A Unique Mechanism of Transfluthrin Action Revealed by Mapping Its Binding Sites in the Mosquito Sodium Channel.

Pyrethroid insecticides exert their toxic action by prolonging the opening of insect voltage-gated sodium channels, resulting in the characteristic tail current during membrane repolarization in voltage clamp experiments. Permethrin (PMT) and deltamethrin (DMT), representative type I and type II pyrethroids, respectively, are predicted to bind to two lipid-exposed pyrethroid receptor sites, PyR1 and PyR2, at the lipid-exposed interfaces of repeats II/III and I/II, respectively. Transfluthrin (TF), a volatile type I pyrethroid and mosquito repellent, has received increased attention in the global combat of vector-borne human diseases. However, the electrophysiological and molecular bases of TF action on insect sodium channels remain unexplored. In this study we discovered that, unlike DMT and PMT, TF barely induces the characteristic tail current of the Aedes aegypti mosquito sodium channel (AaNa_v1-1) expressed in Xenopus oocytes. Instead, TF induces a unique persistent current. We docked TF into the AlphaFold2 model of AaNa_v1-1 and found that the tetrafluorophenyl ring of TF binds to alpha helices S5, P1, and S6, but not to the linker helices S4-S5 within either PyR1 or PyR2. In agreement with the model, functional examination of 15 AaNa_v1-1 mutants demonstrated that substitutions of DMT/PMT-sensing residues in helices S5, P1, and S6, but not in the linker- helices S4-S5, altered channel sensitivity to TF. These results revealed the unique action of TF on channel gating and suggest a distinct subtype of type I pyrethroids with a previously uncharacterized pattern of interactions with residues at the dual pyrethroid receptor sites.