The influence of the transmembrane protein EAT-18 upon nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of neurotransmitter-gated ion channels, which are associated with many physiological and pathological conditions. Numerous investigations have been carried out since it was first identified about 100 years ago. However, the mechanism by which this receptor function in variety of species still not entirely clear. A major current problem is that it is difficult to express functional recombinant nicotinic receptor on the surface of expression systems (e.g. in cultured mammalian and insect cell lines). Recent genetic studies have identified some promising candidates molecules, which facilitate more efficient trafficking, assembling or folding of nAChRs. EAT-18 is a recent identified small molecule with a single transmembrane domain and a short extracellular region, which has previously been reported, associated with a defect in C. elegans feeding behaviour. Evidence shows that eat-18 is required for α-bungarotoxin binding to most or all pharyngeal nicotinic receptors. The current project aims to examine the influence of co-expression of eat-18 with a variety of nAChRs subunit combinations, eat-18 was subcloned into plasmid vector pRmHa3 and co-transfected with a variety of human (H), rat (R) and Drosophila (D) nAChRs subunit combinations (Hα7, Rα7, Rα4/β2, Rα3/β4, Dα7, Dα6, Dα1/β2, Dα3/β2, Dα2/β2, Dα1/α2/β2, Dα3/α2/β2 and Dα3/α1/β2) in Drosophila S2 cells. Radioactive non-selective agonist epibatidine and 7-selective antagonist methyllyacconitine (MLA) were used for ligand binding studies. Increase in specific radioligand binding were observed when eat-18 was co-expressed with some subunit combination, but were not statistically significant