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. Photos 101-115Photos 101-115 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 and Alexa Fluor 555-α-bungarotoxin fluorescence in Neuro2a cells expressing human α7 nAChR (nicotinic acetylcholine receptor); Photos 201-210 – Cytochemical fluorescent and bright field images were generated for quantification of Alexa Fluor 555-α-bungarotoxin fluorescence and background fluorescence (in green channel) in Neuro2a cells expressing human α7 nAChR; Photos 301-316 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 fluorescence and background fluorescence (in red channel) in Neuro2a cells expressing human α7 nAChR; Photos 401-413 – Cytochemical fluorescent and bright field images were generated for quantification of background fluorescence (in green and red channels) in Neuro2a cells expressing human α7 nAChR; Photos 501-505 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 and Alexa Fluor 555-α-bungarotoxin fluorescence in Neuro2a cells expressing mouse muscle (WT nAChR; Photos 604-612 – Cytochemical fluorescent and bright field images were generated for quantification of Alexa Fluor 555-α-bungarotoxin fluorescence and background fluorescence (in green channel) in Neuro2a cells expressing mouse muscle (WT) nAChR; Photos 701-710 – Cytochemical fluorescent and bright field images were generated for quantification of TMRE (Tetramethylrhodamine, ethyl ester; 20 nM) labeling of Neuro2a cells expressing human α7 nAChR; Photos 801-805 – Cytochemical fluorescent and bright field images were generated for quantification of PI (Propidium iodide; 50 ng/ml) labeling of Neuro2a cells expressing human α7 nAChRCytochemical photos 101-805_Alexa555-aBgt_Case12_TMRE_PI.zipVideo_Calcium response of N2A cells_a7 nAChRVideos show calcium response of Neuro2a cells expressing human α7 nAChR (nicotinic acetylcholine receptor), a chaperone NACHO and a fluorescent genetically-encoded calcium sensor Case12 to different concentration of acetylcholine in the presence of a positive allosteric modulator PNU120596. Controls in the presence of α-cobratoxin are presented as well.Electrophysiology and Ca_imaging normalized dataThe file contains the normalized electrophysiological (TEVC (two-electrode voltage clamp)) and calcium imaging (Ca-img) data for oocyte and Neuro2a cells responses, respectively. WT human α7 nAChR (nicotinic acetylcholine receptor) and mouse muscle nAChR were expressed in oocytes and Neuro2a cells. Besides, their mutants (at positions 117-119, 184, 185, 187 and 189 in α7 nAChR and at positions 153 and 190 in muscle nAChR) were heterologously expressed as well and their responses were estimated accordingly. Responses to different concentrations of acetylcholine (Ach) and epibatidine (Epi) were measured. For calcium imaging studies two calcium sensors (genetically encoded Case12 and low-molecular weight Fluo-4) were used. Calcium imaging analysis was carried out in the presence of a positive allosteric modulator PNU120596.pdbPDB file WT.pdb contains the last frame from 10 ns molecular dynamics simulation alpha7-AChBP chimera (PDB 3SQ6). Only two adjacent subunits were subjected to simulations. PDB files with names E185V, E189G, F187S, L119D, Q117T, R186I, S184N and Y118W contain the last frame from molecular dynamics simulation of alpha7-AChBP chimera with the respective mutations (residues numbered according to human alpha7 nAChR). PDB files F104.pdb and V104_mutant.pdb contain structures in which 104th residue of alpha7 AChBP chimera was changed either to phenylalanine (as in actual human alpha7 nAChR) or to valine (as in alpha9 nAChR).