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

Calcium Imaging With Genetically Encoded Sensor Case12: Facile Analysis of α7/α9 nAChR Mutants

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Free PMC article

Calcium Imaging With Genetically Encoded Sensor Case12: Facile Analysis of α7/α9 nAChR Mutants

Irina Shelukhina et al. PLoS One. .
Free PMC article

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structural basis for point mutagenesis.
(a) Alignments of the loop C region of α-subunits (principal side) and complementary regions of α7, α9, β-, and ɛ-subunits. Mutated residues in the α7-subunit are highlighted in red, substituting amino acids from the α9-subunit are highlighted in green, conserved cysteines in loop C are highlighted in orange. (b) Overall view of the epibatidine (purple) binding site located at the subunit interface of the α7 nAChR extracellular part under the loop C. The principal part (“+”) of the α7 nAChR binding site is highlighted in dark green, the complementary one (“-“) in cyan; the residues picked for mutagenesis are shown in orange. Pictures were rendered in UCSF Chimera using PDB 3SQ6. (c) Molecular dynamics study of possible effects of the chosen point mutations on the α7 extracellular domain-epibatidine complex structure (PDB 3SQ6). Among all tried mutations L119D showed the most drastic changes in epibatidine positioning. Other mutations had tiny effects on epibatidine positioning with the exception of F187S that favored an unusual turn of the epibatidine amine group.
Fig 2
Fig 2. Functional expression of human α7 nAChR in the presence of the chaperone NACHO and the genetically-encoded fluorescent calcium ion sensor Case12 in mouse neuroblastoma Neuro2a cells.
(a, b) Cytochemical detection of α7 nAChR with 50 nM Alexa Fluor 555-α-bungarotoxin (αBgt, red) and (c, d) its co-expression with Case12 (green) in Neuro2a cells. (e) Pie charts represent the percentage of transfected Neuro2a cells labeled with Alexa Fluor 555-α-bungarotoxin (αBgt) in the absence (n = 3, 2521 (total) and 696 (αBgt) cells) or in the presence of Case12 (n = 3, 3141 (total), 2464 (Case12), and 745 (αBgt) cells). (f) Box chart of fluorescence intensity of Alexa Fluor 555-α-bungarotoxin (αBgt) cellular labeling in comparison to background level (n = 3, 1215 and 7641 cells, respectively, Student’s t-test, *p<0.05). (g) Box chart of fluorescence intensity of Case12 in the total cell population and in αBgt-positive cells, and intensity of background cellular fluorescence (n = 3, 8315, 1217, and 7643 cells, respectively, one-way ANOVA, *p<0.05). (h) Correlation between fluorescence intensities of Alexa Fluor 555-α-bungarotoxin (αBgt) and Case12 in co-labeled Neuro2a cells (black points, weak correlation, r = 0.4, Pearson correlation test, p = 2.2e-16, n = 3, 1217 cells). Red and green points represent fluorescence intensities of αBgt and Case12 in mono-labeled cell populations, respectively (n = 3, 1209 and 8315 cells, respectively). Blue points demonstrate background level of fluorescence intensities (n = 3, 7643 cells). Representative (i) microscopic images (scale bar, 100 μm) and (k) a plot of the intracellular calcium response ([Ca2+]i rise) of Neuro2a cell expressing human α7 nAChR, the chaperone NACHO, and the fluorescent calcium ion sensor Case12 to different concentrations of acetylcholine, and (l) corresponding dose-response curve of [Ca2+]i rise amplitude. (i) 73.5±1.0% (n = 3, mean±SEM, 1191 cells in total and 880 ACh-responding cells) of Case12-positive cells responded to 100 μM acetylcholine. Neuro2a cells were preincubated with 10 μM PNU120596, a positive allosteric modulator of α7 nAChR, before acetylcholine application. fl.u.–fluorescence units.
Fig 3
Fig 3. Electrophysiological whole-cell patch clamp recordings of acetylcholine action on α7 nAChR (WT), α7 nAChR [Q117T], and α7 nAChR [Y118W] expressed in Neuro2a cells.
(a) Representative currents through α7 nAChR (WT) under application of 3, 10, 30, and 100 μM acetylcholine and (b) dose-response curves. All ligand solutions contained the positive allosteric modulator 10 μM PNU120596, provoking increased and prolonged responses of α7 nAChR to acetylcholine. Each curve was plotted on the basis of averaged data from 6–7 cells (mean ± SD).
Fig 4
Fig 4. Expression of the fluorescent calcium ion sensor Case12 in Neuro2a cells correlates with cell viability markers.
Cytochemistry of Neuro2a cells transfected with plasmids coding human α7 nAChR, the chaperone NACHO, and the calcium sensor Case12 revealed that 93.5±0.7% (mean±SEM) of Case12-positive cells (green) were labeled with a cell viability marker 20 nM tetramethylrhodamine ethyl ester (TMRE, top panel, red, n = 3,1240 cells). Case12 fluorescence was absent in non-viable Neuro2a cells stained with the DNA-binding reagent propidium iodide (50 ng/ml, bottom panel, red, arrow heads, n = 3, 1561 cells). Scale bars, 60 μm.
Fig 5
Fig 5
Dose-response curves of the [Ca2+]i rise amplitude in Neuro2a cells expressing WT and mutant α7 nAChRs in response to different concentrations of (a, b) acetylcholine and (c) epibatidine. The protein calcium sensor Case12 (a, c) and the fluorescent dye Fluo-4 (b) were used to register changes in [Ca2+]i in Neuro2a cells. The cells were preincubated with 10 μM PNU120596, a positive allosteric modulator of α7 nAChR, for 20 minutes before agonist application. Each plot point reflects data obtained from 4 independent experiments (mean ± SEM).
Fig 6
Fig 6
Electrophysiological recordings of (a, b) acetylcholine- and (c) epibatidine-evoked currents mediated by WT and the L119D mutant α7 nAChRs expressed in Xenopus oocytes. (a) Representative current traces and (b, c) dose-response curves of ion currents. Each plot point reflects data obtained from 5–6 oocytes (mean ± SEM).
Fig 7
Fig 7. Cytochemistry and calcium imaging of Neuro2a cells expressing WT and G153S, Y190F mutant muscle nAChRs.
(a, b) Cytochemical labeling of WT muscle nAChR with Alexa Fluor 555-α-bungarotoxin (50 nM, αBgt) in Neuro2a cells. (a) Bright field image, (b) fluorescent image. Scale bar, 50 μm. (c) Pie charts represent percentage of transfected Neuro2a cells labeled with Alexa Fluor 555-α-bungarotoxin (αBgt) in the absence (n = 3, 413 total cells and 311 cells labeled with αBgt) or in the presence of Case12 (n = 3, 1233 total cells, 1005 cells expressing Case12, and 834 cells labeled with αBgt), respectively. (d, e) Dose-response curves of the [Ca2+]i rise amplitude in cells expressing WT and G153S, Y190F mutant muscle nAChRs in response to different concentrations of acetylcholine. The protein calcium sensor Case12 (d) and the fluorescent dye Fluo-4 (e) were used to register changes in [Ca2+]i. Each plot point reflects data obtained from 4 independent experiments (mean ± SEM).

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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.
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