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

Abstract

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.

The seizure‐inducing plastic explosive RDX inhibits the α1β2γ2 GABAA receptor

“…Using the Rosetta molecular modeling suite 13 with membrane environment‐specific energy functions 14,15 we generated a model of the α 1 β 2 γ 2 GABA A receptor in the resting state based on the cryo‐EM structure of the α 1 β 3 γ 2 receptor 16 with picrotoxinin bound (pdb id: 6X40). Structural refinement and docking of RDX with the RosettaLigand application 17 using the Talaris2014 energy function was performed as previously described in detail 18 . Molecular graphics were rendered with the UCSF Chimera software (Resource for Biocomputing, Visualization, and Informatics, San Francisco, CA) (Pettersen et al., 2004).…”

Perampanel, a potent AMPA receptor antagonist, protects against tetramethylenedisulfotetramine-induced seizures and lethality in mice: comparison with diazepam

“…In addition to its activity in the MES test, pre-treatment with perampanel protects against seizures in mice induced by the GABA A receptor blocker pentylenetetrazol (Hanada et al 2011 ). Similarly, we found that pre-treatment with perampanel protected against seizures and lethality induced by TETS, which is also a GABA A receptor antagonist (Pressly et al 2021 ). The potencies for protection against TETS-induced lethality and the reported activity in the PTZ mouse model (0.94 mg/kg, p.o.…”

“…Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison (Zolkowska et al 2012 ; Rice et al 2017 ; Lauková et al 2020 ) considered to be a chemical threat agent by the National Institutes of Health (Jett and Spriggs 2020 ) and other health authorities (Patocka et al 2018 ). TETS is believed to induce lethal seizures by blocking brain GABA A receptors (Bowery et al 1975 ; Large 1975 ; Dray 1975 ; Roberts et al 1981 ; Nik et al 2017 ; Pressly et al 2021 ). No specific antidote for TETS intoxication is known.…”

The efficacy of γ-aminobutyric acid type A receptor (GABA AR) subtype-selective positive allosteric modulators in blocking tetramethylenedisulfotetramine (TETS)-induced seizure-like behavior in larval zebrafish with minimal sedation

“…From these experiments, the major target of TETS was proposed to be α2β3γ2 ( Pressly et al, 2018 ), which makes up 15–20% of the GABA A Rs in the mammalian brain ( Rudolph and Knoflach, 2011 ). TETS inhibits α2β3γ2 receptors by binding to the non-competitive antagonist site in the channel pore ( Pressly et al, 2020 ). All other tested GABA A Rs were significantly less sensitive except the α6β3γ2 isoform, which was blocked by sub-micromolar TETS concentrations ( Pressly et al, 2018 ).…”