Distinct roles of the DmNav and DSC1 channels in the action of DDT and pyrethroids

Abstract

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (para(ts)) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a para(ts1) allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in para(ts1) mutant flies was almost completely abolished in para(ts1);DSC1(-/-) double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w(1118A)), and the para(ts1);DSC1(-/-) double mutant flies were even more resistant to DDT compared to the DSC1 knockout or para(ts1) mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance.

Keywords: DDT; DSC1; Pyrethroids insecticide resistance; Voltage-gated sodium channel.

Figure 1

The para^ts1 (I265N) channels are less sensitive to the pyrethroids permethrin and deltamethrin than DmNa_v22. A and B. Tail-current induced by permethrin (A) or deltamethrin (B) from DmNa_v22 or I265N channels. The I265N mutation is located in the linker connecting S4 and S5 of domain I. C. Percent channel modification by pyrethroids. The number of oocytes for each channel was >5. The protocols and quantitative analysis of pyrethroid-induced tails are described in the Materials and Methods. Data are the average ± SEM. * indicates significantly different from DmNa_v22 channels (Student’s t-test, P < 0.05).

Figure 2

The para^ts1 (I265N) channels are less sensitive to DDT than DmNa_v22 channels. A. Representative sodium current traces from oocytes expressing DmNa_v22 and I265N channels. B. Representative sodium current traces from DmNa_v22 and I265N channels after overnight incubation in 100 µM DDT. C. Inhibition of fast inactivation by DDT. The number of oocytes for each channel was >15. The protocol and quantitative analysis of DDT inhibition are described in the Materials and Methods. Data are the average ± SEM. * indicates significantly different from DmNa_v22 channels (Student’s t-test, P < 0.05).

Figure 3

The time course of knockdown of D. melanogaster exposed to deltamethrin (A) and DDT (B). DSC1^−/− exhibited increased knockdown by deltamethrin, but demonstrated delayed knockdown by DDT. There was no significant knockdown of para^ts1 or para^ts1;DSC1^−/− by DDT. Data are the average ± SEM. Letters above each data point at every 5 minute intervals indicate significant differences at each time point (One-Way ANOVA, Fishers LSD, P<0.05).

Figure 3

The time course of knockdown of D. melanogaster exposed to deltamethrin (A) and DDT (B). DSC1^−/− exhibited increased knockdown by deltamethrin, but demonstrated delayed knockdown by DDT. There was no significant knockdown of para^ts1 or para^ts1;DSC1^−/− by DDT. Data are the average ± SEM. Letters above each data point at every 5 minute intervals indicate significant differences at each time point (One-Way ANOVA, Fishers LSD, P<0.05).

Figure 4

K_v1.2-based model of the open DmNa_v channel phenotype N^1k12 (N265) with a deltamethrin molecule docked to PyR2 as described before (Du et al., 2013). Helices in domains I, II, III and IV are shown, respectively, by yellow, red, green, and white ribbons. Pyrethroid-sensing residues in the I/II domain interface, as well as N^1k12 and its possible H-bonding partners V^1k8 (A), S^1o2 (B) and V^1o3 (C) are shown as sticks. Asparagine N^1k12 is far from deltamethrin and does not contribute to the pyrethroid binding site. However, an H-bond between the N^1k12 side chain and a backbone atom can deform the backbone and thus affect the mutual disposition of deltamethrin and pyrethroid-sensing residues in PyR2.