Characterizing functional alpha 6 beta 2 nicotinic acetylcholine receptors in vitro: Mutant beta 2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells

• Published in: Biochemical pharmacology 2011-10, Vol.82 (8), p.852-861
• Main Authors: Xiao, Cheng, Srinivasan, Rahul, Drenan, Ryan M, Mackey, Elisha DW, McIntosh, JMichael, Lester, Henry A
• Format: Article
• Language: English
• ISSN: 0006-2952
• DOI: 10.1016/j.bcp.2011.05.005

alpha 6* nicotinic acetylcholine receptors (nAChRs) are highly expressed in mesostriatal and nigrostriatal dopaminergic systems, and participate in motor control, reward, and learning and memory. In vitro functional expression of alpha 6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged- alpha 6 and beta 2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 mu M ACh in 26% (5/19) of cells with evenly expressed alpha 6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional alpha 6-eGFP beta 2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into beta 2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased alpha 6-eGFP beta 2 nAChR current amplitude. alpha 6-eGFP beta 2 nAChRs were also activated by nicotine and by TC-2403. The alpha 6-eGFP beta 2 currents were desensitized by 1 mu M nicotine, blocked by alpha -conotoxin MII, partially inhibited by dihydro- beta -erythroidine, and potentiated by extracellular Ca super(2+. Single-channel recordings showed that alpha 6-eGFP beta 2 nAChRs had similar single-channel conductance to, but longer open time than, alpha 4-eGFP beta 2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of alpha 6* nAChRs for both pharmacological study and drug screening.)