Insect Biochem Mol Biol 2025 Oct 30;:104430. doi: 10.1016/j.ibmb.2025.104430.

Heterologous Expression of Nilaparvata lugens (Stål) Voltage-Gated Sodium Channels in Sf9 Cells for Electrophysiological and Pyrethroid Modulation Studies.

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Voltage-gated sodium channels (VGSCs) are essential for neuronal excitability and serve as primary targets of pyrethroid insecticides. Research on insect VGSCs has relied predominantly on the Xenopus laevis oocytes, yet splice variants containing exon b often fail to generate measurable currents, limiting functional and pharmacological characterization. In this study, we cloned two exon b-containing splice variants from the brown planthopper (Nilaparvata lugens), NlNav7 (harboring exon l) and a previously uncharacterized NlNav25 (harboring exon k), and established an Sf9 insect cell expression system enabling robust functional expression of these challenging variants. NlNav25 exhibited high expression efficiency, reliable gating kinetics, and tetrodotoxin sensitivity even in the absence of NlTipE. Co-expression of NlTipE further increased current amplitude and slightly shifted voltage dependence, indicating a conserved auxiliary role in promoting channel stability and gating modulation. We further evaluated the sensitivity of NlNav7 and NlNav25 to three pyrethroids. Deltamethrin delayed tail currents with comparable efficacy in both variants. In contrast, transfluthrin delayed inactivation and tail currents: at a saturating concentration 50 μM, the I45ms/Ipeak and tail current modification ratios were 103.0% and 93.2% for NlNav7, but only 65.5% and 56.3% for NlNav25, despite similar apparent EC50. At 100 μM, etofenprox modified NlNav7 tail currents (62.5%) more effectively than NlNav25 (34.7%). These finding indicate that exon k/l critically determines the efficacy of transfluthrin and etofenprox, but not deltamethrin. Collectively, this study establishes a high-efficiency Sf9-based heterologous expression system for N. lugens VGSCs, suitable for rapid functional analysis, high-throughput insecticide screening, and provide insights into splice variant-dependent pyrethroid resistance mechanisms.

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