Chronic benzodiazepine treatment of cells expressing recombinant GABA_A receptors uncouples allosteric binding: studies on possible mechanisms

Abstract

Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure 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 allosteric enhancement of [ 3 H]¯unitrazepam binding by GABA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist,¯umazenil.Osmotic-shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immuno¯uorescence after chronic exposure to either BZ or the PKA inhibitor H-89.

Mechanisms of Reversible GABAAReceptor Plasticity after Ethanol Intoxication

“…However, phosphorylation (protein kinase C)-dependent subunit-selective receptor endocytosis and/or recycling may be involved (Ali and Olsen, 2001;Kumar et al, 2003;Kittler et al, 2005). GABA A R assembly may be partly based on subunit availability, especially in the unnatural environment of recombinant expression in heterologous cells; however, distinct assembly signals in certain subunits appear to drive formation of preferred receptor subunit compositions (Bollan et al, 2003).…”

“…However, selective endocytosis and/or recycling of receptors based on subunit composition and sensitivity to protein kinase C phosphorylation may be involved (Ali and Olsen, 2001;Kumar et al, 2003;Kittler et al, 2005). We hypothesized that the rapid development of tolerance to acute EtOH potentiation of the tonic current in CA1 neurons may be attributable to the internalization of the ␣4␤␦ extrasynaptic GABA A Rs, which are sensitive to low [EtOH] (SundstromPoromaa et al, 2002;Wallner et al, 2003).…”

Imidazenil: A low efficacy agonist at α1- but high efficacy at α5-GABAA receptors fail to show anticonvulsant cross tolerance to diazepam or zolpidem

“…Several published work support the hypothesis that selective changes in the expression of different α subunits is associated with BZ tolerance (Ali and Olsen, 2001;Bateson, 2002, Costa et al, 2002Wafford, 2005); however the role of specific subunits in the mechanism of BZ tolerance development is still not clear. Using knock-in mice in which the α1-, α2-, α3-or α5-GABA A receptor subunits were rendered insensitive to diazepam by histidine-arginine point mutation, van Rijnsoever et al (2004) demonstrated that simultaneous and long-term activation of both α1 and α5 GABA A receptor subunit by diazepam is necessary for the development of tolerance to its sedative action.…”

Differential effects of short- and long-term zolpidem treatment on recombinant α1β2γ2s subtype of GABAA receptors in vitro

“…The exact molecular mechanism(s) leading to functional uncoupling between GABA and benzodiazepine binding sites remain unknown. Although uncoupling of the benzodiazepine and GABA binding sites could be produced by drugs inhibiting protein kinase A, it is supposed that direct phosphorylation of GABA A receptors is not involved in coupling/ uncoupling processes [26] . The same authors proposed that prolonged benzodiazepine treatment induces internalization of surface GABA A receptors into intracellular vesicles, where the potentiation by GABA is impaired but the normal benzodiazepine binding can occur.…”

“…However, long-term administration of non-selective full positive allosteric modulator of GABA action at GABA A receptors leads to alterations in receptor expression and/or function, resulting in the development of tolerance and dependence. Many authors working either on animals [29] , neuronal cultures [30][31][32] or recombinant receptors [13,14,20,25,26,33,34] have found reduced allosteric linkage between GABA and benzodiazepine binding sites as a result of prolonged benzodiazepine action. Moreover, animals and humans treated for prolonged period of time with drugs acting as full positive modulators of GABA action at GABA A receptors developed tolerance characterized by a decreased ability of the drug to produce its pharmacological effect.…”