Department of Biology, Duke University, Durham, NC, USA; College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Department of Entomology, Michigan State University, East Lansing, MI, USA.
Insect Biochem Mol Biol. 2022 Sep;148:103814. doi: 10.1016/j.ibmb.2022.103814. Epub 2022 Aug 3.
Pyrethroid insecticides prolong the opening of insect sodium channels by binding to two predicted pyrethroid receptor sites (PyR), PyR1 and PyR2. Many naturally-occurring sodium channel mutations that confer pyrethroid resistance (known as knockdown resistance, kdr) are located at PyR1. Recent studies identified two new mutations, V253F and T267A, at PyR2, which co-exist with two well-known mutations F1534C or M918T, at PyR1, in pyrethroid-resistant populations of Aedes aegypti and Nilaparvata lugens, respectively. However, the role of the V253F and T267A mutations in pyrethroid resistance has not been functionally examined. Here we report functional characterization of the V253F and T267A mutations in the Ae. aegypti sodium channel AaNa2-1 and the N. lugens sodium channel NlNa1 expressed in Xenopus oocytes. Both mutations alone reduced channel sensitivity to pyrethroids, including etofenprox. We docked etofenprox in a homology model of the pore module of the NlNa1 channel based on the crystal structure of an open prokaryotic sodium channel NavMs. In the low-energy binding pose etofenprox formed contacts with V253, T267 and a previously identified L1014 within PyR2. Combining of V253F or T267A with F1534C or M918T results in a higher level of pyrethroid insensitivity. Furthermore, both V253F and T267A mutations altered channel gating properties. However, V253F- and T267A-induced gating modifications was not observed in the double mutant channels. Our findings highlight the first example in which naturally-found combinational mutations in PyR1 and PyR2 not only confer higher level pyrethroid insensitivity, but also reduce potential fitness tradeoff in pyrethroid-resistant mosquitoes caused by kdr mutation-induced sodium channel gating modifications.
拟除虫菊酯杀虫剂通过与两个预测的拟除虫菊酯受体位点（PyR）结合，延长昆虫钠离子通道的开放时间。许多导致拟除虫菊酯抗性（称为击倒抗性，kdr）的天然存在的钠离子通道突变位于 PyR1。最近的研究在埃及伊蚊和褐飞虱的抗药性种群中分别在 PyR2 上发现了两个新的突变 V253F 和 T267A，这两个突变与 PyR1 上的两个著名突变 F1534C 或 M918T 共存。然而，V253F 和 T267A 突变在拟除虫菊酯抗性中的作用尚未进行功能研究。在这里，我们报道了在埃及伊蚊钠离子通道 AaNa2-1 和褐飞虱钠离子通道 NlNa1 中 V253F 和 T267A 突变的功能特征，这些通道是在非洲爪蟾卵母细胞中表达的。这两个突变单独降低了通道对拟除虫菊酯的敏感性，包括乙氰菊酯。我们根据开放原核钠离子通道 NavMs 的晶体结构，将乙氰菊酯对接在 NlNa1 通道孔模块的同源模型中。在低能量结合构象中，乙氰菊酯与 PyR2 中的 V253、T267 和先前鉴定的 L1014 形成了接触。V253F 或 T267A 与 F1534C 或 M918T 的组合导致拟除虫菊酯敏感性降低的程度更高。此外，V253F 和 T267A 突变改变了通道门控特性。然而，在双突变通道中没有观察到 V253F 和 T267A 诱导的门控修饰。我们的研究结果突出了第一个例子，即 PyR1 和 PyR2 中天然发现的组合突变不仅赋予了更高水平的拟除虫菊酯抗性，而且降低了 kdr 突变引起的钠离子通道门控修饰对抗药性蚊子造成的潜在适应性权衡。
