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. 2004 Aug 16;166(4):537-48.
doi: 10.1083/jcb.200404079. Epub 2004 Aug 9.

Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion

Susan Treves  1 , Clara Franzini-Armstrong, Luca Moccagatta, Christophe Arnoult, Cristiano Grasso, Adam Schrum, Sylvie Ducreux, Michael X Zhu, Katsuhiko Mikoshiba, Thierry Girard, Sophia Smida-Rezgui, Michel Ronjat, Francesco Zorzato
Affiliations

Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion

Susan Treves et al. J Cell Biol. .

Abstract

In many cell types agonist-receptor activation leads to a rapid and transient release of Ca(2+) from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP(3)) receptors (InsP(3)Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP(3)R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion-induced calcium entry, whereas its NH(2) terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells.

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Figures

Figure 1.
Figure 1.
Coimmunoprecipitation of InsP 3 R with antijunctate Ab. (Top) Proteins present in the total microsomal fraction (lane 1, 10 μg protein) or integral membrane proteins extracted with 100 mM Na2CO3 (lane 2, 10 μg protein) of bovine cerebella were stained with antijunctate Ab. Solubilized microsomal proteins were incubated in the absence (lanes 3 and 4) and in the presence of affinity-purified antijunctate Ab (lanes 5 and 6). Proteins complexing with junctate were coimmunoprecipitated with protein-A Sepharose beads and stained with anti-InsP3R Ab (lanes 3–6). Lanes 3 and 5, void; lanes 4 and 6, proteins bound to Protein–A Sepharose beads. (Bottom) Peptide pull-down experiments. (A) Triton X-100–solubilized rabbit brain microsomes stained with antibodies against type 1 InsP3R (lane 1, void; lane 2, last wash; lane 3, beads) and type 2 InsP3R (lane 4, void; lane 5, last wash; lane 6, beads). (B) Lung microsomes stained with type 3 InsP3R Ab (lane 1, void; lane 2, last wash; lane 3, beads). (C) Brain microsomes were incubated with a peptide corresponding to the scrambled sequence of the NH2-terminal domain of junctate and stained with anti-InsP3R Abs (lane 1, void; lane 2, last wash; lane 3, beads). (D) Brain microsomes were incubated with a GST fusion protein containing the COOH-terminal domain of junctate and stained with anti-InsP3R Ab (lane 1, void; lane 2, last wash; lane 3 glutathione-Sepharose beads).
Figure 2.
Figure 2.
Junctate affects agonist-induced peak calcium release, receptor-activated calcium entry and t-BuBHQ-sensitive calcium pools. (A–D) Indo-1–loaded COS-7 cells were flushed with a solution containing 100 μM ATP in nominally Ca2+-free Krebs-Ringer; once the [Ca2+]i had returned to resting levels, calcium influx was stimulated by addition of 2 mM Ca2+ in the presence of 100 μM ATP in Krebs-Ringer. Each record is from a single cell. Calcium influx was blocked by exposing the cell to a solution containing 2 mM lanthanum. (E and F) Calcium released and calcium entry peak amplitudes are presented as a percentage of the same changes in mock-transfected cells (mean ± SEM; n = single cells from four different transfections). (G) COS-7 cells were loaded with 5 μM fura-2/AM and then perfused with 50 μM t-BuBHQ in nominally Ca2+-free Krebs-Ringer. Total amount of calcium released was measured by calculating the integral of the calcium transient, using the Origin computer program software. Values are mean ± SEM; n = number of cells from three different transfections; **, indicates statistically significant difference from mock-transfected cells. a.u., arbitrary units.
Figure 3.
Figure 3.
Junctate requires activation of PLC to modulate calcium entry. Cells were first stimulated with ATP; after the intracellular Ca2+ concentration returned to basal the cells were flushed for 40 s with the PLC-inhibitor U73122 and with 2-APB. Traces show the recordings of the calcium influx stimulated by addition of 2 mM calcium in the presence of ATP plus 10 μM of PLC-inhibitor U73122 or 75 μM of 2-APB.
Figure 4.
Figure 4.
Junctate modulates calcium entry mediated by depletion of the ER independently of receptor activation. (A–D) Transfected cells were loaded with 5 μM indo-1/AM in the presence of 1 mM EGTA. Cells were exposed to 50 μM t-BuBHQ in the presence of nominally Ca2+-free Krebs-Ringer. Calcium influx was stimulated by addition of 2 mM Ca2+. (E) Values are mean ± SEM; n = number of cells from three different transfections. *P < 0.0001.
Figure 5.
Figure 5.
Junctate-positive network in junctate overexpressing T3-HEK293 cells. (A) An example of a COS-7 cell transfected with GFP. The cell shows an amorphous cytoplasmic fluorescence. (B and C) Examples of COS-7 cells transfected with the full-length junctate-GFP. An extensive GFP-positive network extends from the perinuclear region throughout the cytoplasm. Approximately 25% junctate-GFP–positive COS-7 cells had the spindle-like appearance as shown in C. (D) GFP-transfected T3-HEK293 cells have a fairly extensive amount of smooth ER near the cell center (left portion of the image) but little of it elsewhere (at right). L, lysosome. (E) This junctate-GFP overexpressing cell exhibits a very extensive ER network, continuous with the nuclear envelope and reaching out to the cell periphery. Such an extensive network was seen in a minority of cell profiles in sections from junctate-GFP expressing cells, but never in the control cells expressing GFP. Other cells from the same cultures show variable amounts of ER.
