Chronic benzodiazepine treatment of cells expressing recombinant GABA(A) receptors uncouples allosteric binding: studies on possible mechanisms

`Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposur e has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABA( A) receptors (GABARs) expressed in Sf9 cells lose the normally observed all osteric enhancement of [H-3]flunitrazepam binding by GAGA agonists, which i s measured in homogenized membranes after a few hours exposure to pharmacol ogical doses of agonist BZ. Treatment of Sf9 cells expressing recombinant G ABAR with various drugs that inhibit protein kinase A (PKA), but not protei n kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding site s; whereas promotion of phosphorylation by PKA, but not PKC, favored coupli ng and recoupling. However, mutation of the only PKA phosphorylation site e xpressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reve rsal of uncoupling after exposure to the competitive BZ antagonist, flumaze nil. Osmotic-shock of cell membrane homogenates to lyse intracellular compa rtments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, sugge sting that GABARs become internalized into an acidic intracellular environm ent where normal BZ binding occurs, but that potentiation by GABA is hinder ed. The internalization of receptors was shown by immunofluorescence after chronic exposure to either BZ or the PKA inhibitor H-89.