Molecular characterisation of insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric multi-subunit proteins that form ligand-gated ion channels in the central nervous systems of vertebrates and invertebrates and at the neuromuscular junction of vertebrate species. In insects, they play a major role in excitatory synaptic transmission and are the primary target site for neonicotinoid insecticides such as imidacloprid. Despite their commercial importance as insecticide targets and the recent cloning of nAChR subunit genes from a range of insect species, the insect nAChR is not well characterized at the molecular level. This study investigates the molecular diversity of nAChR subunit genes of two important agricultural pests, the peach-potato aphid Myzus persicae and the tobacco whitefly Bemisia tabaci. Five genes have already been cloned as full-length cDNAs from M. persicae and one from B. tabaci. Heterologous expression of M. persicae subunit genes in Drosophila S2 cells has revealed some evidence for co-assembly of certain a/p subunit combinations, although functional reconstitution has so far been dependant on co-expression of insect a subunits with a vertebrate β subunit. This suggests that one or more key subunits required for functional reconstitution of the native receptor have yet to be cloned. Heterologous expression of insect nAChR subunits has demonstrated that the binding affinity and agonist potency of imidacloprid is influenced markedly by nAChR subunit composition and only certain M. persicae a/rat β2 complexes display high affinity binding of 3H-imidacloprid, indicating selectivity of this ligand for different a subunits. In this study additional novel nAChR subunit genes have been cloned from both M. persicae and B. tabaci, utilising the recently sequenced D. melanogaster genome to help target a degenerate PCR approach. The contribution of individual amino acids in M. persicae a subunits to imidacloprid binding has been examined by site-directed mutagenesis, heterologous expression and radioligand binding studies.