Figure 6.
Figure 6.
Overexpression of junctate results in larger peripheral couplings. The ER forms junctions with the plasma membrane (peripheral couplings, between arrows). The area of close apposition is smaller in the control (A) than in junctate overexpressing (B–D) T3-HEK293 cells (see also Table II).
Figure 7.
Figure 7.
A peptide encompassing the cytoplasmic domain of junctate complexes with the InsP3R via TRPC3 and inhibits TRPC3 current induced by carbachol in HEK293 cells stably expressing TRPC3. (A) Microsomal membrane fraction from T3-HEK293. Lane 1, Ponceau red staining; lane 2, antijunctate Ab immunostaining. Arrow indicates the band corresponding to junctate. The microsomal proteins of T3-HEK293 were solubilized, pulled down with Streptavidine beads coated either with a biotinylated peptide corresponding to the NH2 terminus of junctate (lane 4) or a biotinylated peptide corresponding to the scrambled sequence of the NH2- terminal domain of junctate (lane 3), and stained with anti–HA-tag Ab (lanes 3 and 4, respectively). (B) Model depicting the experimental approach used in C. (C) Peptide pull-down reactions analyzed by flow cytometry. Beads used in the pull down were coated with either an irrelevant peptide (gray lines, unshaded histograms) or junctate-derived peptide (shaded histograms). After incubation with cell lysates, beads were stained with goat anti-InsP3R type 3, followed by anti–goat IgG-FITC (x axis, log scale of geometric mean fluorescence intensity; y axis, number of events counted) and analyzed by flow cytometry. The geometric mean fluorescence intensity readout values are noted above each peak. (D) Immunoreactivity of total lysates prepared from HEK293 cells (lanes 1, 3, and 5) and T3-HEK293 cells (lanes 2, 4, and 6). Total lysates from 5 × 104 (lanes 1 and 2), 105 (lanes 3 and 4), and 1.5 × 105 cells (lanes 5 and 6) were stained with anti-InsP3R type 3 Ab. (E–G) Cells were clamped at −40 mV and stimulated for 40 s with 200 μM carbachol (CCh, black bar) 8 min after establishment of the whole cell configuration (t = 0 min). Recordings of TRPC3 currents in control cells (E) and peptide dialyzed cells (F). Histogram (G) showing mean ± SD of control and peptide-treated current densities of seven different sets of experiments performed with wild-type HEK293 and T3-HEK293 cells. *P < 0.02.
Figure 8.
Figure 8.
Transfection of T3-HEK293 cells with RNAi-junctate plasmid reduces the junctate transcript and protein level, affects intracellular store calcium content and diminishes store depletion-mediated calcium entry. (A) 60 h after transfection cells were sorted and YFP-positive cells were selected; the boxed scattergram represents a typical experiment in which ∼33% of all sorted cells were YFP positive. The purity of the YFP-positive population of cells was >90% as determined by reanalysis of the sorted cells by FACS® as well as fluorescence microscopy. (B) Total RNA was extracted from the YFP-positive cells. RNA was converted into cDNA and semiquantitative RT-PCR was performed on 50 and 100 ng RNA. The figure is representative of three different transfection experiments. (C) Total cellular protein extracts were prepared from the indicated number of YFP-positive cells as described in the methods section. Proteins were stained with affinity-purified antijunctate antibodies, anti-HA antibodies, and anti-InsP3R antibodies. (D) Calcium content of the stores was determined by perfusing T3-HEK393 transfected with 50 μM t-BuBHQ in nominally Ca2+-free Krebs-Ringer; mean ± SEM; n = cells from two transfections; a.u., arbitrary units. (E and F) YFP-positive T3-HEK293 cells were selected and intracellular Ca2+ measurements were performed. (E) pEYFP-transfected (continuous line) and in pEYFP+pShag-RNAi-junctate–transfected cells (dashed line). (F) T3-HEK293 cells transfected with pEYFP (unshaded histogram) and pEYFP+pShag-RNAi-junctate (shaded histogram). Values are mean ± SEM; n = number of cells from three different transfections.
Figure 9.
Figure 9.
Intracellular calcium measurements of T3-HEK293 cells depleted of junctate by RNAi. Ca2+ measurements in fura-2 loaded T3-HEK293 cells were performed 60 h after transfection with pEYFP (A–F) and with pEYFP+pShag-RNAi-junctate plasmids (G–L). (B and H) Resting [Ca2+]i; (C and I) peak [Ca2+]i after addition of 100 μM ATP in nominally Ca2+-free Krebs-Ringer; (D and J) [Ca2+]i returned to resting level 25 s after addition of ATP; (E and K) calcium entry induced by replacement of EGTA-containing extracellular medium, to Krebs-Ringer containing 2 mM CaCl2, in the continuous presence of 100 μM ATP; (F and L) [Ca2+]i returned to resting levels by adding 0.5 mM La3+ to Krebs-Ringer containing 100 μM ATP and 2 mM Ca2+. Images were acquired at 340/380 nm and ratioed every 0.75 s. (M and N) Statistical analysis of mean (±SEM) peak [Ca2+]i induced by the addition of 100 μM ATP and refilling of calcium pools induced by the readdition of 2 mM CaCl2 in YFP-transfected T3-HEK293 cells (unshaded histograms) and YFP+RNAi-junctate–transfected cells (shaded histograms); n = number of cells analyzed. At least three different transfection experiments were performed and cells from two coverslips were analyzed per transfection experiment.
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