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

• Published in: British journal of pharmacology Vol. 178; no. 24; pp. 4842 - 4858
• Main Authors: McGrath, Megan, Hoyt, Helen, Pence, Andrea, Forman, Stuart A., Raines, Douglas E.
• Format: Journal Article
• Language: English
• Published: London Blackwell Publishing Ltd 01.12.2021
• Subjects: Affinity; Benzodiazepines; Binding sites; Diazepam; Etomidate; Flumazenil; Flurazepam; Locomotion; Midazolam; Mutation; γ-Aminobutyric acid A receptors
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/bph.15662

Background and Purpose In addition to binding to the classical high‐affinity extracellular benzodiazepine binding site of the GABAA 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 GABAA 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 α1β3γ2L GABAA 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 GABAA receptor currents. β3N265M (M2‐15′ in the β+/α− sites) and α1S270I (M2‐15′ in the α+/β− site) mutations reduced or abolished potentiation by these drugs. In contrast, the γ2S280W 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 GABAA receptor can produce positive or negative modulation manifesting as decreases or increases in locomotion, respectively. Selectivity for these sites may contribute to the distinct GABAA receptor and behavioural actions of different benzodiazepines, particularly at high (i.e. anaesthetic) concentrations.