Structural basis of noncompetitive inhibition by the phenylpyrazole insecticide fipronil in an ELIC-α1 GABAAR chimera

Fipronil (FIP) is a phenylpyrazole insecticide that acts as a noncompetitive pore blocker of insect type A γ-aminobutyric acid chloride channels (GABA A Rs). Although genetic and mutagenesis studies have long implicated the pore-lining TM2 helix, particularly the 2′ and 6′ positions, in FIP action, the structural basis by which this asymmetric phenylpyrazole is accommodated within the pseudo-fivefold symmetric chloride-channel pore has remained unknown. Here, we determined a 3.7 Å cryo-EM structure of FIP bound to an ELIC-based chimera (ELIC-α1) containing the transmembrane domain of the human GABA A R α1 subunit, providing the first structural view of phenylpyrazole recognition within the GABA A receptor pore. A single FIP molecule was modeled within the lower TM2-lined ion-conduction pathway in an elongated asymmetric pose. The phenyl group is positioned near the hydrophobic 2′ layer, whereas the trifluoromethylsulfinyl moiety extends toward the polar 6′ threonine ring. Molecular dynamics simulations support the stability of this configuration and reveal repeated formation of a hydrogen bond between the sulfoxide group and the 6′T side chain. The A2′S/T6′M double substitution in the Drosophila melanogaster RDL receptor reduced FIP sensitivity, supporting the functional relevance of the equivalent pore positions in an insect receptor. Structural comparison with picrotoxin- and pregnenolone sulfate-bound GABA A Rs further suggests that FIP exploits a conserved lower-TM2 inhibitory site shared by chemically diverse pore blockers. Together, these findings support a layered pore-binding model for FIP inhibition in ELIC-α1, link equivalent pore positions to FIP sensitivity in insect RDL, and provide a framework for the rational design of next-generation phenylpyrazole insecticides.