Research ArticleCalcium signaling

All three IP3 receptor isoforms generate Ca2+ puffs that display similar characteristics

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Science Signaling  18 Dec 2018:
Vol. 11, Issue 561, eaau0344
DOI: 10.1126/scisignal.aau0344
  • Fig. 1 Protein expression of IP

    3R isoforms and global carbachol- and i-IP3–evoked Ca2+ signals in HEK WT cells and CRISPR-Cas9–modified HEK-293 cells that endogenously express exclusively type 1, 2, or 3 IP3Rs. (A) Equivalent amounts of lysate proteins from HEK WT, triple IP3R-knockout (3KO) and single IP3R isoform–expressing cells were immunoblotted with isoform-specific antibodies as indicated; data are representative of three independent experiments. Bar graphs show the relative expression of each IP3R isoform in single isoform–expressing cells normalized to their respective expression in WT cells. MW, molecular weight. (B to M) Fluorescence records and analyses were made from regions of interest encompassing individual cells loaded with Ca2+ indicator Cal-520. (B to E) Traces show representative, superimposed records of global fluorescence ratio signals (ΔF/F0) evoked by bath application of 100 μM CCH (denoted by line) in WT cells (B; n = 131) and in cells solely expressing type 1 (C; n = 130), type 2 (D; n = 186), or type 3 (E; n = 148) IP3Rs. (F) Bars show the mean peak amplitudes of the fluorescence signals (ΔF/F0) evoked by 100 μM CCH in WT and single isoform-expressing cells; n = 3 imaging fields for each cell line with cell totals of 392 (WT), 383 (IP3R1), 508 (IP3R2), and 494 (IP3R3). (G) Bars show the mean maximum rate of rise of the fluorescence signal (ΔF/F0 s−1), determined from 100 randomly chosen cells for each cell line with n = 3 imaging fields. (H to K) Superimposed traces of i-IP3–mediated global fluorescence signals (ΔF/F0) evoked by a 1000-ms duration photolysis flash (marked by an arrow) in WT cells (G; n = 134) and in cells solely expressing type 1 (H; n = 117), type 2 (I; n = 140), and type 3 (J; n = 112) IP3Rs. The delayed individual-cell signals in (H) and (J) are long-latency initial responses, rather than recurrent signals in cells that had already responded. (L) Dose-response curves of the mean peak amplitudes of the fluorescence signals (ΔF/F0) after photolysis flashes of different durations for WT cells and cells expressing single isoforms (depicted by different symbols and colors as indicated). For all cell lines, data points are means ± 1 SEM of n = 3 imaging fields with totals of 336 to 465 cells. Statistical comparisons between cell lines for a given UV flash duration were determined by analysis of variance (ANOVA) with Tukey post hoc tests: (300 ms) P < 0.01 for WT versus R1, WT versus R2, and WT versus R3; (700 ms) P < 0.01 for WT versus R1, WT versus R3, R1 versus R2, and R2 versus R3; (1000 ms) P < 0.01 for WT versus R1, WT versus R3, R1 versus R2, and R2 versus R3; (2000 ms) P < 0.01 for WT versus R1, WT versus R3, R1 versus R2, and R2 versus R3. (M) Bars show the mean maximum rate of rise (ΔF/F0 s−1) of fluorescence signals after a 1000-ms photolysis flash, determined from 100 randomly chosen cells for each cell line with n = 3 imaging fields. Traces in (B) to (E) and (H) to (K) are shown on identical magnitude (ΔF/F0), but different time scales. Statistically significant differences in (F), (G), and (M) were determined by ANOVA with Tukey post hoc tests; *P < 0.05 and **P < 0.01.

  • Fig. 2 Local, subcellular Ca

    2+ puffs evoked by photoreleased i-IP3 in WT cells and CRISPR-Cas9–modified HEK-293 cells exclusively expressing type 1, 2, or 3 IP3Rs. (A to D) Traces show Cal-520 fluorescence ratio measurements (ΔF/F0) from 1.5 μm by 1.5 μm regions of interest centered on puff sites in WT cells (A) and cells exclusively expressing IP3R1 (B), IP3R2 (C), and IP3R3 (D). Each panel shows records that are representative of puff sites exhibiting low and high frequencies of Ca2+ puffs; arrows and gaps in the traces mark the time of the photolysis flash. Traces in (A) were obtained using a 250-ms photolysis flash duration, and those in (B) to (D) with a flash duration of 500 ms. (E to H) Histograms depict the distributions of Ca2+ puff frequencies (number of puffs per site per 30-s recording) occurring at discrete puff sites in WT cells and cells expressing individual IP3R isoforms. (I) Mean numbers of puff sites per imaging field (37 μm by 19 μm) for WT cells and cells expressing individual IP3R isoforms; *P < 0.05 when assessed by ANOVA with Tukey post hoc tests. (J) Mean numbers of puffs per imaging field for WT cells and cells expressing individual IP3R isoforms; **P < 0.01 when assessed by ANOVA with Tukey post hoc tests. Data in (E) to (J) are from totals of 480 (WT), 272 (IP3R1), 338 (IP3R2), and 304 (IP3R3) puff sites, from 13, 8, 9, and 11 cells, respectively. Error bars in (I) and (J) depict 1 SEM. (K) Dose-response relationship for puffs generated by different IP3R isoforms. The data are from experiments in which records were acquired at a slower frame rate (151 frames s−1) than in (A) to (J) and from larger imaging fields (53 μm by 53 μm) that typically contained two to three cells. The plot shows the numbers of puffs detected throughout the imaging field in 10 s after photolysis flashes of various durations in cells solely expressing type 1, 2, or 3 IP3Rs (depicted by different symbols and colors, as indicated). Data points are means ± 1 SEM from n = 4 to 7 imaging fields.

