Structural model for γ-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site

Abstract

Several major insecticides, including ␣-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human ␤3 homopentameric GABAA receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the ␤3 homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1 C, A2 C, T6 C, and L9 C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands ␤3 homopentamer ͉ transmembrane 2 ͉ insecticide ͉ disulfide trapping ͉ receptor model P est insect control in the past 60 years was achieved, in part, by application of Ͼ3 billion (3 ϫ 10 9 ) pounds of polychlorocycloalkane insecticides, including cyclodienes (e.g., ␣-endosulfan and dieldrin), lindane and its isomers, and others, which are now highly restricted or banned except for endosulfan and some uses of lindane (1-3). One of the replacement compounds is the phenylpyrazole fipronil. All of these insecticides and the botanical picrotoxinin (PTX) have widely diverse chemical structures but appear to act at the same nerve target. It is therefore important to understand how these compounds work in mammals and insects, or how they do not work when resistant insect strains appear.The GABA-gated chloride channel is the target for the insecticides and toxicants referred to above based on radioligand binding and electrophysiology studies (3)(4)(5)(6)(7)(8)(9)(10) (Fig. 1A). All of these compounds act in mammals and insects as noncompetitive antagonists (NCAs) to block chloride flux so the target is referred to as the GABA receptor NCA-binding site. Vertebrate GABA receptors consist of ␣, ␤, ␥, , and other subunits in various combinations, for example, ␣ 1 ␤ 2 ␥ 2 as a heteropentamer and 1 as a homopentamer (13-15). The molecular localization of the NCA site defined here (Fig. 1B) was first indicated by mutagenesis studies (16) as A2Ј (17-20), T6Ј (21, 22), and L9Ј (23, 24) in the cytoplasmic half of the transmembrane 2 domain of the channel (Fig. 2). Drosophila resistant to dieldrin (RDL) have a mutation conferring GABA receptor insensitivity identified as A2ЈS (17). The NCA target of the GABA A receptor requires a ␤ subunit, and a ␤ 3 homopentamer is sufficient for binding (9,26). Importantly, the ␤ 3 subunit from human brain, when expressed in insect Sf9 cells, assembles to form a receptor sensitive to all of the important GABAergic insecticides (9) and, surprisingly, reproduces the insecticide sensitivity and structureactivit...

Insecticide interactions with .GAMMA.-aminobutyric acid and nicotinic receptors: predictive aspects of structural models

“…2, with the addition of BIDN and the EBOB analog TBPS in the original report. 7) The interacting residues are Ala-2Ј (or more generally the Ala-2Ј to Ile-5Ј hydrophobic pocket) and Thr-6Ј (the most important molecular determinant) with a supplemental role for Leu-9Ј. The more compact NCAs require only Ala-2Ј and Thr-6Ј, whereas the potency of long or extended NCA molecules is enhanced by the Leu-9Ј In comparison, the affinity ratio for EBOB is 5.0.…”

“…Favorable GABAR-NCA interactions (hydrophobic and hydrogen bonding) are illustrated in Fig. 2 and detailed by Chen et al 7) The model provides a template for ligand design, structure optimization and improved potency. Consideration of known SARs shows that high affinity corresponds to binding in a more stable configuration with Ͼ30 kJ/mol differences for the more potent g (lindane) than the less active a, b and g isomers of hexachlorocyclohexane, and the more potent a than the less active b-endosulfan.…”

“…Molecular interactions are shown for five NCAs at the cytoplasmic end of the TM2/a-helix of the human b3 homopentameric channel (Lower). 7) Two of the five b3 subunits are illustrated. These schemes do not allow visualization of three-dimensional positions and geometries for NCAs and their interacting residues.…”

“…the importance of 9Ј for fipronil and EBOB. Several other mutated b3 homopentamer TM2 sites also reduce NCA binding 7) and a TM3 mutation in Drosophila confers resistance to fipronil. 22) A nAChR leafhopper mutation associated with lower imidacloprid mortality and receptor sensitivity than the wild type involves Tyr-151 to Ser-151 (adjacent to loop B Trp-149) of a subunits.…”

Mechanisms for Picrotoxinin and Picrotin Blocks of α2 Homomeric Glycine Receptors

“…The number of binding sites of PTX, the location of these sites, and the inhibitory mechanism of this compound are still under debate (8), but there are several lines of evidence to indicate that PTX can bind within the channel pore. The 2Ј-6Ј amino acid residues that line the pore in the M2 domain are likely to be involved in the PTX effect (5, 7, 10 -12) or to form the binding site for PTX at least in ionotropic GABA receptors (13). The proposal that the location of the PTX-binding site is within the channel pore of GlyRs was recently reinforced.…”