Characterizing functional 62 nicotinic acetylcholine receptors in vitro: mutant 2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells.
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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 6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-6 and 2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 M ACh in 26% (5/19) of cells with evenly expressed 6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional 6-eGFP2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into 2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased 6-eGFP2 nAChR current amplitude. 6-eGFP2 nAChRs were also activated by nicotine and by TC-2403. The 6-eGFP2 currents were desensitized by 1M nicotine, blocked by -conotoxin MII, partially inhibited by dihydro--erythroidine, and potentiated by extracellular Ca(2+). Single-channel recordings showed that 6-eGFP2 nAChRs had similar single-channel conductance to, but longer open time than, 4-eGFP2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of 6* nAChRs for both pharmacological study and drug screening.
author list (cited authors)
Xiao, C., Srinivasan, R., Drenan, R. M., Mackey, E., McIntosh, J. M., & Lester, H. A.