Selective actions of benzodiazepines at the transmembrane anaesthetic binding sites of the GABA 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 receptor, some benzodiazepines occupy transmembrane inter-subunit anaesthetic sites that bind etomidate (β /α sites) or the barbiturate derivative R-mTFD-MPAB (α /β and γ /β sites). We aimed to define the functional effects of these interactions on GABA 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 α β γ GABA 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 receptor currents. β N265M (M2-15' in the β /α sites) and α S270I (M2-15' in the α /β site) mutations reduced or abolished potentiation by these drugs. In contrast, the γ S280W mutation (M2-15' in the γ /β 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 μM and suppressed at 100 μM, suppressed by midazolam and enhanced by flurazepam.

Conclusions And Implications: Benzodiazepine binding to transmembrane anaesthetic binding sites of the GABA 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 receptor and behavioural actions of different benzodiazepines, particularly at high (i.e. anaesthetic) concentrations.