Different Residues in the GABA_AReceptor Benzodiazepine Binding Pocket Mediate Benzodiazepine Efficacy and Binding

Abstract

Benzodiazepines (BZDs) exert their therapeutic actions by binding to the GABA A receptor (GABA A R) and allosterically modulating GABA-induced chloride currents (I GABA ). A variety of ligands with divergent structures bind to the BZD site, and the structural mechanisms that couple their binding to potentiation of I GABA are not well understood. In this study, we measured the effects of individually mutating 22 residues throughout the BZD binding pocket on the abilities of eszopiclone, zolpidem, and flurazepam to potentiate I GABA . Wild-type and mutant ␣ 1 ␤ 2 ␥ 2 GABA A Rs were expressed in Xenopus laevis oocytes and analyzed using a two-electrode voltage clamp. GABA EC 50 , BZD EC 50 , and BZD maximal potentiation were measured. These data, combined with previous radioligand binding data describing the mutations' effects on BZD apparent binding affinities (J Neurosci 28:3490 -3499, 2008; J Med Chem 51:7243-7252, 2008), were used to distinguish residues within the BZD pocket that contribute to BZD efficacy and BZD binding. We identified six residues whose mutation altered BZD maximal potentiation of I GABA (BZD efficacy) without altering BZD binding apparent affinity, three residues whose mutation altered binding but had no effect on BZD efficacy, and four residues whose mutation affected both binding and efficacy. Moreover, depending on the BZD ligand, the effects of some mutations were different, indicating that the structural mechanisms underlying the ability of BZD ligands with divergent structures to potentiate I GABA are distinct.

Desformylflustrabromine Modulatesα4β2 Neuronal Nicotinic Acetylcholine Receptor High- and Low-Sensitivity Isoforms at Allosteric Clefts Containing theβ2 Subunit

“…Previous mutations, specifically a1(S205) and a1(S206) [equivalent to nAChR b2(D217) and b2(D218)], g2(Y72), g2(D75), and g2(F78) [equivalent to nAChR a4(M83), a4(N860), and a4(V89)] located in the GABA A R BZD-binding site have been shown to increase or decrease GABA potency (Teissere and Czajkowski, 2001;Morlock and Czajkowski, 2011). Similarly, several recent studies have shown that mutation of residues a3(E173), a3(L158), a3(A179), a3(K183), b2(R46), and b2(A127) located in nonorthosteric interfaces of the rat a3-and b2-nAChR subunits also increase or decrease ACh potency from 2-to 60-fold compared with wild-type a3b2-nAChR (Chrisman et al, 2014;Short et al, 2015).…”

Structural basis for cooperative interactions of substituted 2-aminopyrimidines with the acetylcholine binding protein

“…In the case of the predominant heteromeric receptors where the binding interfaces will differ, such ligands may possibly serve as positive or negative allosteric modulators at sites distinct from those occupied by agonist and competitive antagonist (36). Such appears to be the case for the benzodiazepines (36,37) and other sedative agents (38) that act in this manner with the GABA receptor (39,40). Accordingly, new dimensions for achieving pharmacologic selectivity for particular nAChR subtypes may result with the cooperative nAChR ligands possessing electron-rich substituted 2-aminopyrimidines.…”

Agonist-Specific Conformational Changes in the α1-γ2 Subunit Interface of the GABAA Receptor

“…Mutations to this interface can have large effects on binding affinity and/or efficacy of benzodiazepine ligands Padgett and Lummis, 2008;Morlock and Czajkowski, 2011). In particular, residues of loop F of the ␥2 subunit appear to play an important role in defining the efficacy of benzodiazepine ligands Padgett and Lummis, 2008).…”

“…Benzodiazepines potentiate the GABA A receptor through interactions with the ␣-␥ subunit interface at which several loop F residues have been shown to affect the efficacy of several benzodiazepine ligands (Padgett and Lummis, 2008;Morlock and Czajkowski, 2011). We tested the possibility that the MTSES molecule occupies the benzodiazepine binding cavity, thereby imitating the effect of a bound benzodiazepine and underlying the enhanced current response after exposure to MTSES.…”