Lees K et al. (2014), Functional characterisation of a nicotinic acet...

XB-ART-47748

Int J Parasitol 2014 Jan 01;441:75-81. doi: 10.1016/j.ijpara.2013.11.002.

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Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus.

Lees K , Jones AK , Matsuda K , Akamatsu M , Sattelle DB , Woods DJ , Bowman AS .

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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.

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Species referenced: Xenopus laevis
Genes referenced: actl6a chrna4 tbx2

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	Fig. 1. Protein sequence alignment of the Rhipicephalus sanguineus α1 (Rsanα1) nicotinic acetylcholine receptor (nAChR) subunit. Drosophilia melanogaster (Dm) α1, Tetranychus urticae (Tu) α2 and Homo sapiens (Hs) α7 nAChRs are included for comparison. The N-terminal signal peptides are underlined. The positions of loops (LpA-F) which are implicated in ligand binding and the four transmembrane domains (TM1–4) are indicated. The sites of cysteine residues involved in the cys-loop are marked with asterisks and the vicinal cysteine residues characteristic of α-type nAChR are highlighted in black. Potential glycosylation and putative phosphorylation sites are highlighted in grey.
	Fig. 2. Phylogenetic analysis of the Rhipicephalus sanguineus α nicotinic acetylcholine receptor (nAChR) subunit. Unrooted phylogenetic tree containing the identified R. sanguineus nAChR subunit. Sequences were aligned using ClustalX2. The phylogenetic tree was constructed using protein sequences with the Neighbor-Joining (NJ) method using MEGA version 5 (Kumar et al., 2008). Scale bar represents an estimate of the number of amino acid substitutions per site and numbers represent bootstrap values with 1,000 replications. Accession and database sequence identifiers are as follows: Drosophila melanogaster: Dα1 (X07194), Dα2(X53583), Dα3 (CAA75688), Dα4, (AJ272159); Dα5 (AF272778), Dα6 (AF321445), Dα7, (AY036614), Dβ1 (CAA27641), Dβ2, (CAA39211), Dβ3, (CAC48166). Bombyx mori: Bmoriα1, (EU082074); Bmoriα2, (EU082075); Bmoriα3 (EU082076), Bmoriα4, (EU82077); Bmoriα5, (EU082080); Bmoriα6, (EU082082); Bmoriα7, (EU082084), Bmoriα8, (EU082085); Bmoriα9, (EU082087), Bmoriβ1 (EU82071), Bmoriβ2 (EU082072), Bmoriβ3 (EU082073). Tribolium castaneum: Tcasα1 (EF526080), Tcasα2 (EF526081), Tcasα3 (EF526082), Tcasα4 (EF526083), Tcasα5 (EF526085), Tcasα6 (EF526086), Tcasα7 (EF526089), Tcasα8 (EF526090), Tcasα9 (EF526091), Tcasα10 (EF526092), Tcasα11 (EF526093), Tcasβ1 (EF526094). Pardosa pseudoannulata: Ppα1 (HM01780) Ppα2 (ADG63462), Ppβ1 (GQ259335), Ppβ2 (ADG63463).
	Fig. 3. Tissue and temporal distribution of Rsanα1 nicotinic acetylcholine receptor (nAChR) in adult Rhipicephalus sanguineus. The quality of the cDNA was validated by PCR for actin (data not shown) and then the cDNA was adjusted to 1 μg/μl for each tissue. The products were obtained from nested-PCR using gene-specific primers. (A) Tissue expression pattern of Rsanα1 in partially fed adult female R. sanguineus. Lane 1, synganglion tissue; lane 2, salivary glands; lane 3, gut; lane 4, Malpighian tubules; lane 5, oviduct tissue and lane 6, negative control (water). (B) Temporal distribution of Rsanα1. Lane 1, unfed adult synganglion cDNA (mixed male and female); lane 2, partially fed individual female tick adult synganglion cDNA (tick weight = 6.3 mg); lane 3, partially-fed individual female adult tick synganglion (tick weight = 51.2 mg); lane 4, fully engorged female tick synganglion (tick weight = 132 mg) and lane 5, negative control (water).
	Fig. 4. Life-stage distribution of Rhipicephalus sanguineus Rsanα1 nicotinic acetylcholine receptor (nAChR). The quality of the cDNA was validated by PCR for actin (data not shown) and then the cDNA was adjusted to 1 μg/μl for each tissue. Nested PCR using gene-specific primers were used to detect the presence of Rsanα1 across different life stages of R. sanguineus. Lane 1, eggs; lane 2, larval tissue; lane 3, nymphal tissue; lane 4, whole unfed adult cDNA and lane 5, negative control (water).
	Fig. 5. Representative two-electrode voltage clamp current traces from Xenopus oocytes expressing Rsanα1/β2 to (A) 1 mM acetylcholine (ACh), (B) 100 μM imidacloprid (IMI) and (C) 100 μM spinosad following a 5 s exposure (grey bar) to each compound. Only oocytes displaying responses to ACh were then challenged with the test compounds (n > 4 for all compounds).
	Fig. 6. Concentration–response curve for ACh obtained using two-electrode voltage clamp recording from Xenopus oocytes expressing the Rsanα1/β2. Data was normalised to the observed peak amplitude of currents recorded in response to 300 μM ACh. Each point represents the mean ± SEM of 6–10 experiments.
	Fig. 7. Two-electrode voltage clamp current traces (representative, n > 4) from Xenopus oocytes expressing Rsanα1/β2 to the potential ligands (A) acetylcholine (ACh, 1 mM), (B) choline (100 μM) and (C) nicotine (100 μM) following a 5 s exposure (grey bar). Traces are obtained from a single oocyte.

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