Nicotinic acetylcholine receptors (nAChRs) are cysteine-loop ligand-gated ion channels that are the targets of effective insecticides, including neonicotinoids and spinosad. The heterologous expression of nAChRs, in Xenopus laevis oocytes, has enabled the study of functional and pharmacological properties of these receptors by two-electrode voltage clamp electrophysiology. Myzus persicae, the peach potato aphid, is a major global pest of multiple crop species. The nAChR subunits and several protein chaperones from Myzus persicae have been cloned. It was difficult to achieve robust and frequent expression of a nAChR consisting entirely of subunits from Myzus persicae with the chaperones RIC-3, UNC-50 and TMX3. However, use of the α1 subunit from Drosophila melanogaster allowed nAChRs containing Myzus persicae α3, α8 and β1 subunits to be expressed and characterised with an EC50 for acetylcholine of 123 µM. The Drosophila melanogaster α1 and Myzus persicae β1 subunits formed a functional nAChR with an EC50 for acetylcholine of 91.1 µM, upon which neonicotinoids acted as partial agonists. The Apis mellifera, European honeybee, α5 subunit formed a homomeric nAChR in oocytes without any chaperones. This was sensitive to 0.1 μM α-bungarotoxin and responded to both acetylcholine and choline but with low affinity, as shown by EC50 values in the millimolar range (2.65 mM and 9.07 mM respectively). Further testing revealed that the α5 nAChR responded to dopamine (EC50 3.37 μM), 5-hydroxytryptamine (EC50 122 μM), octopamine (EC50 378 μM), tyramine (EC50 91.1 μM) and histamine (EC50 3.36 mM) but not to GABA, glycine or glutamate. Dopamine, 5-hydroxytryptamine, octopamine and tyramine all acted as superagonists relative to acetylcholine and had higher affinities than acetylcholine, as shown by EC50 values in the micromolar range, with dopamine having the highest efficacy and affinity. Histamine was a low-affinity, partial agonist. Phylogenetic analysis indicated that the non-Dipteran α5, including from Apis mellifera, form a distinct group from the Dipteran α5. This thesis contributes to the rapidly growing field of knowledge gained from expressing insect nAChRs in heterologous systems. This is especially useful as a nAChR from a pest species has not been robustly expressed yet. The characterisation of a novel nAChR from the beneficial species, Apis mellifera, potentially indicates a new class of receptor, with unusual pharmacological properties.
Permanent link to this resource: https://doi.org/10.24384/djf6-na69
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Mitchell, Eleanor Louise
Supervisors: Jones, Andrew K.; Bermudez, Isabel; Goodchild, James
Department of Biological and Medical SciencesFaculty of Health and Life Sciences
Year: 2022
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