Selective actions of benzodiazepines at the transmembrane anaesthetic binding sites of the GABAA receptor: In vitro and in vivo studies

Background and Purpose In addition to binding to the classical high-affinity extracellular benzodiazepine binding site of the GABA(A) receptor, some benzodiazepines occupy transmembrane inter-subunit anaesthetic sites that bind etomidate (beta(+)/alpha(-) sites) or the barbiturate derivative R-mTFD-MPAB (alpha(+)/beta(-) and gamma(+)/beta(-) sites). We aimed to define the functional effects of these interactions on GABA(A) receptor activity and animal behaviour. Experimental Approach With flumazenil blocking classical high-affinity extracellular benzodiazepine site effects, modulation of GABA-activated currents by diazepam, midazolam and flurazepam was measured electrophysiologically in wildtype and M2-15 ' mutant alpha(1)beta(3)gamma(2L) GABA(A) receptors. Zebrafish locomotive activity was also assessed in the presence of each benzodiazepine plus flumazenil. Key Results In the presence of flumazenil, micromolar concentrations of diazepam and midazolam both potentiated and inhibited wildtype GABA(A) receptor currents. beta(3)N265M (M2-15 ' in the beta(+)/alpha(-) sites) and alpha(1)S270I (M2-15 ' in the alpha(+)/beta(-) site) mutations reduced or abolished potentiation by these drugs. In contrast, the gamma(2)S280W mutation (M2-15 ' in the gamma(+)/beta(-) site) abolished inhibition. Flurazepam plus flumazenil only inhibited wildtype receptor currents, an effect unaltered by M2-15 ' mutations. In the presence of flumazenil, zebrafish locomotion was enhanced by diazepam at concentrations up to 30 mu M and suppressed at 100 mu M, suppressed by midazolam and enhanced by flurazepam. Conclusions and Implications Benzodiazepine binding to transmembrane anaesthetic binding sites of the GABA(A) receptor can produce positive or negative modulation manifesting as decreases or increases in locomotion, respectively. Selectivity for these sites may contribute to the distinct GABA(A) receptor and behavioural actions of different benzodiazepines, particularly at high (i.e. anaesthetic) concentrations.