Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of alpha-Bungarotoxin (alpha-Btx) and omega-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm alpha-Btx interactions with the Drosophila alpha 5 (D alpha 5), D alpha 6, D alpha 7 subunits. Finally, we report the localisation of fluorescent tagged endogenous D alpha 6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.

Automated summary

This study utilized a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in Drosophila, and investigated their interactions with peptide neurotoxins. The findings have important implications for the development of more effective insecticides.