Abstract
Tetramethylenedisulfotetramine (TETS) is a so-called "caged" convulsant, that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABAA antagonist picrotoxinin, TETS has been proposed to bind to the non-competitive antagonist (NCA) site in the pore of the gamma-aminobutyric acid (GABA) 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 α2β3γ2 GABAA receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α2β3γ2 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, where it shows perfect space-complementarity and is forming hydrogen bonds or making hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α2 or β3 subunit reduces the IC50 of TETS by ~700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the non-competitive antagonist (NCA) site in the pore of the GABAA 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 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 α2β3γ2 GABAA receptor at the so-called T6' ring, where five threonine residues line the permeation pathway of the pentameric receptor channel.
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