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Article |
Address correspondence to Francesco Zorzato, Dipartimento di Medicina Sperimentale e Diagnostica, sez. Patologia Generale Università di Ferrara, Via Borsari 46, 44100 Ferrara, Italy. Tel.: 39-0532-291356 Fax: 39-0532-247278. email: zor{at}unife.it; or fzorzato{at}uhbs.ch
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Abstract |
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Key Words: calcium homeostasis; IP3R; calcium entry channel; calcium binding protein; ER
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Introduction |
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Results |
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Junctate affects agonist-induced calcium release and receptor-activated calcium entry
We investigated the role of junctate (a) on the regulation of InsP3-mediated agonist induced calcium release from intracellular stores and (b) on calcium entry elicited in response to InsP3R activation, i.e., Ca2+-influx mediating refilling of the intracellular calcium stores. ATP stimulation of the PLC/InsP3 pathway in COS-7 cells in the presence of nominally calcium-free Krebs-Ringer, caused an immediate rapid rise in the [Ca2+]i followed by a slow decline to resting levels (Fig. 2, AD). COS-7 cells overexpressing full-length junctate (junctate-EGFP) exhibited a significant increase in peak ATP-induced Ca2+ release (mean ± SEM was 20% ± 2, n = 57; **P < 0.0004; Fig. 2 E). The transient increases in [Ca2+]i of cells transfected with either NH2-terminal (NH2-TM-EGFP) or COOH-terminal (EGFP-TM-COOH) domains of junctate were not significantly different from those observed in mock-transfected cells, excluding the possibility that the increases in calcium release observed in cells transfected with full-length junctate, is due to nonspecific cellular responses secondary to overexpression of recombinant proteins. Junctate overexpression did not affect the amount of InsP3R expressed, as determined by Western blot analysis (unpublished data). To establish if the effect of the different junctate constructs on calcium transients were due to increases of releasable calcium from ER, we determined the amount of calcium released from the stores by treating transfected COS-7 cells with the sarcoplasmic and ER Ca2+ATPase (SERCA) inhibitor Di-tert-butylhydroquinone (t-BuBHQ) in the presence of nominally calcium-free medium. Overexpression of either full-length junctate, or of its COOH-terminal calcium binding domain significantly increased the t-BuBHQ releasable Ca2+; on the other hand in cells overexpressing the NH2-terminal domain, the t-BuBHQ releasable-Ca2+ was not different from control COS-7 cells (Fig. 2 G). The ER-free [Ca2+] of mock-transfected cells and cells overexpressing junctate were not significantly different (the means ± SD were 456 ± 54 and 412 ± 27 µM, respectively; n = 47; t test, P = 0.497). These results indicate that overexpression of the COOH-terminal Ca2+-binding domain of junctate increases the releasable calcium present in the intracellular stores of COS-7 cells.
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We also determined whether the potentiation of receptor activated calcium entry by junctate requires PLC activation and/or generation of diacylglycerol by studying the characteristics of calcium entry after application of U73122, which is a specific inhibitor of PLC (Smith et al., 1990; Fig. 3 A). The presence of 10 µM U73122 almost completely abolished calcium influx in both mock-transfected COS-7 cells and cells transfected with the full-length junctate-EGFP (Fig. 3, A and C). Under similar experimental conditions, the inactive analogue U73343 did not block calcium influx (not depicted).
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Junctate affects calcium entry stimulated by depletion of ER stores independently of receptor-coupled events
2-APB has been reported to block the calcium entry pathways activated either by receptor agonist-activation or by store-depletion (Bootman et al., 2002). Therefore, we examined the effect of junctate on calcium entry mediated by depletion of ER calcium stores. To ensure store depletion, transfected COS-7 cells were loaded with 4 µM indo-1/AM in the presence of 1 mM EGTA (Fig. 4). This procedure depletes intracellular calcium stores because addition of 50 µM of the SERCA inhibitor t-BuBHQ (Fig. 4) failed to induce a [Ca2+]i transient. No difference in peak amplitude calcium entry was observed between mock-transfected cells and cells overexpressing either the NH2- or the COOH-terminal domains of junctate. On the contrary, cells transfected with the full-length junctate construct exhibited a 51 ± 6% increase (mean ± SEM; n = 48; *P < 0.0001) of peak amplitude calcium entry compared with mock-transfected cells.
