Combinatorial mutations in loops D and F strongly influence responses of the α7 nicotinic acetylcholine receptor to imidacloprid

A Role for Leu118 of Loop E in Agonist Binding to the α7 Nicotinic Acetylcholine Receptor

“…Conversely, several other insect nAChR subunits, such as D. melanogaster D␣1, D␣3, and D␣4, as well as Myzus persicae ␣2 and ␣3, possess either an arginine or lysine at the position corresponding to Leu118 (Table 2), which in our site-directed mutagenesis experiments increased the efficacy of imidacloprid (Table 1). It is noteworthy that studies have indicated that these subunits play a role in determining sensitivity to the insecticide, imidacloprid (Huang et al, 1999;Lansdell and Millar, 2000;Matsuda et al, 2001a;Shimomura et al, 2002Shimomura et al, , 2003Shimomura et al, , 2004Shimomura et al, , 2006. In heteromeric nAChRs consisting of ␣ and ␤ subunits, it is believed that loop E from the ␤ subunit contributes to the agonist binding site (Corringer et al, 2000b); however, it is important to note that the subunit composition and stoichiometry of native insect nAChRs have yet to be determined.…”

Responses to Neonicotinoids of Chicken .ALPHA.7 Nicotinic Acetylcholine Receptors: Effects of Mutations of Isoleucine 191 in Loop F to Aromatic Residues

“…3,8) Using a combination of voltage-clamp electrophysiology and site-directed mutagenesis, we have shown that loops C, D and F play important roles in such interactions. 3,[9][10][11][12] Mutations to acidic residues of G189 in loop F 9) and Q79 in loop D 10) of the chicken a7 nAChR were found to markedly reduce the responses of the nAChR to neonicotinoids, whereas such changes were not observed for the responses to desnitro-imidacloprid (DN-IMI, Fig. 1), an imidacloprid derivative lacking the nitro group.…”

“…1), an imidacloprid derivative lacking the nitro group. In addition, mutations of Q79 to basic residues enhanced the sensitivity of the a7 nAChR to neoni-cotinoids, 10) suggesting that the changes in the responses of the nAChR to neonicotinoids resulting from site-directed mutagenesis are due to electronic interactions of the nitro group of the insecticides with the amino acids newly added to loops D and F. Based on these results, together with findings obtained using a three-dimensional model constructed for a complex of imidacloprid with the ligand-binding site of the a7 nAChR, 11) it is postulated that loops D and F are likely to be located in proximity to the nitro group of neonicotinoids tested when the insecticides bind to the nAChR. On the other hand, we have also found that replacement of the chicken a4 subunit with Drosophila ALS and SAD subunits of the chicken a4b2 nAChR expressed in Xenopus oocytes resulted in shifts of the dose-response curves for neonicotinoids to lower concentrations.…”

“…A series of mutations were introduced by PCR as described earlier. [9][10][11] Oligonucleotides I191F sense and I191F antisense (Table 2) were prepared to generate the a7 I191F mutation on the a7 subunit containing the I191F mutation, while KM001 (5Ј-TGTCCACTCCCAGGTCCAACTG-3Ј) and KM012 (5Ј-CTCCATGCTTGACAGGCTGCATC-3Ј) were designed, respectively, on the basis of the sequence flanking the multiple Vol. 29, No.…”

Neonicotinoids Show Selective and Diverse Actions on Their Nicotinic Receptor Targets: Electrophysiology, Molecular Biology, and Receptor Modeling Studies

“…70) This model of the 7 nAChR-imidacloprid complex has been further refined on the basis of the crystal structure of the AChBP-nicotine complex, 58) the 58) The structure of imidacloprid was superposed on the crystal structure of nicotine, and the water molecules within 6 A radius of the centrally located nicotine in the ligand-binding site of AChBP were merged to the 7 nAChR model. Next, the whole model was energyminimized using molecular modeling software package SYBYL ver.…”