Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor β subunits influence neonicotinoid selectivity

Nicotinic acetylcholine (ACh) receptors (nAChRs) are ligand-gated ion channels which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A-C present in alpha subunits together with loops D-F present in either non-a subunits or homomer-forming a subunits. To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of altering insect-specific loops D, E and F in hybrid nAChRs containing insect and mammalian subunits (N1 alpha 1 from the brown planthopper Nilaparvata lugens and 2 from rat). Introduction of the insect-specific loops D, E and F, singly or together. into rat 2 subunit resulted in a leftward shift of the imidacloprid dose-response curves for nAChRs N1 alpha 1-beta 2 chimeras, reflecting decreases in EC50, compared to wildtype nAChRs N1 alpha 1-beta 2. By contrast, the influences on ACh potency were minimal or negligible. The effects of loop D could be interpreted by the earlier findings of Shimomura et al. [2006. Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist-binding site. Mol. Pharmacol. 70,1255-1263.], in which T77R and E79V were shown to be responsible for neonicotinoid selectivity. In the present study, S131Y(R) and D133N in loop E and T191W and P192K in loop F were found to contribute to the neonicotinoid selectivity of insect-specific loops E and F. These results indicated the insect-specific loops D, E and F each play important roles in neonicotinoids selectivity. This study contributes to our understanding of the molecular mechanism underlying selectivity of neonicotinoids against insects over vertebrates. (c) 2008 Elsevier Ltd. All rights reserved.