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, N(1575)Y, in the cytoplasmic loop linking domains III and IV (LIII/IV) was found to coexist with a PyR2 mutation, L(1014)F in IIS6, in pyrethroid-resistant populations of Anopheles gambiae. To examine the role of this mutation in pyrethroid resistance, N(1575)Y alone or N(1575)Y + L(1014)F were introduced into an Aedes aegypti sodium channel, AaNav1-1, and the mutants were functionally examined in Xenopus oocytes. N(1575)Y did not alter AaNav1-1 sensitivity to pyrethroids. However, the N(1575)Y + L(1014)F double mutant was more resistant to pyrethroids than the L(1014)F mutant channel. Further mutational analysis showed that N(1575)Y could also synergize the effect of L(1014)S/W, but not L(1014)G or other pyrethroid-resistant mutations in IS6 or IIS6. Computer modeling predicts that N(1575)Y allosterically alters PyR2 via a small shift of IIS6. Our findings provide the molecular basis for the coexistence of N(1575)Y with L(1014)F in pyrethroid resistance, and suggest an allosteric interaction between IIS6 and LIII/IV in the sodium channel.