A Mutation in the Intracellular Loop III/IV of Mosquito Sodium Channel Synergizes the Effect of Mutations in Helix IIS6 on Pyrethroid Resistance

Activation and inactivation of voltage-gated sodium channels are critical for proper electrical signaling in excitable cells. Pyrethroid insecticides promote activation and inhibit inactivation of sodium channels, resulting in prolonged opening of sodium channels. They preferably bind to the open state of the sodium channel by interacting with two distinct receptor sites, pyrethroid receptor sites PyR1 and PyR2, formed by the interfaces of domains II/III and I/II, respectively. Specific mutations in PyR1 or PyR2 confer pyrethroid resistance in various arthropod pests and disease vectors. Recently, a unique mutation, (NY)-Y-1575, in the cytoplasmic loop linking domains III and IV (LIII/IV) was found to coexist with a PyR2 mutation, (LF)-F-1014 in IIS6, in pyrethroid-resistant populations of Anopheles gambiae. To ex-amine the role of this mutation in pyrethroid resistance, (NY)-Y-1575 alone or (NY)-Y-1575 1 (LF)-F-1014 were introduced into an Aedes aegypti sodium channel, AaNa(v)1-1, and the mutants were functionally examined in Xenopus oocytes. (NY)-Y-1575 did not alter AaNa(v)1-1 sensitivity to pyrethroids. However, the (NY)-Y-1575 + (LF)-F-1014 double mutant was more resistant to pyrethroids than the (LF)-F-1014 mutant channel. Further mutational analysis showed that (NY)-Y-1575 could also synergize the effect of (LS)-S-1014/W, but not L(1014)G or other pyrethroid-resistant mutations in IS6 or IIS6. Computer modeling predicts that (NY)-Y-1575 allosterically alters PyR2 via a small shift of IIS6. Our findings provide the molecular basis for the coexistence of (NY)-Y-1575 with (LF)-F-1014 in pyrethroid resistance, and suggest an allosteric interaction between IIS6 and LIII/IV in the sodium channel.