Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published October 15, 2011 | Accepted Version
Journal Article Open

Characterizing functional α6β2 nicotinic acetylcholine receptors in vitro: Mutant β2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells

Abstract

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 Ca^(2+). 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.

Additional Information

© 2011 Elsevier Inc. Received 8 April 2011. Accepted 9 May 2011. Available online 17 May 2011. 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).

Attached Files

Accepted Version - nihms-303292.pdf

Files

nihms-303292.pdf
Files (1.4 MB)
Name Size Download all
md5:fa4b2b1f2a4e05b3fd71b1f6277ff1be
1.4 MB Preview Download

Additional details

Created:
August 22, 2023
Modified:
October 24, 2023