GABAA receptor open-state conformation determines non-competitive antagonist binding

► The β1 and β3 subunits were compared by chimeragenesis, mutagenesis and modulators. ► Low β1 NCA binding was rescued by replacing its transmembrane helices with those of β3. ► GABA at 3 µM stimulated heteromers α1β1 and α1β3 binding levels more than 2-fold. ► Mutation at 15' position in TM2 reduced GABA stimulation of NCA binding. ► The open-state conformation largely determines GABAA receptor sensitivity to NCAs.