  • Fig. 3 Amplitudes of Ca

    2+ puffs evoked in HEK WT cells and cells exclusively expressing type 1, 2, and 3 IP3Rs. (A) Left: The fluorescence traces (ΔF/F0) illustrate representative Ca2+ puffs evoked by photoreleased i-IP3 in WT cells. The panel shows several puffs, superimposed after alignment of the times of peak amplitude. The histogram plots the distribution of peak puff amplitudes (ΔF/F0) measured from 1841 events in 13 cells. (B to D) Panels show corresponding examples of puff traces and amplitude distributions in cells exclusively expressing IP3R1 (B; histogram is from 810 events from 8 cells), IP3R2 (C; 796 events from 9 cells), and IP3R3 (D; 1438 events from 11 cells). (E) Cumulative frequency curves showing the percentage of all detected events as a cumulative function of puff amplitude for WT cells and cells expressing each individual isoform (depicted by different colored symbols as indicated). (F) Bars show mean peak puff amplitudes (ΔF/F0) in WT cells and cells expressing each IP3R isoform, with n = the number of cells examined as listed above; error bars denote 1 SEM. Mean peak puff amplitudes were not significantly different between cell lines when assessed by either ANOVA or Kruskal-Wallis tests.

  • Fig. 4 Quantal analysis of single-channel IP

    3R Ca2+ flux during Ca2+ puffs. (A) Left: Traces show representative puffs in a WT cell, presented on an expanded time scale to illustrate stepwise increments and decrements of fluorescence (ΔF/F0) resulting from the successive openings and closings of individual IP3R channels. Fluorescence measurements were made from 1.5 μm by 1.5 μm regions of interest centered on puff sites. Horizontal lines drawn at integer multiples of fluorescence amplitude indicate the numbers of open channels during dwell times at different step amplitudes. The histogram shows the distribution of amplitudes (ΔF/F0) of visually identified mean dwell levels in WT cells. The curves show individual components of a multi (four)–Gaussian fit to the distribution. Data are from 415 WT dwell states. (B to D) Corresponding examples of stepwise puffs and of step dwell-state amplitude distributions and multi-Gaussian fits to data from cells expressing exclusively IP3R1 (B), IP3R2 (C), and IP3R3 (D). Data are from 665 (IP3R1), 659 (IP3R2), and 696 (IP3R3) dwell states. (E) The graph shows the center values of the four Gaussian distributions against their ordinate number (1 to 4). Different symbols represent data from the different cell lines, as indicated. Symbols overlap where not visible. The line is a regression fit to data from all cell types, constrained to pass through the origin, with a slope of ΔF/F0 = 0.126 per unitary step level.

  • Fig. 5 Temporal characteristics of Ca

    2+ puffs in WT cells and cells exclusively expressing type 1, 2, or 3 IP3Rs. (A to D) Histograms show the distributions of Ca2+ puff rise times (from 20 to 80% of peak value; left), fall times (fall from 80 to 20% of peak; center), and FDHM (right) for the different cell lines, as indicated. (E) Cumulative frequency curves show the percentage of all detected events as a function of puff rise time (left), fall time (center), and duration (right) for WT cells and cells expressing each isoform (depicted as indicated by different colored symbols). (F) Bars show mean puff rise times (left), fall times (center), and durations (right); error bars denote 1 SEM, with n = the number of cells examined. *P < 0.05 and **P < 0.01 when determined by ANOVA with Tukey post hoc tests. Data in this figure were derived from the same cells and recordings as in Fig. 3: WT, 1841 events from 13 cells; IP3R1, 810 events from 8 cells; IP3R2, 796 events from 9 cells; IP3R3, 1438 events from 11 cells.

