Drug target discovery by transcriptome analysis of the brown dog tick, Rhipicephalus sanguineus, synganglion : initial characterisation of a nicotinic acetylcholine receptor

A normalised cDNA library was constructed from the synganglia of unfed adult R. sanguineus. Of interest from a drug discovery perspective were ESTs encoding a chitinase enzyme and four transmembrane receptors including two glutamate-gated chloride receptors, a leucokinin-like receptor and a nicotinic acetylcholine receptor (nAChR) α subunit. The nAChR α subunit was chosen for further study. This is the first identified full-length arachnid nAChR subunit. Phylogenetically it is most similar to insect α1 nAChR group and has been called Rsα1. This subunit is expressed in larval, nymphal and adult stages and within partially fed adult females. Rsα1 was most abundant in the synganglion and also present in Malpighian tubules and oviduct yet was undetected in the salivary glands and gut tissues. Using two-electrode voltage clamp in Xenopus laevis oocytes, Rsα1 did not functionally express as a homomer or when co-expressed with the vertebrate chicken β2 subunit using either RNA or DNA. Chimeric constructs consisting of the tick N-terminal and the transmembrane portion of the D. melanogaster α2 nAChR (Dα2) (Rsα1-Dα2) and vice-versa were prepared. Responses to acetylcholine were obtained from the Dα2-Rsα1 chimera showing that the Rsα1 nAChR can form a functional pore region. However, no responses were obtained from the Rsα1-Dα2 chimera. This preliminary data suggests that the Rsα1 nAChR cannot form a functional ligand binding region when expressed in Xenopus oocytes. The present study has proven that the tick synganglion is a rich source of transmembrane receptor targets for exploitation in future drug discovery.EThOS - Electronic Theses Online ServiceGBUnited Kingdo