Identification of α6-Containing Nicotinic Acetylcholine Receptors as the Primary Target of Nereistoxin Insecticides...

Nereistoxin (NTX) and its derivative insecticides cartap and monosultap have been used for decades to control lepidopteran pests by targeting nicotinic acetylcholine receptors (nAChRs). Unlike neonicotinoid agonists that induce excitatory neurotoxicity, nereistoxin insecticides act as antagonists, blocking cholinergic neurotransmission. However, the molecular target and mechanism of action of these compounds remain incompletely understood. Here, we elucidated the mode of action of cartap and monosultap in Drosophila melanogaster through integrated genetic and computational approaches. Bioassays with Drosophila nAChR subunit mutants demonstrated that the α6 subunit is critically required for insecticidal activity of both compounds, with α6 knockout conferring approximately 10-fold resistance to monosultap and 7-fold resistance to cartap. Molecular docking of protonated NTX into an α6 homopentameric channel model revealed a "dual-anchor" blocking mechanism: the protonated amine forms electrostatic interactions with residue Glu267, while the dithiolane ring creates steric hindrance at residue Thr270. Pore diameter measurements showed an optimal binding cavity of 5.96-6.22 Å in the 267-270 region, narrowing dramatically to 1.64 Å at the deep gate (Ser278), explaining how NTX binding physically occludes the channel. Collectively, these results identify α6-containing nAChRs as the primary target of nereistoxin insecticides and provide a structural framework for understanding channel blockade, with important implications for resistance monitoring and the development of next-generation channel-blocking insecticides.