Structural and dynamic mechanisms of GABAA receptor modulators with opposing activities

Abstract

Abstractγ-Aminobutyric acid type A (GABAA) receptors are pentameric ligand-gated ion channels abundant in the central nervous system and are prolific drug targets for treating anxiety, sleep disorders and epilepsy. Diverse small molecules exert a spectrum of effects on γ-aminobutyric acid type A (GABAA) receptors by acting at the classical benzodiazepine site. They can potentiate the response to GABA, attenuate channel activity, or counteract modulation by other ligands. Structural mechanisms underlying the actions of these drugs are not fully understood. Here we present two high-resolution structures of GABAA receptors in complex with zolpidem, a positive allosteric modulator and heavily prescribed hypnotic, and DMCM, a negative allosteric modulator with convulsant and anxiogenic properties. These two drugs share the extracellular benzodiazepine site at the α/γ subunit interface and two transmembrane sites at β/α interfaces. Structural analyses reveal a basis for the subtype selectivity of zolpidem that underlies its clinical success. Molecular dynamics simulations provide insight into how DMCM switches from a negative to a positive modulator as a function of binding site occupancy. Together, these findings expand our understanding of how GABAA receptor allosteric modulators acting through a common site can have diverging activities.

Benzodiazepine Modulation of GABAA Receptors: A Mechanistic Perspective

“…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 ]. The site is homologous to the two GABA binding sites at the β+/α− subunit interfaces, with the ligand binding in a pocket behind loop C, where it interacts with residues from both subunits ( Figure 2 ).…”

“…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. Advances in cryo-electron microscopy (cryo-EM) have recently enabled the resolution of structures of heteromeric GABA A (α1) 2 (βX) 2 (γ2) 1 receptors in complex with BZDs [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ]. Together, these observations aid in the continued search for a complete molecular understanding of the mechanisms of action of BZDs on GABA A Rs.…”

“…The critical histidine residue discussed above lies towards the back of the pocket, where it forms a hydrogen bond with the chlorine atom in PAMs such as diazepam and alprazolam (Xanax), the fluorine atom in the antagonist flumazenil, or the twin methoxy groups of the NAM DMCM ( Figure 2 i ). Z-drugs such as zolpidem (Ambien), with similar sedative and hypnotic effects as BZDs, also bind at this site [ 48 , 60 ].…”

“…Advances in cryo-EM have begun to provide structural snapshots of heteromeric GABA A (α1) 2 (βX) 2 (γ2) 1 receptors in complex with both agonists and BZDs [ 42 , 43 , 44 , 45 , 46 , 47 , 48 ]. These studies validate the canonical high-affinity BZD site in the ECD, as identified by functional studies, and reveal details as well as subunit specificity of lower-affinity BZD sites in the TMD ( Figure 1 A–C).…”

Cys-loop receptors on cannabinoids: All high?

“… Cys-loop receptor structure and selected binding sites. For each family, a representative dimer is depicted in grey ribbon: GABAAR (8DD2— Zhu et al, 2022 ), GlyR (7M6O— Kumar et al, 2022 ), nAChR (7EKT— Zhao et al, 2021 ), 5-HT3 (6HIO— Polovinkin et al, 2018 ). Ligand bound structures have been superposed to render multiple sites.…”