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

Abstract

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that present a target for insecticides. However, a better understanding of receptor subunit composition is needed for effective design of insecticides. Peptide neurotoxins are known to block nAChRs by binding to its target subunits. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes. 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 Dα5, Dα6, Dα7 subunits. Finally, we report the localization of fluorescent tagged endogenous Dα6 during nervous system development. Taken together this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins.

Gcm counteracts Toll-induced inflammation and impacts hemocyte number through cholinergic signaling

“…The larva contains on average 1.05% ± 0.49% of nAchRalpha6-positive hemocytes (n = 3, estimated by cytometry on 10 larvae per replicate). Because of the highest effect observed in the double mutant larvae, we focused on nAchRalpha6 and confirmed its expression profile with a transgenic line expressing a nAchRalpha6-YFP fusion protein (46) (Supplementary Figures S3D-S3F'''). The nAchRalpha6-positive hemocytes express strongly the plasmatocyte markers Nimrod C1 (NimC1 or P1) and Hemese (He) (Supplementary Figures S3C-S3F''').…”