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 α6 subunits (α6^*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6^*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6^*-nAChRs remain poorly understood because of the lack of selective agonists for α6^*-nAChRs and the challenging heterologous expression of functional α6^*-nAChRs in mammalian cell lines. In particular, the α6 subunit is commonly co-expressed with α4^*-nAChRs in the midbrain, which masks α6^*-nAChR (without α4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of α6^*-nAChRs and compared these properties with those of α4β2 nAChRs expressed in the same cell line. Heterologously expressed human α6/α3 chimeric subunits (α6 N-terminal domain joined with α3 trans-membrane domains and intracellular loops) with β2 and β3 subunits in the human SH-EP1 cell line (α6^*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed α6^*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with α4β2-nAChRs. Pharmacologically, α6^*-nAChR was highly sensitive to the α6 subunit-selective antagonist α-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-β-erythroidine. Nicotine and acetylcholine were found to be full agonists for α6^*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed α6^*-nAChRs exhibited pharmacology and function distinct from those of α4β2-nAChRs, suggesting that α6^*-nAChRs may mediate different cholinergic signals. Our α6^*-nAChR expression system can be used as an excellent cell model for future investigations of α6^*-nAChR function and pharmacology.