GABA A receptor subunit M2-M3 linkers have asymmetric roles in pore gating and diazepam modulation.

GABAA receptors (GABAA Rs) 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 α1 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 β2 (I275A) and γ2 (V290A) subunits, which together with α1 comprise typical heteromeric α1β2γ2 synaptic GABAA Rs. 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 α1(V279A) or γ2(V290A) bias the channel toward a closed conformation, whereas β2(I275A) biases the channel toward an open conformation to the extent that the channel becomes leaky and opens spontaneously in the absence of agonist. In contrast, only α1(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 α1β2γ2 GABAA Rs contribute asymmetrically to the intrinsic closed-open equilibrium and its modulation by DZ.