Structure-activity relationships of acetylcholine derivatives with Lucilia cuprina nicotinic acetylcholine receptor alpha 1 and alpha 2 subunits in chicken beta 2 subunit hybrid receptors in comparison with chicken nicotinic acetylcholine receptor alpha 4/ beta 2.

摘要

Insect nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several insecticide classes. In the present study, we report the gene identification and cloning of nAChR alpha 1 and alpha 2 subunits (Lc alpha 1 and Lc alpha 2) from the sheep blowfly Lucilia cuprina. Xenopus oocytes voltage clamp experiments as hybrids with the chicken beta 2 nAChR (Gg beta 2) subunit resulted in ACh-gated ion channels with distinct dose-response curves for Lc alpha 1/Gg beta 2 (effective concentration 50% [EC₅₀]=80 nM; n_H=1.05), and Lc alpha 2/Gg beta 2 (EC₅₀=5.37 micro M, n_H=1.46). The neonicotinoid imidacloprid was a potent agonist for the alpha -bungarotoxin-sensitive Lc alpha 1/Gg beta 2 (EC₅₀ ~20 nM), while the alpha -bungarotoxin-resistant Lc alpha 2/Gg beta 2 showed a 30-fold lower sensitivity to this insecticide (EC₅₀=0.62 micro M). Thirteen close derivatives of ACh were analysed in EC₅₀, Hill coefficient and maximum current (relative to ACh) determinations for Lc alpha 1/Gg beta 2 and Lc alpha 2/Gg beta 2 and the chicken Gg alpha 4/Gg beta 2 nAChRs, and comparisons relative to ACh allowed the definition of novel structure-activity and structure-selectivity relationships. In the case of N-ethyl-acetylcholine, the EC₅₀ of the chicken Gg alpha 4/Gg beta 2 rose by a factor of 1000, while for both Lc alpha 1/Gg beta 2 and Lc alpha 2/Gg beta 2, potency remained unchanged. Further derivatives with insect nAChR selectivity potential were acetyl- alpha -methylcholine and trimethyl-(3-methoxy-3-oxopropyl)ammonium, followed by acetylhomocholine and trimethyl-(4-oxopentyl) ammonium. Our results may provide guidance for the identification or design of insect-specific nAChR agonists using structure-based or in silico methods.