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

Benzodiazepines are positive allosteric modulators of the GABA receptor (GABAR), acting at the α-γ subunit interface to enhance GABAR function. GABA or benzodiazepine binding induces distinct conformational changes in the GABAR. The molecular rearrangements in the GABAR following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABAR, 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 oocytes, we investigated these interactions by substituting one or both amino acids of each potential pair. We found that Lys in the α subunit forms an electrostatic bond with Asp of the γ 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 GABAR that resembles a benzodiazepine-bound WT GABAR. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The α Lys and γ Asp interaction did not play a role in ethanol or neurosteroid modulation of GABAR, 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 GABAR.