Nicotinic acetylcholine receptors: Ex-vivo expression of functional, non-hybrid, heteropentameric receptors from a marine arthropod, Lepeophtheirus salmonis.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mostly located in the post-synaptic membrane of cholinergic synapses. The natural neurotransmitter is acetylcholine, but they are also the direct targets for neonicotinoids, chemicals widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects. In arthropods, functional nAChRs made of alpha subunits have been expressed from Drosophila genes, and hybrid receptors (sometimes also referred to as chimeric receptors) using species-specific alpha subunits and vertebrate beta subunits have been expressed ex-vivo. Arthropod-specific nAChRs made of both alpha and beta subunits from the target species have not been expressed ex-vivo. The aim of the current study was to express such receptors in Xenopus oocytes using only genes from Lepeophtheirus salmonis, to characterize them and study their modulation. Genes encoding alpha and beta subunits of the nAChRs and three ancillary proteins, RIC-3, UNC-50 and UNC-74 were identified in the L. salmonis genome, subjected to RACE-PCR, cloned into an expression vector and the cRNA produced was then injected into Xenopus laevis oocytes. Co-expression of the ancillary proteins was essential for the successful expression of the L. salmonis nAChRs with both alpha and beta subunits. Two functional nAChRs were identified: Lsa-nAChR1 consisting of alpha 1, alpha 2, beta 1 and beta 2 subunits, reconstituted to one distinct receptor, while Lsa-nAChR2, consisting of alpha 3, beta 1 and beta 2 subunits reconstitutes receptors with two distinct characteristics. Out of seven neonicotinoids tested, six worked as partial agonist of Lsa-nAChR1 while only three did so for Lsa-nAChR2. Four non-neonicotinoid compounds tested had no effect on either of the nAChRs. The study demonstrated that fully functional, non-hybrid nAChRs containing both alpha and beta subunits from an arthropod can be reconstituted ex-vivo by co-expression of essential ancillary proteins. Such models would be valuable for in-depth studies of effects by neonicotinoids and other compounds on target pests, as well as for studies of adverse effects on non-target arthropods. Author summary Nicotinic acetylcholine receptors, nAChRs, respond to the neurotransmitter acetylcholine or drugs like nicotine. These receptors are targets for neonicotinoids, the most commonly used compounds against ectoparasites and agricultural pests. In-depth studies of the function of these channels in arthropods are sparse, as no groups managed to reconstitute functional nAChRs made of both alpha and beta subunits using genes only from the target arthropod in an ex-vivo system. We report the successful assembly of non-hybrid, fully functional nAChRs containing both alpha and beta subunits from a marine arthropod, assembled and expressed in Xenopus laevis oocytes. We identified three possible combinations of alpha and beta subunits producing functional receptors. We found ancillary proteins to be essential for successful expression and assembly of both alpha and beta subunits into a functional receptor. The findings of the present study provide a basis for studying native nAChRs from arthropods, with a switch from hybrid nAChRs to species-specific native nAChRs.