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

Xiao, Cheng and Srinivasan, Rahul and Drenan, Ryan M. and Mackey, Elisha D. W. and McIntosh, J. Michael and Lester, Henry A. (2011) Characterizing functional α6β2 nicotinic acetylcholine receptors in vitro: Mutant β2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells. Biochemical Pharmacology, 82 (8). pp. 852-861. ISSN 0006-2952. PMCID PMC3162078. https://resolver.caltech.edu/CaltechAUTHORS:20110922-073823540

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.bcp.2011.05.005DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162078PubMed CentralArticle
ORCID:
AuthorORCID
Xiao, Cheng0000-0001-9649-7450
Drenan, Ryan M.0000-0002-8141-8577
Lester, Henry A.0000-0002-5470-5255
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).
Funders:
Funding AgencyGrant Number
NIHDA17279
NIHNS-11756
NIHAG033954
NIHDA19375
NIHDA12242
NIHMH53631
NIHGM48677
Targacept Inc.UNSPECIFIED
Louis and Janet FletcherUNSPECIFIED
California Tobacco-Related Disease Research Program18FT-0066
NIH Predoctoral FellowshipDA021492
NIHDA030396
Subject Keywords:Nicotinic acetylcholine receptor; α6; β2; Fluorescent protein; Neuro2a cell; Endoplasmic reticulum exit sites
Issue or Number:8
PubMed Central ID:PMC3162078
Record Number:CaltechAUTHORS:20110922-073823540
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20110922-073823540
Official Citation:Cheng Xiao, Rahul Srinivasan, Ryan M. Drenan, Elisha D.W. Mackey, J. Michael McIntosh, Henry A. Lester, Characterizing functional α6β2 nicotinic acetylcholine receptors in vitro: Mutant β2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells, Biochemical Pharmacology, Volume 82, Issue 8, 15 October 2011, Pages 852-861, ISSN 0006-2952, 10.1016/j.bcp.2011.05.005. (http://www.sciencedirect.com/science/article/pii/S000629521100298X)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:25394
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:22 Sep 2011 15:08
Last Modified:08 Oct 2019 21:25

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