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

Cheng Xiao, Rahul Srinivasan, Ryan M. Drenan, Elisha D W MacKey, J. Michael McIntosh, Henry A. Lester*

*Corresponding author for this work

Research output: Contribution to journalArticle

30 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)852-861
Number of pages10
JournalBiochemical Pharmacology
Volume82
Issue number8
DOIs
StatePublished - Oct 15 2011

Keywords

  • Endoplasmic reticulum exit sites
  • Fluorescent protein
  • Neuro2a cell
  • Nicotinic acetylcholine receptor
  • α6
  • β2

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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