Una interacción electrostática entre subunidades en el receptor GABAA facilita sus respuestas a las benzodiazepinas.
An inter-subunit electrostatic interaction in the GABAA receptor facilitates its responses to benzodiazepines.
J Biol Chem. 2018 Apr 5. pii: jbc.RA118.002128. doi: 10.1074/jbc.RA118.002128. [Epub ahead of print]
Abstract
Benzodiazepines are positive allosteric modulators of the GABAA receptor (GABAAR), acting at the α-γ subunit interface to enhance GABAAR function. GABA or benzodiazepine binding induces distinct conformational changes in the GABAAR. The molecular rearrangements in the GABAAR following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABAAR, we identified electrostatic interactions between specific amino acids at the α-γ subunit interface which were broken by, or formed after, benzodiazepinebinding. 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 Lys-104 in the α1 subunit forms an electrostatic bond with Asp-75 of the γ2subunit 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 GABAAR that resembles a benzodiazepine-bound wildtype GABAAR. Breaking this bond restored sensitivity to GABA to wildtype levels and increased the receptor's response to benzodiazepines. The α1 Lys-104 and γ2 Asp-75 interaction did not play a role in ethanol or neurosteroid modulation of GABAAR, 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 GABAAR.
KEYWORDS: Cys-loop receptor; GABA receptor; Xenopus; allosteric regulation; benzodiazepines; cysteine-mediated cross-linking; electrophysiology; electrostatics
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