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On the basis of these data, we would expect that intracellular perfusion in T3-HEK293 cells of the peptide encompassing the NH2 terminus of junctate would compete with the endogenous junctate and affect calcium influx via the TRPC3 channels. We performed patch clamp experiments in the whole cell configuration on T3-HEK293 cells. Bath application of 200 µM of the muscarinic agonist carbachol produced an inward current, previously characterized as being mostly carried by TRPC3 channels (Hurst et al., 1998). To test the effect of the NH2-domain of junctate, the corresponding peptide (5 µM) was introduced into the pipette solution and allowed to diffuse into the cytoplasm for 8 min after establishment of the whole cell configuration. Peptide dialysis produced a significant decrease (70%; P < 0.02; Fig. 7 G) of the peak inward current density induced by carbachol (Fig. 7 E). Fig. 7 (E and F) shows representative recordings of inward current induced by carbachol in control cells and in cells dialyzed with the junctate peptide, respectively. It is important to note that dialysis of peptide alone did not induce the appearance of any current (Fig. 7 F). Furthermore, dialysis with the corresponding scrambled peptide (10 µM) did not induce significant changes of the inward current amplitude (not depicted). Wild-type HEK293 cells showed negligible current densities (<0.05 pA/pF; Fig. 7 G).
Effect of junctate knockdown in T3-HEK293 cells
To investigate the role of endogenous junctate on [Ca2+]i of T3-HEK293 we cotransfected cells with the pShag-1 RNAi construct and the reporter plasmid p enhanced YFP (EYFP). Control cells were transfected with the pEYFP plasmid. From both groups, YFP-positive cells were sorted (Fig. 8 A). In RNAi-treated cells the mRNA encoding junctate was less abundant than in pEYFP-transfected control cells (Fig. 8 B). The presence of residual mRNA for junctate could be due either to the presence of a small subpopulation of cells which had been transfected only with the marker plasmid, or to the inability of the construct to fully eliminate the expression of junctate. The amount of ß-actin transcript did not differ between cells transfected with the two constructs (Fig. 8 B, compare lanes 1 and 2 to lanes 3 and 4, top and bottom). Depletion of junctate in pShag-1-RNAi-junctatetreated cells was also confirmed at the protein level by Western blot analysis (Fig. 8 C). The RNAi construct we used specifically degraded the mRNA specific for junctate because (a) the level of ß-actin mRNA was unchanged and (b) the expression of other key proteins involved in calcium signaling such as the InsP3R and the TRPC3 channel was not affected (Fig. 8 C). We also performed an additional control to ensure the specificity of the RNAi protocol. In mammalian cells RNAi has been reported to induce an interferon response, which leads to nonspecific mRNA degradation (Sledz et al., 2003). We tested for such a response in our cellular model by measuring the production of and ß interferons. We did not observe significant differences in the production of either interferon in RNAi-treated T3-HEK293 cells (unpublished data).
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To further substantiate the role of junctate on calcium signaling, we also monitored the changes in [Ca2+]i induced by ATP in pShag-1-RNAi-junctatetransfected cells by studying the [Ca2+]i of single T3-HEK293 cells. Based on the presence YFP expressing cells, we measured Ca2+ fluxes in groups of 510 cells. Stimulation of cells with 100 µM ATP induced a rapid and transient increase in [Ca2+]i (Fig. 9, C and I) which was reduced in T3-HEK293 cells in which endogenous junctate had been knocked down. The decrease in the amplitude of calcium release is evident not only in all the cells cotransfected with the pEYFP, but also in two adjacent cells which apparently did not pick up the pEYFP plasmid (Fig. 9, G and I). This is probably due to the fact that transfection efficiency is lower with larger plasmids, thus it is likely that the cells which did not exhibit the YFP fluorescence were transfected with the smaller construct carrying the RNAi cassette. Once the calcium signal returned to basal level (Fig. 9, D and J) calcium entry was measured (Fig. 9, E and K). Cells transfected with pEYFP alone exhibited a larger agonist-activated calcium entry compared with cells transfected with pShag-1-RNAi-junctate (Fig. 9 N).
