Benzodiazepines act on GABAA receptors via two distinct and separable mechanisms.

Benzodiazepines (BZs) act on gamma-aminobutyric acid type A (GABAA) receptors such as alpha1beta2gamma2 through key residues within the N-terminal region of alpha 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 alpha1beta2gamma2-receptor channel, with distinct components in the nanomolar and micromolar concentration ranges. Mutations at equivalent residues within the second transmembrane domains (TM2) of alpha, beta and gamma subunits, proven important for the action of other anesthetics, abolish the micromolar, but not the nanomolar component. Converse mutation of the corresponding TM2 residue and a TM3 residue within rho1 subunits confers diazepam sensitivity on homo-oligomeric rho1-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 GABAA-receptor channel through two separable mechanisms.