Benzodiazepines act on GABAA receptors via two distinct and separable mechanisms

Benzodiazepines (BZs) act on γ-aminobutyric acid type A (GABA A ) receptors such as α 1 β 2 γ 2 through key residues within the N-terminal region of α subunits, to render their sedative and anxiolytic actions. However, the molecular mechanisms underlying the BZs' other clinical actions are not known. Here we show that, with low concentrations of GABA, diazepam produces a biphasic potentiation for the α 1 β 2 γ 2 -receptor channel, with distinct components in the nanomolar and micromolar concentration ranges. Mutations at equivalent residues within the second transmembrane domains (TM 2 ) of α, β and γ subunits, proven important for the action of other anesthetics, abolish the micromolar, but not the nanomolar component. Converse mutation of the corresponding TM 2 residue and a TM 3 residue within ρ 1 subunits confers diazepam sensitivity on homo-oligomeric ρ 1 -receptor channels that are otherwise insensitive to BZs. Thus, specific and distinct residues contribute to a previously unresolved component (micromolar) of diazepam action, indicating that diazepam can modulate the GABA A -receptor channel through two separable mechanisms.