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Discussion |
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Role of junctate in agonist-activated calcium release
The COOH-terminal domain of junctate is a moderate affinity, high capacity calcium binding domain. As expected, overexpression either of the full-length protein or of the lumenal COOH-domain induces an increase in the amount of releasable calcium from the stores sensitive to the SERCA inhibitor t-BuBHQ. However, although overexpression of full-length junctate also increases the amount of calcium released by ATP-mediated receptor activation, the COOH-terminal domain does not. Thus, the COOH domain acts similarly to calreticulin, a high capacity calcium binding protein uniformly distributed within the lumen of the ER. Overexpression of calreticulin increases the calcium content of ER calcium stores, but does not affect InsP3-mediated calcium release (Bastianutto et al., 1995; Xu et al., 2000).
The free calcium concentration in the endoplasmic reticulum lumen has been calculated to be 500600 µM (Elliot, 2001). In the presence of physiological concentrations of KCl, junctate binds calcium with an affinity of 217 µM. Thus, under resting conditions the calcium binding sites of junctate are saturated by calcium. Our results clearly demonstrate that in vivo junctate interacts with the InsP3R and though we do not exactly know how many junctate binding sites there are in the InsP3R complex, the simplest stoichiometric ratio is one junctate binding site per InsP3R complex. This implies that under resting conditions one mole of the junctate-InsP3R complex binds at least 21 moles of calcium and these are adjacent to the inner leaflet of the ER membrane. Thus, the moderate affinity calcium binding sites of junctate are close to the lumenal mouth of the InsP3R and it is tempting to speculate that an interaction between junctate's NH2 terminus and InsP3R causes a local increase of releasable Ca2+ upon agonist-receptor activation. This hypothesis is in agreement with the results obtained by the junctate RNAi experiments: knocking down junctate induced a decrease in the amplitude of agonist-induced calcium release. Measurements of [Ca2+]ER during receptor-mediated stimulation of calcium release have shown that the amplitude of InsP3R-mediated calcium release declines to near zero below a lumenal calcium concentration of 100 µM (Barrero et al., 1997), and at such a free [Ca2+]ER only
10% of junctate's calcium binding sites would be occupied (Treves et al., 2000). Under these conditions, the junctate-associated calcium would be too low to drive further release of calcium via the InsP3R and would thus result in the decline in calcium release, which has been experimentally demonstrated.
These data suggest that the local releasable calcium adjacent to the lumenal mouth of the ER calcium release channel, as opposed to the global ER calcium content, is the major limiting factor influencing InsP3-induced calcium release events triggered by activation of surface receptors.
Role of junctate on calcium entry
The elucidation of store- and receptor-activated calcium entry has emerged as a major theme in the field of cellular signaling in a variety of cell types. According to the conformational coupling hypothesis the InsP3R localized next to the plasma membrane interacts with the calcium channel responsible for calcium entry (Irvine, 1990; Kiselyov et al., 2000). The membrane compartment containing the InsP3R and the calcium entry channel may be endowed with other protein components that could be involved in the calcium entry mechanism. The ultrastructural and functional data reported here not only support this model, but also identify junctate as a candidate directly involved both in the assembly of the supramolecular complex and in the modulation of its function. In T3-HEK293 cells a small amount of ER extends throughout the cytoplasm and approaches the plasma membrane, where it forms discrete, small contacts with the plasma membrane (peripheral couplings). In the presence of extra amounts of junctate, the ER of T3-HEK293 cells is much more extensive, and peripheral couplings are more frequent and larger. Considering the dynamics of the ER, one likely explanation is that the ER is continuously reaching toward the plasma membrane and retracting from it and that junctate either induces an association between the ER and the plasma membrane or stabilizes it once it is formed. Coimmunoprecipitation and pull-down assay are consistent with such a view and indicate that the molecular basis of the peripheral coupling in T3-HEK293 cells is a heteroligomeric complex formed by junctate, IP3R and TRPC3 channels.
