Relative Positioning of Classical Benzodiazepines to the γ₂-Subunit of GABA_A Receptors

Abstract

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60, and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors.

The GLP-1 Receptor Agonist Exendin-4 and Diazepam Differentially Regulate GABAA Receptor-Mediated Tonic Currents in Rat Hippocampal CA3 Pyramidal Neurons

“…The drugs enhance the GABA signaling by different mechanisms. Diazepam interacts directly with the receptor by binding to a binding site on the GABA A receptors [ 24 – 26 ]. Upon application of diazepam alone, the tonic current doubled in amplitude and the frequency and the amplitude of the sIPSCs increased in comparison to control sIPSCs values.…”

Metabolic Products of Linalool and Modulation of GABAA Receptors

“…Thus, GABA A Rs represent excellent drug targets for anticonvulsant and sedative agents (Middendorp et al, 2014; Mendonca Junior et al, 2015). …”

GABA A Receptor Family: Overview on Structural Characterization

“…γ 2 Met130, ubicada en la parte posterior y superior del sitio, no está en contacto directo con diazepam, pero sí lo está con residuos que interactúan directamente con el ligando como α 1 Tyr210 y Los valores de precisión y sensibilidad son altos para el mejor modelo. Además, esta configuración concuerda con aquellas propuestas en estudios in silico previos [260,261]. La pose encontrada para diazepam se corresponde con una de las propuestas por Richter et.al.…”

“…• Estudios de SCAM mostraron que elátomo de cloro de diazepam apunta hacia este residuo. [259,261,298] • Es marcado por foto-afinidad por flunitrazepam. [256] • Fue identificado a partir de estudios de mutagénesis dirigida e intercambio de dominio, como necesario para la unión de diazepam y zolpidem.…”

“…• Estudios de Proximity-accelerated chemical coupling han demostrado su cercanía alátomo C3 del anillo de las benzodiazepinas. [259,261] • Su mutación por Alanina no afecta la potenciación de BDZs clásicas.…”