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

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.

Looking for Novelty in an “Old” Receptor: Recent Advances Toward Our Understanding of GABAARs and Their Implications in Receptor Pharmacology

“…Given that the high affinity binding site exists at the α–γ interface, a multitude of side effects can occur following administration due to their non-selective targeting of GABA A Rs. Classical benzodiazepines interact non-selectively with all GABA A Rs containing α1-, α2-, α3-, α5-, and γ2 subunits ( Moody and Jenkins, 2018 ). Whereas α1-containing GABA A Rs have the greatest distribution throughout the brain ( Möhler et al, 2002 ), α2-, α3-, and α5-containing GABA A Rs are expressed with greater confinement to specific brain regions ( Engin et al, 2018 ).…”

“…This can prove to be problematic when considering off-target drug effects and unforeseen complications from drug administration due to drug binding across many GABA A R subtypes in different brain regions. Considering the nature of current GABA A R pharmaceuticals, the design of these drugs is based on previously characterized binding sites, such as the high affinity benzodiazepine binding site which exists between the α and γ subunits ( Moody and Jenkins, 2018 ). However, there is still a lack of new and clinically relevant subunit-specific GABA A R-targeting drugs despite scientific success in furthering our understanding of GABA A R structure and function ( Figure 1 ).…”

Benzodiazepine Modulation of GABAA Receptors: A Mechanistic Perspective

“…Thereafter, multiple variants of the α (1–6), β (1–3) and γ (1–3) subunits were discovered [ 28 ], whose combinations confer differential effects of BZDs [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ]. The cloning of GABA A R subunits enabled the molecular mechanism of BZD modulation to be probed via mutagenesis, which has led to numerous studies that identified not only the binding site but also the regions contributing to the drug’s modulatory effect.…”

“…Mutagenesis and photoaffinity labeling identified a region in the extracellular domain (ECD) at the α+/γ− subunit interface as the location of the high-affinity BZD binding site where mutations often impaired BZD binding or the modulation of GABA-evoked responses [ 34 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ] ( Figure 1 A,B). Cryo-EM structures confirmed this as a recognition site for BZDs [ 42 , 44 , 45 , 46 , 47 , 48 ].…”

Midazolam at Low Nanomolar Concentrations Affects Long-term Potentiation and Synaptic Transmission Predominantly via the α1–γ-Aminobutyric Acid Type A Receptor Subunit in Mice

“…20 Interestingly, even though midazolam potentiates α 5 -containing GABA A receptors in a similar concentration range as α 1 - and α 2 -subunits, it is most efficacious on α 1. 21 This may imply that a complex interplay of all three subunits in the hippocampus is involved in the drug’s amnesic properties. In the current study, we first investigated the contribution of α 1 -, α 2 -, and α 5 -subunits on midazolam’s actions on long-term potentiation evoked in hippocampal brain slices and finally detailed the midazolam effect on synaptic inhibition in wild-type and α 2/3/5 –knock-in mice.…”