Characterizing functional alpha6beta2 nicotinic acetylcholine receptors in vitro: mutant beta2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells.

摘要

alpha6* 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 alpha6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-alpha6 and beta2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 muM ACh in 26% (5/19) of cells with evenly expressed alpha6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional alpha6-eGFPbeta2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into beta2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased alpha6-eGFPbeta2 nAChR current amplitude. alpha6-eGFPbeta2 nAChRs were also activated by nicotine and by TC-2403. The alpha6-eGFPbeta2 currents were desensitized by 1muM nicotine, blocked by alpha-conotoxin MII, partially inhibited by dihydro-beta-erythroidine, and potentiated by extracellular Ca(2+). Single-channel recordings showed that alpha6-eGFPbeta2 nAChRs had similar single-channel conductance to, but longer open time than, alpha4-eGFPbeta2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of alpha6* nAChRs for both pharmacological study and drug screening.