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The γ-aminobutyric acid (GABA) type A receptor (GABAAR) is one of the most important targets for insecticide action. The human recombinant β3 homomer is the best available model for this binding site and 4-n-[3H]propyl-4′-ethynylbicycloorthobenzoate ([3H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the β3 homomer relative to the much-less-active but structurally very-similar β1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The β1 and β3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the α1 subunit and modulators. Chimera β3/β1 with the β3 subunit extracellular domain and the β1 subunit transmembrane helices retained the high [3H]EBOB binding level of the β3 homomer while chimera β1/β3 with the β1 subunit extracellular domain and the β3 subunit transmembrane helices had low binding activity similar to the β1 homomer. GABA at 3 μM stimulated heteromers α1β1 and α1β3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the α1 subunit rescued the inactive β1/β3 chimera close to wildtype α1β1 activity. EBOB binding was significantly altered by mutations β1S15′N and β3N15′S compared with wildtype β1 and β3, respectively. However, the binding activity of α1β1S15′N was insensitive to GABA and α1β3N15′S was stimulated much less than wildtype α1β3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation β3N15′S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABAA receptor sensitivity.