Characterization of the Picrotoxin Site of GABA<sub>A</sub> Receptors

Summary Type-A γ-aminobutyric receptors (GABA A Rs) are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and common substances of abuse. Without reliable structural data, the mechanistic basis for pharmacological modulation of GABA A Rs remains largely unknown. Here we report high-resolution cryoEM structures of the full-length human α1β3γ2L GABA A R in lipid nanodiscs, bound to the channel blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA and the classical benzodiazepines alprazolam (Xanax) and diazepam (Valium), respectively. We describe the binding modes and mechanistic impacts of these ligands, the closed and desensitised states of the GABA A R gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding, and the transmembrane, pore-forming, regions. This work provides a structural framework to integrate decades of physiology and pharmacology research and a rational basis for development of novel GABA A R modulators.

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GABAA receptor signalling mechanisms revealed by structural pharmacology

Masiulis

Desai

Uchański

et al. 2019

Nature

444

517

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Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α₂β₃γ₂ GABA_A Receptor

et al. 2020

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Tetramethylenedisulfotetramine (TETS) is a so-called “caged” convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABA A ) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α 2 β 3 γ 2 GABA A receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α 2 β 3 γ 2 receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6′ ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6′ in either the α 2 or β 3 subunit reduces the IC 50 of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABA A receptor at a location that is overlapping but not identical to the picrotoxinin binding site. SIGNIFICANCE STATEMENT Our study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α 2 β 3 γ 2 GABA receptor type A receptor at the so-called T6′ ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel.

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Characterization of the Picrotoxin Site of GABA_A Receptors

Cited by 2 publications

References 18 publications

GABAA receptor signalling mechanisms revealed by structural pharmacology

GABAA receptor signalling mechanisms revealed by structural pharmacology

Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α₂β₃γ₂ GABA_A Receptor

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Characterization of the Picrotoxin Site of GABAA Receptors

Cited by 2 publications

References 18 publications

GABAA receptor signalling mechanisms revealed by structural pharmacology

GABAA receptor signalling mechanisms revealed by structural pharmacology

Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α2β3γ2 GABAA Receptor

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Product

Resources

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Characterization of the Picrotoxin Site of GABA_A Receptors

Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α₂β₃γ₂ GABA_A Receptor