Voltage-gated sodium channels are responsible for electrical signaling in the nervous system and other excitable cells. Insects take advantage of extensive alternative splicing and RNA editing of a single gene to achieve functional diversity of sodium channels. TipE and four TipE-homologous genes (TEH1-4) in Drosophila melanogaster are thought to encode auxiliary subunits of sodium channels. However, limited information is available on how TipE and TEH proteins modulate the gating of sodium channels. Sodium channels are the primary target site of pyrethroid insecticides. Due to intensive use of pyrethroids, resistance has become a serious problem for pest control. A major mechanism of pyrethroid resistance, known as knockdown resistance (kdr), is caused by mutations in the sodium channel. In addition to kdr mutations, naturally occurring sodium channel variants exhibit different sensitivities to pyrethroids. The objectives of my Ph.D. dissertation are: (1) to investigate the effects of TipE and TEH1-4 on the gating properties and sensitivity of sodium channel variants (DmNav) from D. melanogaster to pyrethroid insecticides; (2) to explore the involvement of RNA editing in regulating channel gating and sensitivity to pyrethroids; and (3) to determine whether a naturally occurring mutation contributes to pyrethroid resistance in mosquitoes. For the first objective, TipE or TEH1-4 was co-expressed individually with three different DmNav variants in Xenopus oocytes, and gating properties were determined by two-electrode voltage clamp. TEH1 and TEH4 induce hyperpolarizing and depolarizing shifts, respectively, in the voltage-dependence of activation, fast inactivation, and slow inactivation; TEH2 does not modulate the gating properties; and TipE modulates only the voltage-dependence of fast inactivation of one variant. TEH3 enhances an outward current of an endogenous channel(s) in Xenopus oocytes, which prevented further analysis of its effect on the sodium channel variants. For the second objective, the role of RNA editing in gating and channel sensitivity to pyrethroids was investigated by individually introducing four RNA editing sites into an unedited variant DmNav26. One RNA editing event, N1587S, causes a hyperpolarizing shift in the voltage-dependence of slow inactivation of the DmNav26 channel. Another RNA editing event, Q1296R, enhances the sensitivity of DmNav 26 channels to a pyrethroid insecticide, deltamethrin. For the third objective, whether a naturally occurring mutation N1575Y, detected from pyrethroid-resistant populations of Anopheles gambiae mosquitoes, contributes to resistance to pyrethroids was investigated. I found that the N1575Y mutation alone does not alter the gating or sensitivity of a mosquito sodium channel to deltamethrin. However, the N1575Y mutation augmented channel sensitivity to deltamethrin induced by two kdr mutations L1014F/S located in pyrethroid receptor site 2, but not by a kdr mutation, V1016G, located in pyrethroid receptor site 1. These results suggest that the N1575Y mutation functions as an enhancer of site-2 kdr mutations. In conclusion, my research shows critical roles of TipE/TEH proteins and RNA editing in the regulation of insect sodium channel function. Furthermore, identification of both the role of RNA editing in regulating channel sensitivity to pyrethroids and the N1575Y mutation as an enhancer for pyrethroid resistance in mosquitoes provide new insights into the molecular mechanisms of pyrethroid resistance.
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