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

Abstract

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 α subunits have been expressed from Drosophila genes, and hybrid receptors (sometimes also referred to as chimeric receptors) using species-specific α subunits and vertebrate β subunits have been expressed ex-vivo. Arthropod-specific nAChRs made of both α and β 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 α and β 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 α and β subunits. Two functional nAChRs were identified: Lsa-nAChR1 consisting of α1, α2, β1 and β2 subunits, reconstituted to one distinct receptor, while Lsa-nAChR2, consisting of α3, β1 and β2 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 α and β 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.