Asymmetric roles for M2-M3 linkers of specific GABAA receptor subunits in the intrinsic closed-open equilibrium and its modulation by the benzodiazepine diazepam

GABAA receptors (GABAARs) are neurotransmitter-gated ion channels critical for inhibitory synaptic transmission as well as the molecular target for benzodiazepines (BZDs), one of the most widely prescribed class of psychotropic drugs today. Despite structural insight into the conformations underlying functional channel states, the detailed molecular interactions involved in conformational transitions and the physical basis for their modulation by BZDs are not fully understood. We previously identified that alanine substitution at the central residue in the 1 subunit M2-M3 linker (V279A) enhances the efficiency of linkage between the BZD site and the pore gate. Here, we expand on this work by investigating the effect of alanine substitutions at the analogous positions in the M2-M3 linkers of {beta}2 (I275A) and {gamma}2 (V290A) subunits, which together with 1 comprise typical heteromeric 1{beta}2{gamma}2 synaptic GABAARs. We find that these mutations confer subunit-specific effects on the intrinsic pore closed-open equilibrium and its modulation by the BZD diazepam (DZ). The mutations 1(V279A) or {gamma}2(V290A) bias the channel toward a closed conformation, whereas {beta}2(I275A) biases the channel toward an open conformation to the extent that the channel becomes leaky and opens spontaneously in the absence of agonist (e.g., GABA). In contrast, only 1(V279A) enhances the efficiency of DZ-to-pore linkage, whereas mutations in the other two subunits have no effect. These observations show that the central residue in the M2-M3 linkers of distinct subunits in synaptic 1{beta}2{gamma}2 GABAARs contribute asymmetrically to the intrinsic closed-open equilibrium and its modulation by DZ.