DOI: 10.5061/dryad.4k831
title: Data from: Calcium imaging with genetically encoded sensor Case12: facile analysis of α7/α9 nAChR mutants

description: 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.
description: 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).