Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants

PLoS One. 2017 Aug 10;12(8):e0181936. doi: 10.1371/journal.pone.0181936. eCollection 2017.


Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117-119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs.

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Cell Line, Tumor
  • Humans
  • Mutagenesis, Site-Directed
  • Rats
  • Receptors, Nicotinic / genetics*
  • alpha7 Nicotinic Acetylcholine Receptor / genetics*


  • Receptors, Nicotinic
  • alpha7 Nicotinic Acetylcholine Receptor
  • nAChR alpha9
  • Calcium

Grant support

The work of E. Spirova, С. Methfessel, M. Werner, and Prof. M. Hollmann was supported by ERA-Net EuroTransBio 12614r/9282. The work of Shelukhina I. was supported by a grant of the President of the Russian Federation (project No. МК-6216.2016.4). The work of Kudryavtsev D. was supported by RFBR grant (project No. 16-34-00627). The work of Ojomoko L. and Prof. Tsetlin V. was supported by Russian Science Foundation grant 16-14-00215. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.