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.