Calcium imaging with genetically encoded sensor Case12: Facile analysis of [alpha]7/[alpha]9 nAChR mutants

• Published in: PloS one Vol. 12; no. 8; p. e0181936
• Main Authors: Shelukhina, Irina, Spirova, Ekaterina, Kudryavtsev, Denis, Ojomoko, Lucy, Werner, Markus, Methfessel, Christoph, Hollmann, Michael, Tsetlin, Victor
• Format: Journal Article
• Language: English
• Published: Public Library of Science 10.08.2017
• Subjects: Amino acids; Chemical properties; Fluorescence microscopy; Nicotinic receptors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0181936

Elucidation of the structural basis of pharmacological differences for highly homologous [alpha]7 and [alpha]9 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 [alpha]7 and [alpha]9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of [alpha]7/[alpha]9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of [alpha]7/[alpha]9 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 [alpha]7 nAChRs, and for their mutants with homologous residues of [alpha]9 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 [alpha]7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between [alpha]7 and [alpha]9 nAChRs.