Fig 1.
Gating properties of AaNav1-1 and V1016I channels.
(A) Membrane topology. (B) Voltage dependence of activation. (C) Voltage dependence of fast inactivation. (D) Voltage dependence of slow inactivation. (E) Recovery from fast inactivation. (F) Recovery from slow inactivation. Voltage step protocols used to generate the curves are shown above each panel.
Fig 2.
Gating properties of the AaNav1-1, T1520I and T1520I/F1534C channels.
(A) Positions of T1520I and F1534C. (B) Voltage dependence of activation. (C) Voltage dependence of fast inactivation. (D) Voltage dependence of slow inactivation. (E) Recovery from fast inactivation. (F) Recovery from slow inactivation. Voltage step protocols are the same as in Fig 1.
Table 1.
Voltage dependence of activation and fast inactivation of AaNav1-1 and mutant sodium channels.
Table 2.
Voltage dependence of slow inactivation of AaNav1-1 and mutant sodium channels.
Fig 3.
Effects of mutations V1016I and F1534C on the channel sensitivity to permethrin (PMT) and deltamethrin (DMT).
(A) Representative tail currents induced by 1.0 μM PMT. (B) Representative tail currents induced by 1.0 μM DMT. (C) Dose-response curves of the channels modification by PMT. (D) Dose-response curves of the channels modification by DMT. The dose-response curve was fitting with Hill equation. Statistical significance was determined by using one-way ANOVA with Scheffé's post hoc analysis, and significant values were set at p < 0.05. The number of oocytes for each mutant construct was more than 6.
Fig 4.
Effects of mutations T1520I and F1534C on the channel sensitivity to pyrethroids.
(A) Channel modification by 1.0 μM PMT. (B) Channel modification by 1.0 μM DMT. (C) Channel modification by 1.0 μM cypermethrin, cyfluthrin, NRDC 157 and bifenthrin. The number of oocytes for each mutant was > 6. The asterisks indicate significant differences from the AaNav1-1 channel as determined by using the one way ANOVA with Scheffé's post hoc analysis (p < 0.05). The pound sign indicates a significant difference in sensitivity to PMT between mutants as determined using one-way ANOVA with Scheffé's post hoc analysis (p < 0.05).
Fig 5.
Effects of mutations on the channel sensitivity to DDT.
(A) Representative traces from AaNav1-1, V1016G, V1016I, T1520I, F1534C, T1520I+F1534C and V1016I+F1534C channels after incubation with DDT (100 μM). (B) Percentages of channel inactivation inhibited by DDT (100 μM). The number of oocytes for each mutant was more than 8. Error bars indicate mean ± s.e. The asterisks indicate significant differences in sensitivity of mutants versus wildtype to DDT as determined by using one-way ANOVA with Scheffé's post hoc analysis (p < 0.05). The pound sign indicates a significant difference in sensitivity to DDT between mutants as determined using one-way ANOVA with Scheffé's post hoc analysis (p < 0.05).
Fig 6.
(A) Sequence alignment of sodium channel segments involved in the proposed mechanism by which mutation T1520/3i(-1)I allosterically induces small changes in the PyR1 site geometry. Highlighted are residues (except position 2i18), which are shown in panels B and C. Residue numbers in NavPaS and EeNav1.4 are sequential numbers in the PDB files of the cryo-EM structures where some segments are lacking. (B) and (C), Cryo-EM structures of eukaryotic sodium channels NavPaS (B) and EeNav1.4 (C). The pore-module helices in repeats I, II, III, and IV are magenta, yellow, green and gray, respectively. Side chains in positions 2i18 and 3i13, which correspond to AaNav1-1 residues 1016 and 1534, are space-filled. In both channels, these residues are in the II/III repeat interface that harbors the pyrethroid receptor site PyR1. Threonine T3(i-1) in the extracellular loop of NavPaS, which correspond to T1520/3(i-1) in AaNav1-1, is close to the N-terminus of IIIS6 and in AaNav1-1 it cannot directly interact with PyR1-bound pyrethroids.
Fig 7.
Sequential selection of kdr mutations for pyrethroid resistance in Ae. aegypti.
Darker background colors indicate higher levels of resistance. V1016 and T1520I are selected in mosquitoes carrying F1534C. F1534C (and probably V1016I) emerged under the DDT pressure prior to usage of pyrethroids. T1520I was selected under pressure of Type I pyrethroids.