The role of loops B and C in determining the potentiation of GABAA receptors by midazolam.

Olivia A Moody,Andrew Jenkins

doi:10.1002/prp2.433

Abstract

Many benzodiazepines are positive allosteric modulators (PAMs) of GABAA receptors that cause sedation, hypnosis, and anxiolysis. Benzodiazepines bind GABAA receptors at the extracellular interface of the α and γ subunits. Within the α subunit, the benzodiazepine binding site is defined by three highly conserved structural loops, loops A‐C. Although previous mutagenesis studies have identified His102 in Loop A as important for benzodiazepine modulation of GABAA receptors, the functional roles of many of the other conserved residues in loops A‐C remain incompletely understood. In this study, we made single mutations in loops A‐C of the benzodiazepine binding‐site across all six α subunits. We used whole‐cell patch clamp recording to measure the functional effects of these mutations on midazolam potentiation. The results showed that mutating the threonine in loop B and serine in loop C (Thr163 and S206 in human α1) did not abolish the receptors’ responsiveness to midazolam, as the α1(H102R) mutation did. The loop C mutations exhibited a novel array of α‐isoform specific effects on midazolam potentiation. The α3(S230I) and α5(S209I) mutations had the largest effect on midazolam potentiation, increasing the efficacy of midazolam. Novel benzodiazepines targeting loop C may represent a future direction for designing new drugs that specifically alter the activity of α3‐ and α5‐containing GABAA receptors.

Highlights

Benzodiazepines can induce sedation, anxiolysis, amnesia, seizure reduction, and muscle relaxation by enhancing inhibitory GABAergic neurotransmission through the γ‐aminobutyric acid type A (GABAA) receptors.[1,2] GABAA receptors are cys loop ligand‐gated ion channels assembled from five subunits (α1‐6, β1‐3, γ1‐3, δ, ε, θ, π, ρ1‐3) around a central pore.[3]
We hypothesized that mutating single residues in the conserved loops A‐C of the benzodiazepine binding site (Figure 1C) would alter the modulation of GABAA receptors by midazolam
positive allosteric modulator (PAM) benzodiazepines were initially thought to enhance the activity of GABAA receptors by altering the γ-aminobutyric acid (GABA) binding steps,[29] but more recent models have focused on gating mechanisms.[30,31]

Summary

| INTRODUCTION

Benzodiazepines can induce sedation, anxiolysis, amnesia, seizure reduction, and muscle relaxation by enhancing inhibitory GABAergic neurotransmission through the γ‐aminobutyric acid type A (GABAA) receptors.[1,2] GABAA receptors are cys loop ligand‐gated ion channels assembled from five subunits (α1‐6, β1‐3, γ1‐3, δ, ε, θ, π, ρ1‐3) around a central pore.[3]. A combination of mutagenesis with functional or binding assays has been used to determine the role of specific amino acids within the structural loops A‐F of the benzodiazepine site.[11,12,13,14,15,16] The conserved histidine in loop A (His[101] in rodents and His[102] in bovine and human cDNAs) is important for the molecular and behavioral actions of diazepam using in vitro experiments[6,11] and knock‐in mice.[2,17] Other residues in loops A‐C have been studied, but most mutagenesis experiments were constrained to mutating less than three α subunit isoforms. These subunit‐selective observations will be useful for the design of α3‐ and α5‐selective benzodiazepines

| MATERIALS AND METHODS

| RESULTS

| DISCUSSION