Mutations of two acidic residues at the cytoplasmic end of segment IIIS6 of an insect sodium channel have distinct effects on pyrethroid resistance

Abstract

Mutations in sodium channels are known to confer knockdown resistance to pyrethroid insecticides, such as permethrin and cypermethrin, in various agricultural pests and disease vectors. Double mutations, D³ⁱ²⁸V and E³ⁱ³²G, were detected in cypermethrin-resistant Helicoverpa armigera and Heliothis virescens populations. However, the role of the two mutations in pyrethroid resistance remains unclear. In this study, we introduced the mutations into the cockroach sodium channel, BgNa_v1-1a, and examined their effects on channel gating and pyrethroid sensitivity in Xenopus oocytes. D³ⁱ²⁸V alone and the double mutation, D³ⁱ²⁸V/E³ⁱ³²G, shifted the voltage dependence of activation in the depolarizing direction by 15 mV and 20 mV, respectively, whereas E³ⁱ³²G had no significant effect. D³ⁱ²⁸V reduced the amplitude of tail currents induced by permethrin and NRDC 157 (Type I pyrethroids) and deltamethrin and cypermethrin (Type II pyrethroids), whereas E³ⁱ³²G alone had no effect. Intriguingly, the amplitude of Type II pyrethroid-induced tail current from D³ⁱ²⁸V/E³ⁱ³²G channels was similar to that of BgNa_v1-1a channels, but the decay of the tail currents was accelerated. Such effects were not observed for Type I pyrethroid-induced tail currents. Computational analysis based on the model of dual pyrethroid receptors on insect sodium channels predicted D³ⁱ²⁸V and E³ⁱ³²G exert their effects on channel gating and pyrethroid action via allosteric mechanisms. Our results not only illustrate the distinct effect of the D³ⁱ²⁸V/E³ⁱ³²G double mutations on Type I vs. Type II pyrethroids, but also reinforce the concept that accelerated decay of tail currents can be an effective mechanism of pyrethroid resistance to Type II pyrethroids.

Keywords: Knockdown resistance; Pyrethroid receptor; Sodium channel; Type I pyrethroids; Type II pyrethroids.

Fig. 1

A. Sodium channel topology indicating two mutations from Helicoverpa armigera and Heliothis virescens that were resistant to cypermethrin (Head et al., 1998). The positions of D³ⁱ²⁸V and E³ⁱ³²G are indicated in brackets. We use a residue-labeling scheme that is universal for P-loop channels (Du et al., 2013; Zhorov and Tikhonov, 2004). A residue is labeled by the domain number (1–4), segment type (k, the linker-helix between S4 and S5; i, the inner helix S6; and o, the outer helix S5), and relative number of the residue in the segment. B. The sequence alignment of segments involved in state-dependent contacts of residues in position i28 and i32. Underlined residues are mutations explored in this work.

Fig. 2

Voltage dependence of activation and inactivation of BgNa_v1-1a and mutant sodium channels. A. Voltage dependence of activation. B. Voltage dependence of inactivation. Voltage step protocols used to generate these curves are indicated above each panel.

Fig. 3

Effects of D³ⁱ²⁸V and E³ⁱ³²G mutations on the sensitivity of BgNa_v1-1a channels to permethrin (PMT) and deltamethrin (DMT). A and B. Tail currents induced by PMT or DMT from oocytes expressing BgNa_v1-1a and mutant channels. C. Percentage of channel modification by PMT and DMT. The values of EC₂₀ for permethrin were 0.13, 0.63, 0.12 and 0.49 μM for BgNa_v1-1a, D³ⁱ²⁸V, E³ⁱ³²G and D³ⁱ²⁸V/E³ⁱ³²G channels, respectively. The values of EC₂₀ for deltamethrin were 0.09, 0.59, 0.07 and 0.08 μM for BgNa_v1-1a, D³ⁱ²⁸V, E³ⁱ³²G and D³ⁱ²⁸V/E³ⁱ³²G channels, respectively. The number of oocytes for each mutant construct was >5. Error bars indicate mean ± SEM. The asterisks indicate significant differences from the BgNa_v1-1a channel as determined by using one-way analysis of variance (ANOVA) with Scheffé’s post hoc analysis (p < 0.05).

Fig. 4

Effects of D³ⁱ²⁸V and E³ⁱ³²G mutations on the sensitivity of BgNa_v1-1a channels to cypermethrin (CPMT) and NRDC 157. A and B. Tail currents induced by NRDC 157 or CPMT from oocytes expressing BgNa_v1-1a and mutant channels. C. Percentage of channel modification by 1.0 μM NRDC 157 and 1.0 μM CPMT. The number of oocytes for each mutant construct was >5. Error bars indicate mean ± SEM. The asterisks indicate significant differences from the BgNa_v1-1a channel as determined by using one-way ANOVA with Scheffé’s post hoc analysis (p < 0.05).

Fig. 5

Side views of the closed-state (A) and open-state (B) models of the wildtype BgNa_v1-1a channel and the open-state modes of the double-mutant D³ⁱ²⁸V/E³ⁱ³²G (lower panes at B). Repeats I, II, III and IV are colored pink, yellow, green and gray. In the open channel, E³ⁱ³² lacks inter-segment contacts, while D³ⁱ²⁸ forms unfavorable contact with the hydrophobic part of S^3o5. In the open channel, both D³ⁱ²⁸ and E³ⁱ³² are engaged in the inter-segment H-bonds with Q^3o1 and N^3k9. In the double mutant these H-bonds are lost, which would increase mobility of the cytoplasmic part of IIIS6. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)