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

Abstract

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.

Alpha6-containing nicotinic acetylcholine receptor is a highly sensitive target of alcohol

“…3A), this approach reproducibly increases expression compared to that seen for native α6 subunits while retaining α6-like pharmacology (Kuryatov et al, 2000). More detailed information of the process of expression of this α6Nα3Cβ2β3-nAChR and maintenance of SH-EP1-α6/3 cells was as previously described (Chen et al, 2018).…”

“…The homology model of the α6/α3 chimera was built using the sequences described previously (Chen et al, 2018) without signal peptide submitted to the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). One of the five top scored models was used for presentation.…”

“…Recently, we have found that low concentrations of EtOH (1-10 mM) enhance GABA A receptor (GABA A R)-mediated spontaneous and evoked inhibition of IPSCs via processes blocked by the α6*-nAChR-selective antagonist α-conotoxin MII (α-Ctx MII) and that there is lowered EtOH sensitivity and reward in nAChR α6 subunit knock-out (KO) mice (Steffensen et al, 2017). Moreover, we have established functional, heterologous expression of α6*-nAChRs by co-transfection of an α6/3 subunit chimera together with β2 and β3 subunits into the human SH-EP1 cell line (Chen et al, 2018). This transfected α6*-nAChR exhibits robust function and has been used for drug screening, and use of the chimera avoids the enhanced agonist potency and efficacy effects associated with including a 9'S mutant subunit (Letchworth and Whiteaker, 2011).…”

Rapid ‘multi-directed’ cholinergic transmission at central synapses

“…However, these results are generally difficult to interpret due to space-clamp considerations 54 ; and as such, do not provide an indication of the patterns of excitation on single dendrites of the DSGC, where direction is computed 28,55-57 . To circumvent these issues, we assessed ACh dynamics using a G-protein-coupled receptor activation-based sensor (ACh 3.0), which has an ACh binding affinity of ~2 µM, that is roughly comparable to that of the endogenous α6* nAChRs 58,59 .…”

Cocaine potently blocks neuronal α3β4 nicotinic acetylcholine receptors in SH-SY5Y cells

“…The human epithelial cell line expressing α 6 /α 3 chimera β 2 β 3 nAChRs (α 6 N/α 3 Cβ 2 β 3 nAChRs) was developed by Dr. Whiteaker’s laboratory [ 31 ], and its pharmacological response properties have been profiled recently [ 32 ]. α 6 /α 3 denotes a chimeric subunit composed of the extracellular, ligand-binding domain of the human α 6 subunit fused to the first transmembrane domain and followed by the sequence of the human α 3 nAChR subunit; this approach reproducibly increases expression compared to that of native α 6 subunits while retaining α 6 -like pharmacology.…”