Pharmacological and functional comparisons of α6/α3β2β3-nAChRs and α4β2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line

Neuronal nicotinic acetylcholine receptors containing alpha 6 subunits (alpha 6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of alpha 6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of alpha 6*-nAChRs remain poorly understood because of the lack of selective agonists for alpha 6*-nAChRs and the challenging heterologous expression of functional alpha 6*-nAChRs in mammalian cell lines. In particular, the alpha 6 subunit is commonly co-expressed with alpha 4*-nAChRs in the midbrain, which masks alpha 6*-nAChR (without alpha 4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of alpha 6*-nAChRs and compared these properties with those of alpha 4 beta 2 nAChRs expressed in the same cell line. Heterologously expressed human alpha 6/alpha 3 chimeric subunits (alpha 6 N-terminal domain joined with alpha 3 trans-membrane domains and intracellular loops) with beta 2 and beta 3 subunits in the human SH-EP1 cell line (6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed alpha 6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with alpha 4 beta 2-nAChRs. Pharmacologically, alpha 6*-nAChR was highly sensitive to the alpha 6 subunit-selective antagonist a-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-beta-erythroidine. Nicotine and acetylcholine were found to be full agonists for alpha 6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed alpha 6*-nAChRs exhibited pharmacology and function distinct from those of alpha 4 beta 2-nAChRs, suggesting that alpha 6*-nAChRs may mediate different cholinergic signals. Our alpha 6*-nAChR expression system can be used as an excellent cell model for future investigations of 6*-nAChR function and pharmacology.