Electrophysiology and Ca_imaging normalized data

The 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

Photos 101-115

Photos 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 nACh

Video_Calcium response of N2A cells_a7 nAChR

Videos 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

Expression of the fluorescent calcium ion sensor Case12 in Neuro2a cells correlates with cell viability markers.

<p>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 (<i>green</i>) were labeled with a cell viability marker 20 nM tetramethylrhodamine ethyl ester (TMRE, top panel, <i>red</i>, 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, <i>red</i>, <i>arrow heads</i>, n = 3, 1561 cells). Scale bars, 60 μm.</p

Cytochemistry and calcium imaging of Neuro2a cells expressing WT and G153S, Y190F mutant muscle nAChRs.

<p>(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).</p

Mutagenic primers.

<p>Mutagenic primers.</p

Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants - Fig 6

<p><b>Electrophysiological recordings of (a, b) acetylcholine- and (c) epibatidine-evoked currents mediated by WT and the L119D mutant α7 nAChRs expressed in <i>Xenopus</i> oocytes.</b> (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).</p

Agonist affinity to WT and mutant muscle nAChRs measured by calcium imaging of cell populations with calcium ion indicators Case12 and Fluo-4.

<p>Agonist affinity to WT and mutant muscle nAChRs measured by calcium imaging of cell populations with calcium ion indicators Case12 and Fluo-4.</p

Agonist affinity to WT and mutant α7 nAChRs measured by calcium imaging of cell populations, and electrophysiology with <i>Xenopus oocytes</i>.

<p>Agonist affinity to WT and mutant α7 nAChRs measured by calcium imaging of cell populations, and electrophysiology with <i>Xenopus oocytes</i>.</p

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.

<p>(a, b) Cytochemical detection of α7 nAChR with 50 nM Alexa Fluor 555-α-bungarotoxin (αBgt, <i>red</i>) and (c, d) its co-expression with Case12 (<i>green</i>) 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 <i>t</i>-test, *<i>p</i><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, *<i>p</i><0.05). (h) Correlation between fluorescence intensities of Alexa Fluor 555-α-bungarotoxin (αBgt) and Case12 in co-labeled Neuro2a cells (<i>black points</i>, weak correlation, <i>r</i> = 0.4, Pearson correlation test, <i>p</i> = 2.2e^-16, n = 3, 1217 cells). <i>Red</i> and <i>green</i> points represent fluorescence intensities of αBgt and Case12 in mono-labeled cell populations, respectively (n = 3, 1209 and 8315 cells, respectively). <i>Blue</i> 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.</p