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABAA and benzodiazepine receptors as well as benzodiazepine tolerance.