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

Abstract

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.

A critical residue in the α1M2–M3 linker regulating mammalian GABAA receptor pore gating by diazepam

“…However, other regions including the 4-5 linker in the channel's outer vestibule also affect BZD modulation of agonist-evoked currents (Pflanz et al, 2018;Venkatachalan and Czajkowski, 2012). Thus, BZD modulation likely involves larger domain fluctuations in addition to any specific molecular pathways involving  1 V279.…”

“…Thus, a stronger intersubunit coupling in this area could potentially both reduce unliganded opening and enhance coupling with rearrangements of the γ 2 subunit upon BZD binding. However, other regions including the β4-β5 linker in the channel’s outer vestibule also affect BZD modulation of agonist-evoked currents ( Pflanz et al, 2018 ; Venkatachalan and Czajkowski, 2012 ). Thus, BZD modulation likely involves larger domain fluctuations in addition to any specific molecular pathways involving α 1 V279.…”

Analysis of GABRG2 C588T polymorphism in genetic epilepsy and evaluation of GABRG2 in drug treatment

“…[9][10][11] The GABA A receptor has multiple binding sites for anti-epileptic drugs in the brain. [12][13][14][15] The main GABA A receptor in the brain is composed of a1, ß2, and γ2 subunits. The γ2 subunit was reported to change the kinetics of GABA A related to channels and to the synaptic and postsynaptic clustering and maintenance.…”

A critical residue in the α1M2-M3 linker regulating GABAAreceptor pore gating by diazepam

“…linker in the channel's outer vestibule also affect BZD modulation of agonist-evoked currents (Pflanz et al, 2018;Venkatachalan and Czajkowski, 2012). Thus, BZD modulation likely involves larger domain fluctuations in addition to any specific molecular pathways involving a1V279.…”