Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α(2)β(3)γ(2) GABA(A) Receptor

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.