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

Abstract

<jats:p><jats:italic>Drosophila</jats:italic> 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 <jats:italic>nAChR</jats:italic> 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 α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the <jats:italic>Drosophila</jats:italic> α5 (D<jats:italic>α</jats:italic>5), Dα6, D<jats:italic>α</jats:italic>7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during <jats:italic>Drosophila</jats:italic> CNS development. Taken together, this study elucidates native <jats:italic>Drosophila</jats:italic> nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.</jats:p>