TY - JOUR
T1 - Characterizing functional α6β2 nicotinic acetylcholine receptors in vitro
T2 - Mutant β2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells
AU - Xiao, Cheng
AU - Srinivasan, Rahul
AU - Drenan, Ryan M.
AU - MacKey, Elisha D W
AU - McIntosh, J. Michael
AU - Lester, Henry A.
N1 - Funding Information:
This work was supported by grants from the US National Institutes of Health (DA17279, NS11756, AG033954, DA19375, DA12242, MH53631, and GM48677); from Targacept Inc. ; from the California Tobacco-Related Disease Research Program (TRDRP); and from Louis and Janet Fletcher. R.S. was supported by a postdoctoral fellowship from TRDRP (18FT-0066), and R.D. by an NIH National Research Service Award (DA021492) and an NIH Pathway to Independence Award (DA030396).
PY - 2011/10/15
Y1 - 2011/10/15
N2 - α6* 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 α6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-α6 and β2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 μM ACh in 26% (5/19) of cells with evenly expressed α6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional α6-eGFPβ2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into β2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased α6-eGFPβ2 nAChR current amplitude. α6-eGFPβ2 nAChRs were also activated by nicotine and by TC-2403. The α6-eGFPβ2 currents were desensitized by 1 μM nicotine, blocked by α-conotoxin MII, partially inhibited by dihydro-β-erythroidine, and potentiated by extracellular Ca2+. Single-channel recordings showed that α6-eGFPβ2 nAChRs had similar single-channel conductance to, but longer open time than, α4-eGFPβ2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of α6* nAChRs for both pharmacological study and drug screening.
AB - α6* 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 α6* nAChRs is essential for full pharmacological characterization of these receptors and for drug screening, but has been challenging. We expressed eGFP-tagged-α6 and β2 nAChR subunits in Neuro-2a cells, leading to functional channels. Inward currents were elicited with 300 μM ACh in 26% (5/19) of cells with evenly expressed α6-eGFP in cytoplasm and periphery. We dramatically increased chances of detecting functional α6-eGFPβ2 nAChRs by (i) introducing two endoplasmic reticulum (ER) export-enhancing mutations into β2 subunits, and (ii) choosing cells with abundant Sec24D-mCherry-labeled ER exit sites. Both manipulations also modestly increased α6-eGFPβ2 nAChR current amplitude. α6-eGFPβ2 nAChRs were also activated by nicotine and by TC-2403. The α6-eGFPβ2 currents were desensitized by 1 μM nicotine, blocked by α-conotoxin MII, partially inhibited by dihydro-β-erythroidine, and potentiated by extracellular Ca2+. Single-channel recordings showed that α6-eGFPβ2 nAChRs had similar single-channel conductance to, but longer open time than, α4-eGFPβ2 nAChRs. These methods provide avenues for developing cell lines expressing subtypes of α6* nAChRs for both pharmacological study and drug screening.
KW - Endoplasmic reticulum exit sites
KW - Fluorescent protein
KW - Neuro2a cell
KW - Nicotinic acetylcholine receptor
KW - α6
KW - β2
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U2 - 10.1016/j.bcp.2011.05.005
DO - 10.1016/j.bcp.2011.05.005
M3 - Article
C2 - 21609715
AN - SCOPUS:80052027375
SN - 0006-2952
VL - 82
SP - 852
EP - 861
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 8
ER -