In addition to an indirect role mediated by an increase in peripheral couplings, our results suggest a more direct effect of junctate on calcium entry and indicate that this role may differ depending on how entry is elicited in an activated cell. Modulation of receptor-activated calcium entry is affected by the NH2 terminus of junctate, i.e., the domain of the protein interacting with the InsP3R. This interaction is not sufficient per se to modulate calcium entry, because the PLC inhibitor U73122 blocked the modulatory effect. This suggests that junctate affects receptor-activated calcium entry by modulating the steps which link the ligand-bound InsP3R and the plasma membrane calcium channel. It is plausible that initiation of the retrograde signal for stimulation of the receptor-activated calcium entry requires ligand-bound InsP3R. Binding of InsP3 to its receptor unmasks the effect on calcium entry, which derives from the interaction between the InsP3R and the NH2-terminal domain of junctate. In contrast to receptor-activated calcium entry, modulation of calcium entry induced by store depletion requires the entire junctate molecule: the overexpression of either the NH2-terminal domain or the COOH-terminal domain does not modulate store-depletion activated calcium entry, whereas it is significantly diminished in cells in which endogenous junctate has been knocked down. The retrograde signal activating store-depletion activated calcium entry is probably initiated by the removal of the calcium bound to the calcium binding domain of junctate, which is adjacent to the lumenal mouth of the release channel. Such a signal would be sensed by the calcium entry channel via the ligand-free InsP3R.
In conclusion, our data demonstrate that junctate, a protein of sarco(endo)plasmic reticulum membranes, is a novel component of the supramolecular machinery involved in calcium entry.
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Materials and methods |
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EGFP-junctate constructs
Junctate-EGFP, EGFP-junctate, and EYFP-junctate, were cloned into the MCS of the pEGFPN3, pEGFPC1, and pEYFPC1 plasmids, respectively. NH2-TM-EGFP and NH2-TM-EYFP, in which the first 85 aa of junctate, including the transmembrane domain, were cloned into the MCS of pEGFPN3 and pEYFPN3, respectively. EGFP-TM-COOH and EYFP-TM-COOH in which the transmembrane and COOH domain of junctate were cloned into the MCS of pEGFPC1 and pEYFPC1, respectively. For the latter four constructs cDNA was amplified by PCR using the following forward and reverse primers: 5'-AGAATTCACAAATGGCTGAAG-3' and 5'-GGGATCCCTTTGCTTTGGCTAGA-3' for the NH2-TM-EGFP(EYFP) constructs; and 5'-GGAATTCCACCATGAGGAAAGGCGGACTCTCA-3' and 5'-GAAGCTTTTAGGATCCTGGTG-3' for the EGFP (EYFP)-TM-COOH constructs. All DNA constructs were checked by sequencing.
Expression of recombinant junctate in COS-7 cells and intracellular calcium measurements
Expression of GFP-junctate and single cell intracellular calcium measurements in COS-7 cells were performed as described previously (Treves et al., 2000). Measurements were made on cells from four distinct transfections.
RNAi for junctate with small double-stranded RNA
The RNAi oligo (5'-GATCAAAAAAGTGTACCATTTCATGGAGGGACTGAATTCAAGCTTCAATTCAGTCCCTTCTCCATGAAATGGTACG-3') was annealed with equimolar amounts of the complementary oligo missing nucleotides CG and GATC at the 5' and 3' ends, respectively. The annealed oligos were subcloned into the U6 RNA pol III promoter pShag-1 vector. The nucleotide sequence of the construct was verified by sequencing. RT-PCR on YFP-positive cells using junctate specific primers (5'-TCAAAAAGACTGCCCCTACC-3' and 5'-GGACATGCAGGAATGTAACATGAC-3' for forward and reverse, respectively) or human ß-actin specific primers (5'-TGACGGGGTCACCCAC-3' and 5'-CTAGAAGCATTGCGGT-3' for forward and reverse, respectively). YFP-positive T3-HEK293 cells were sorted in a FACSVantageTM SE sorter (model 621, Becton Dickinson; 488 nm Enterprise laser, filter 530/30).
