NACHO permits functional heterologous expression of an insect homomeric α6 nicotinic acetylcholine receptor.

Insect nicotinic acetylcholine receptors (nAChR) are molecular targets of highly effective insecticides. The use of chaperone proteins has been key to successful functional expression of these receptors in heterologous systems, permitting functional and pharmacological studies of insect nAChRs with particular subunit composition. Here, we report the first use of the chaperone protein, NACHO, to enable functional expression of an insect nAChR, the α6 subunit from Apis mellifera, in Xenopus laevis oocytes. This is also the first report of functional expression of a homomeric insect α6 nAChR. Using two-electrode voltage-clamp electrophysiology we show that the acetylcholine EC50 of the α6 receptor is 0.88 µM and that acetylcholine responses are antagonized by α-bungarotoxin. Spinosad showed agonist actions and kept the ion channel open when co-applied with acetylcholine, reinforcing the α6 nAChR subunit as a key molecular target for the spinosyn class of insecticide. The use of NACHO may provide a basis for future expression studies of insect α6 nAChRs, potentially providing a tool for the discovery of novel insecticides.

The Apis mellifera alpha 5 nicotinic acetylcholine receptor subunit expresses as a homomeric receptor that is sensitive to serotonin.

Insect nicotinic acetylcholine receptors (nAChRs) are molecular targets of highly effective insecticides such as neonicotinoids. Functional expression of these receptors provides useful insights into their functional and pharmacological properties. Here, we report that the α5 nAChR subunit of the honey bee, Apis mellifera, functionally expresses in Xenopus laevis oocytes, which is the first time a homomeric insect nAChR has been robustly expressed in a heterologous system without the need for chaperone proteins. Using two-electrode voltage-clamp electrophysiology we show that the α5 receptor has low sensitivity to acetylcholine with an EC50 of 2.37 mM. However, serotonin acts as an agonist with a considerably lower EC50 at 119 µM that is also more efficacious than acetylcholine in activating the receptor. Molecular modelling indicates that residues in the complementary binding site may be involved in the selectivity towards serotonin. This is the first report of a ligand-gated ion channel activated by serotonin from an insect and phylogenetic analysis shows that the α5 subunit of A. mellifera and other non-Dipteran insects, including pest species, belong to a distinct subgroup of subunits, which may represent targets for the development of novel classes of insecticides.