Functional characterisation of the honeybee Apis mellifera and diamondback moth Plutella xylostella α5 nicotinic acetylcholine receptor identifies a subset of ion channels with unusual pharmacological properties

Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of cysteine-loop ligand-gated ion channels (cysLGICs) and are best known for mediating the fast actions of acetylcholine. These receptors are made up of five subunits belonging to either alpha or non-alpha types that are arranged around a central ion channel. In insects, nAChRs perform important roles in signalling in the nervous system and are also targets of highly effective insecticides such as neonicotinoids. Most of these subunits form heteromers consisting of at least two different subunit types. However, members of the α5-7 group are similar to the vertebrate α7 nAChR in sequence similarity and by forming homomeric receptors consisting of five identical alpha subunits. Phylogenetic analysis of nAChR peptide sequences suggested that α5 subunits of Hymenoptera (e.g. bees) and Lepidoptera (e.g. moths and butterflies) form a clade distinct to that of Diptera (e.g. flies and mosquitoes) α5 subunits. This prompted an investigation into whether Hymenoptera α5 nAChRs show distinct pharmacological properties. Here, we confirm preliminary findings that the α5 nAChR subunit of the western honeybee, Apis mellifera, expresses as a homomeric ion channel in Xenopus laevis oocytes that shows unusual sensitivity to biogenic amines including dopamine whilst being insensitive to acetylcholine. We have taken advantage of this to create mutations in the ligand-binding site of α5 to identify amino acid residues in loop E as being important for determining dopamine potency and efficacy. As yet, the Lepidopteran α5 nAChR has not been functionally characterised. The diamondback moth, Plutella xylostella, is a cosmopolitan pest of cruciferous crops. Here, we report successful heterologous expression of the Plutella α5 nAChR in Xenopus oocytes and show that it is also responsive to biogenic amines, with dopamine and serotonin showing greater potency than acetylcholine. Interestingly, flupyrimin acted as an agonist, increasing our understanding of the mode of action and potential molecular targets of this insecticide. Subsequent molecular docking has highlighted possible ligand interactions with other insecticides including triflumezopyrim and dicloromezotiaz of the mesoionic class. The characterisation of bee and moth α5 nAChRs highlights a novel subclass of receptors particular to certain insects, thereby enhancing our understanding of the evolution of cysLGICs. Findings from this study can prompt further investigation into the role of α5 in invertebrate nervous system function, as well as provide a basis for investigating molecular targets of currently used insecticides as well as the effects of compounds to be used as novel pesticides or biopesticides.

Name dc:type.qualificationname

Ph.D

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

Oxford Brookes University

Author dc:creator

• Armstrong, Emily Beatrice

Advisors dc:contributor.advisor

• Jones, Andrew K
• Bermudez-Diaz, Isabel

DOI dc:identifier.doi

https://doi.org/10.24384/fbtj-z158

OAI record oai:identifier

https://radar.brookes.ac.uk/radar/oai?verb=GetRecord&metadataPrefix=uketd_dc&identifier=tle%3Af611c9fa-a168-4eb9-8a13-f49f10344065%3Ad6bd9758-527a-46cd-bfe2-c433766e8fca%3A1