Differential sensitivity of Ctenocephalides felis and Drosophila melanogaster nicotinic acetylcholine receptor alpha 1 and alpha 2 subunits in recombinant hybrid receptors to nicotinoids and neonicotinoid insecticides

Nicotinic acetylcholine receptors (nAChRs) are the binding sites for nicotinoid drugs, such as nicotine and epibatidine, and are the molecular targets of the selectively insecticidal neonicotinoids. In this study we report the full length cDNA cloning of the three Ctenocephalides (C.) felis (cat flea) nAChR alpha subunits Cf alpha 1, Cf alpha 2, and Cf alpha 3. When expressed in Xenopus oocytes as hybrid receptors with the Gallus gallus (chicken) beta 2 (Gg beta 2) subunit, these cat flea alpha subunits formed acetylcholine-responsive ion channels. Acetylcholine-evoked currents of Cf alpha 2/Gg beta 2 were resistant to alpha -bungarotoxin, while those of Cf alpha 1/Gg beta 2 were sensitive to this snake toxin. The pharmacological profiles of Cf alpha 1/Gg beta 2, Cf alpha 2/Gg beta 2 and the chicken neuronal receptor Gg alpha 4/Gg beta 2 for acetylcholine, two nicotinoids and 6 insecticidal neonicotinoids were determined and compared. Particularly remarkable was the finding that Cf alpha 1/Gg beta 2 was far more sensitive to acetylcholine, nicotine and neonicotinoid agonists than either Cf alpha 2/Gg beta 2 or Gg alpha 4/Gg beta 2: for the anti flea neonicotinoid market compound imidacloprid the respective EC sub(50s) were 0.02 mu M, 1.31 mu M and 10 mu M. These results were confirmed for another insect species, Drosophila melanogaster, where the pharmacological profile of the Dm alpha 1 and Dm alpha 2 subunits as hybrid receptors with Gg beta 2 in Xenopus oocyte expressions resulted in a similar sensitivity pattern as those identified for the C. felis orthologs. Our results show that at least in a Gg beta 2 hybrid receptor setting, insect alpha 1 subunits confer higher sensitivity to neonicotinoids than alpha 2 subunits, which may contribute in vivo to the insect-selective action of this pesticide class.