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

作者：Pflanz Natasha C; Daszkowski Anna W; Cornelison Garrett L; Trudell James R; Mihic S John^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2018, 293(21): 8264-8274.

DOI：10.1074/jbc.RA118.002128

• allosteric regulation
• GABA receptor
• electrostatics
• electrophysiology
• Xenopus
• cysteine-mediated cross-linking
• Cys-loop receptor
• benzodiazepines
• ionotropic receptor
• sedative

Benzodiazepines are positive allosteric modulators of the GABA(A) receptor (GABA(A)R), acting at the - subunit interface to enhance GABA(A)R function. GABA or benzodiazepine binding induces distinct conformational changes in the GABA(A)R. The molecular rearrangements in the GABA(A)R following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABA(A)R, we identified electrostatic interactions between specific amino acids at the - 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 Lys(104) in the (1) subunit forms an electrostatic bond with Asp(75) of the (2) 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(A)R that resembles a benzodiazepine-bound WT GABA(A)R. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The (1) Lys(104) and (2) Asp(75) interaction did not play a role in ethanol or neurosteroid modulation of GABA(A)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 GABA(A)R.

• 出版日期2018-5-25