An intersubunit electrostatic interaction in the GABA_A receptor facilitates its responses to benzodiazepines

Benzodiazepines are positive allosteric modulators of the GABA_A receptor (GABA_AR), acting at the α-γ subunit interface to enhance GABA_AR function. GABA or benzodiazepine binding induces distinct conformational changes in the GABA_AR. The molecular rearrangements in the GABA_AR following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABA_AR, we identified electrostatic interactions between specific amino acids at the α-γ subunit interface that were broken by, or formed after, benzodiazepine binding. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes, we investigated these interactions by substituting one or both amino acids of each potential pair. We found that Lys¹⁰⁴ in the α₁ subunit forms an electrostatic bond with Asp⁷⁵ of the γ₂ subunit after benzodiazepine binding and that this bond stabilizes the positively modified state of the receptor. Substitution of these two residues to cysteine and subsequent covalent linkage between them increased the receptor's sensitivity to low GABA concentrations and decreased its response to benzodiazepines, producing a GABA_AR that resembles a benzodiazepine-bound WT GABA_AR. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The α₁ Lys¹⁰⁴ and γ₂ Asp⁷⁵ interaction did not play a role in ethanol or neurosteroid modulation of GABA_AR, suggesting that different modulators induce different conformational changes in the receptor. These findings may help explain the additive or synergistic effects of modulators acting at the GABA_AR.