  • Fig. 6 Spatial spread of fluorescence signal during Ca

    2+ puffs evoked in WT cells and cell lines exclusively expressing specific IP3R isoforms. (A) Left: Representative, temporally filtered image of a Ca2+ puff, with increasing fluorescence (F/F0) depicted on a pseudocolor scale and by increasing height. Right: 2D Gaussian fit to this puff. Scale bar, 5 μm. (B to E) Plots showing the distributions of puff spatial widths (FWHM of the Gaussian fit) measured, respectively, in WT cells and cells expressing type 1, 2, and 3 IP3Rs. The algorithm used to fit the image data truncated measurements at widths <2.4 μm. (F) Cumulative frequency curves show the cumulative percentage of all detected events as a function of increasing spatial width (FWHM) of puff fluorescence signals for WT cells and cells expressing each isoform (depicted by different colored symbols). (G) Bars show mean spatial widths; error bars denote 1 SEM, with n = the number of cells. **P < 0.01 when determined by ANOVA with Tukey post hoc tests. Data in this figure were derived from the same cells and recordings as in Fig. 3: WT, 1841 events from 13 cells; IP3R1, 810 events from 8 cells; IP3R2, 796 events from 9 cells; IP3R3, 1438 events from 11 cells.

  • Fig. 7 Subcellular localizations of Ca

    2+ puffs in WT cells and cells exclusively expressing type 1, 2, or 3 IP3Rs. (A) Left: Representative example of the locations of Ca2+ puffs recurring at an individual site in a WT cell. Circles mark the centroid localizations of Ca2+ fluorescence evoked by puffs within a 1 μm by 1 μm region of interest (outlined by the box) that was centered on the puff site. Middle: Distribution plot of neighbor-neighbor distances between every puff localization and every other localization. Distances were calculated for all puff localizations within the 37 μm by 19 μm imaging field, out to a radius of 5 μm around each localization. The black line, generated by a linear fit to the counts between 1 and 5 μm and constrained through zero, approximates the distribution expected if puff locations were randomly distributed. Right: Plot of the difference between the observed and predicted random distributions of neighbor-neighbor distances to provide a measure of the clustering of puff locations. (B to D) Corresponding examples of puff localizations and neighbor-neighbor distance distributions for cells exclusively expressing IP3R1, IP3R2, and IP3R3. (E) Neighbor-neighbor distance distributions for WT and each isoform replotted and overlaid from the right panels in (A) to (D). Data from the different cell lines are depicted by different colored symbols and lines and are shown after normalizing to their respective peaks and plotting on an expanded scale to better visualize differences in the spatial localization of puffs mediated by the different IP3R isoforms. Data in this figure were derived from the same cells and recordings as in Fig. 3: WT, 1841 events from 13 cells; IP3R1, 810 events from 8 cells; IP3R2, 796 events from 9 cells; IP3R3, 1438 events from 11 cells.

  • Fig. 8 Distribution of puff sites throughout the interior of WT cells and cells expressing individual IP

    3R isoforms, visualized by lattice light-sheet microscopy. (A and B) The images illustrate a single, diagonal light-sheet section through a cell expressing type 3 IP3Rs. The plasma membrane is depicted in red, and local Ca2+ puffs (ΔF/F0) are depicted in green. Images of puffs arising at different times and locations are superimposed on the membrane image. Representative examples are shown of puffs arising immediately adjacent to the plasma membrane (A) and deep in the interior of the same cell (B). Scale bar, 5 μm. (C) Bar graph summarizing the percentages of puffs arising near the cell edge or deeper (>2 μm) within the cell interior in WT (n = 42), IP3R1 (n = 8), IP3R2 (n = 12), and IP3R3 (n = 9) cells.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/561/eaau0344/DC1

    Fig. S1. Basal Ca2+ homeostasis in HEK WT and KO cell lines.

    Fig. S2. Comparisons of photoreleased i-IP3–evoked global Ca2+ signals and local Ca2+ puffs in HEK WT cells in the absence and presence of 20 μM ryanodine.

    Fig. S3. Comparison of Ca2+ puffs evoked by photoreleased i-IP3 in the presence and absence of extracellular Ca2+ in EGTA-loaded HEK cells solely expressing type 1, 2, or 3 IP3Rs.

  • This PDF file includes:

    • Fig. S1. Basal Ca2+ homeostasis in HEK WT and KO cell lines.
    • Fig. S2. Comparisons of photoreleased i-IP3–evoked global Ca2+ signals and local Ca2+ puffs in HEK WT cells in the absence and presence of 20 μM ryanodine.
    • Fig. S3. Comparison of Ca2+ puffs evoked by photoreleased i-IP3 in the presence and absence of extracellular Ca2+ in EGTA-loaded HEK cells solely expressing type 1, 2, or 3 IP3Rs.

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