An intersubunit electrostatic interaction in the GABA A receptor facilitates its responses to benzodiazepines.

Benzodiazepines are positive allosteric modulators of the GABAA receptor (GABAA R), acting at the α-γ subunit interface to enhance GABAA R function. GABA or benzodiazepine binding induces distinct conformational changes in the GABAA R. The molecular rearrangements in the GABAA R following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABAA R, 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 GABAA R that resembles a benzodiazepine-bound WT GABAA R. 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 GABAA R, 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 GABAA R.