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Proc Natl Acad Sci U S A
2020 Jul 14;11728:16283-16291. doi: 10.1073/pnas.2003667117.
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Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.
Ihara M
,
Furutani S
,
Shigetou S
,
Shimada S
,
Niki K
,
Komori Y
,
Kamiya M
,
Koizumi W
,
Magara L
,
Hikida M
,
Noguchi A
,
Okuhara D
,
Yoshinari Y
,
Kondo S
,
Tanimoto H
,
Niwa R
,
Sattelle DB
,
Matsuda K
.
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The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.
Fig. 1. Colocalization of nAChR subunits and their functional expression. (A) Cartoon of testis, accessory glands, and ejaculatory ducts of the fruit fly and a microscope image of these tissues. (B–E) Microscope images of ejaculatory ducts of male flies carrying UAS-GFP, UAS-mCD8::GFP with Dα1-2A-GAL4 (B), Dα2-2A-GAL4 (C), Dβ1-2A-GAL4 (D), and Dβ2-2A-GAL4 (E). (F) ACh-induced currents recorded from X. laevis oocytes expressing various D. melanogaster nAChR subunits in combination with DmRIC-3, DmUNC-50, and DmTMX3. Boxes show median and 25th to 75th percentiles of ACh response amplitudes with minimum and maximum indicated as whiskers (n = 20). *P < 0.05 (one-way ANOVA, Kruskal–Wallis test). (G) ACh concentration–response relationships. Each plot represents mean ± SEM (n = 5).
Fig. 2. Modulation by neonicotinoids of D. melanogaster nAChRs. (AâD) Concentrationâresponse relationships of neonicotinoids for wild-type fruit fly nAChRs and their R81T mutants in which the Arg81 of the Dβ1 subunit was replaced by threonine. In the mutant nAChRs, the Dβ1 subunit possesses the R81T amino acid substitution. Each plot represents mean ± SEM (n = 5). (EâH) Neonicotinoid modulation of responses to 100 μM ACh of the wild-type and mutant fruit fly nAChRs. Boxes show median and 25th to 75th percentiles with minimum and maximum indicated as whiskers of normalized peak amplitude of response to ACh (n = 5). *P < 0.05 (one-way ANOVA, Bonferroni test).
Fig. 3. Agonist actions of neonicotinoids on honeybee and bumblebee nAChRs. (A) Relationships of D. melanogaster, A. mellifera, and B. terrestris nAChR subunit proteins. DmRDL: D. melanogaster GABAA receptor subunit RDL. Bootstrap values are shown at each node. (B and C) ACh-induced responses of the honeybee (B) and bumblebee (C) nAChRs. The boxes represent median and 25th to 75th percentiles of ACh response amplitudes with minimum and maximum shown as whiskers (honeybee, n = 20; bumblebee, n = 10). (D and E) Concentration–response relationships for neonicotinoids on the honeybee (D) and bumblebee (E) nAChRs. Each plot represents mean ± SEM (n = 5).
Fig. 4. Modulation of honeybee (A. mellifera) and bumblebee (B. terrestris) nAChRs by imidacloprid, thiacloprid, and clothianidin. Neonicotinoid modulation of 100 μM ACh-induced response of the A. mellifera (A) and B. terrestris nAChRs (B) were examined according to the same method as performed for the fruit fly (D. melanogaster) nAChRs (Fig. 2). Bar graphs represent mean ± SEM, and data points are plotted in each bar graph (n = 5). *P < 0.05 (one-way ANOVA, Bonferroni test) compared with untreated control response to ACh.
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