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

Biochem Pharmacol. 2011 Oct 15;82(8):852-61. doi: 10.1016/j.bcp.2011.05.005. Epub 2011 May 17.


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 1μM 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Cloning, Molecular
  • Drug Discovery / methods*
  • Electrophysiological Phenomena
  • Endoplasmic Reticulum / metabolism
  • Green Fluorescent Proteins / genetics*
  • Ligands
  • Mice
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Mutation
  • Patch-Clamp Techniques
  • Receptors, Nicotinic / genetics
  • Receptors, Nicotinic / physiology*
  • Recombinant Fusion Proteins / genetics
  • Transfection


  • Ligands
  • Receptors, Nicotinic
  • Recombinant Fusion Proteins
  • alpha6beta2 nicotinic acetylcholine receptor
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins