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1.  Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus☆ 
•A nicotinic acetylcholine receptor α-subunit (Rsanα1) was identified in Rhipicephalus sanguineus.•Rsanα1 was not restricted to the synganglion (“brain”).•Rsanα1 was functionally expressed in Xenopus oocytes.•Rsanα1 responded to acetylcholine, nicotine and choline.•Rsanα1 was unresponsive to imidacloprid and spinosad.
Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken β2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 μM) and choline (100 μM). Rsanα1/β2 was insensitive to both imidacloprid (100 μM) and spinosad (100 μM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides.
PMCID: PMC4029082  PMID: 24291321
Rhipicephalus sanguineus; Tick; Ion channel; Acaricide; Nicotinic acetylcholine receptor; Xenopus oocytes; Imidacloprid
2.  Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin 
Invertebrate Neuroscience   2008;8(2):71-81.
Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.
PMCID: PMC2413115  PMID: 18338186
Acetylcholine binding protein (Lymnaea stagnalis); Crystal structures; Neonicotinoids; Nicotinic acetylcholine receptors; Ion channels
3.  Effects of mutations of a glutamine residue in loop D of the α7 nicotinic acetylcholine receptor on agonist profiles for neonicotinoid insecticides and related ligands 
British Journal of Pharmacology  2002;137(2):162-169.
Neonicotinoid insecticides are agonists of insect nicotinic acetylcholine receptors (AChRs) and show selective toxicity for insects over vertebrates. To elucidate the molecular basis of the selectivity, amino acid residues influencing neonicotinoid sensitivity were investigated by site-directed mutagenesis of the chicken α7 nicotinic AChR subunit, based on the crystal structure of an ACh binding protein (AChBP).In the ligand binding site of AChBP, Q55 in loop D is close to Y164 in loop F that corresponds to G189 of the α7 nicotinic receptor. Since Q55 of AChBP is preserved as Q79 in the α7 nicotinic receptor and the G189D and G189E mutations have been found to reduce the neonicotinoid sensitivity, we investigated effects of Q79E, Q79K and Q79R mutations on the neonicotinoid sensitivity of the α7 receptor expressed in Xenopus laevis oocytes to evaluate contributions of the glutamine residue to nicotinic AChR–neonicotinoid interactions.The Q79E mutation markedly reduced neonicotinoid sensitivity of the α7 nicotinic AChR whereas the Q79K and Q79R mutations increased sensitivity, suggesting electronic interactions of the neonicotinoids with the added residues.By contrast, the Q79E mutation scarcely influenced responses of the α7 nicotinic receptor to ACh, (−)-nicotine and desnitro–imidacloprid (DN–IMI), an imidacloprid derivative lacking the nitro group, whereas the Q79K and Q79R mutations reduced the sensitivity to these ligands. The results indicate that the glutamine residue of the α7 nicotinic receptor is likely to be located close to the nitro group of the insecticides in the nicotinic receptor–insecticide complex.
PMCID: PMC1573474  PMID: 12208772
Imidacloprid; nitenpyram; neonicotinoid; desnitro-imidacloprid; nicotinic acetylcholine receptor; chicken α7 subunit; loop D; loop F; Xenopus laevis oocyte; two-electrode voltage-clamp

Results 1-3 (3)