For single cell intracellular measurements, transfected T3-HEK293 cells were loaded with 5 µM fura-2/AM; YFP-positive cells were identified using an inverted microscope (model Axiovert S100 TV; Carl Zeiss MicroImaging, Inc.) using a 40x Plan-Neofluar objective (NA = 1.3) and filter (Carl Zeiss MicroImaging, Inc.) set N°44 (BP 475/40, FT 500, BP 530/50). Intracellular calcium measurements were obtained using a 40x plan-Neofluar oil-immersion objective, at 340 and 380 nm excitation and 510 nm emission (filters BP 340/ BP 380; FT 425; 500530). Fluorescence values obtained at 340 and 380 nm were acquired with a multiformat CCD C4880-80 camera (Hamamatsu), ratioed and the [Ca2+]i was analyzed using an Openlab imaging system. Calcium calibration was performed following the instructions provided with the calcium calibration kit. The [Ca2+]ER was determined in cells loaded with 2 µM Mag-Fura-2/AM as described by Solovyova et al. (2002).
Subcellular fractionation of HEK293
Subcellular fractions from T3-HEK293 cells stably transfected with the HA-tagged TRPC channel protein (Zhu et al., 1998) were prepared as described previously (Treves et al., 2000).
Coimmunoprecipitation and pull-down experiments
Total bovine cerebellum microsomal proteins (2 mg/ml) or integral membrane proteins extracted from microsomes with 100 mM Na2CO3 as described previously (Treves et al., 2000) were solubilized in: 1% Triton X-100, 200 mM NaCl, and 50 mM Tris-HCl, pH 7.4, at RT for 60 min in the presence of a protease inhibitor cocktail. Insoluble material was removed by centrifugation. The solubilized fraction was diluted fivefold with 200 mM NaCl, and 50 mM Tris-HCl, pH 7.4. Affinity-purified polyclonal antijunctate antibodies (Treves et al., 2000) were incubated with 200 µg solubilized bovine cerebellum microsomes for 2 h at RT, followed by incubation with 30 µl protein-A Sepharose. After 1 h, the beads were washed three times with 200 mM NaCl, 50 mM Tris-HCl, pH 7.4; bound proteins were separated on a SDS-PAG and blotted onto nitrocellulose.
For the pull-down experiments, microsomal proteins from T3-HEK293 cells, rabbit brain, and lung microsomes were washed with 0.6 M KCl, solubilized as described above, and incubated for 60 min with Streptavidin beads coated with 10 µg of a biotinylated peptide corresponding either to the NH2 terminus of junctate or to its scrambled sequence, and GSTCOOH junctate. The beads were washed three times with 0.1% Triton X-100, 200 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.4, and Western blots of bound protein were stained with antibodies. GSTCOOH-terminal domain of junctate was prepared as described previously (Treves et al., 2000). Protein concentration was measured as described previously (Treves et al., 2000).
Electrophoresis and immunoblotting
SDS-PAGE, protein transfer onto nitrocellulose and immunostaining were performed as described previously (Treves et al., 2000). Blots were probed with a polyclonal anti-InsP3R, or monoclonal anti-InsP3R type 1, 2, and 3 antibodies (Sugiyama et al., 1994), with affinity-purified antijunctate Ab (Treves et al., 2000) and anti-HA antibodies.
Electrophysiology
Recordings were performed on T3-HEK293 cells stably expressing TRPC3, in the whole cell configuration of the patch clamp technique. The pipette solution contained (mM) CaCl2 2, CsCl 120, EGTACs 5, Hepes 10, MgCl2 2, pH 7.2. Cells were bathed in Ringer's solution containing (mM) NaCl 140, KCl 5.4, MgCl2 1, CaCl2 2, D-glucose 10, and Hepes 10, pH 7.4. Pipettes (57 M were pulled from Kimax 51 glass (Kimball). Cells were clamped with an Axopatch 200B amplifier (Axon Instruments) and currents analyzed with pClamp 6.0. Current was filtered at 1 kHz. Holding potential was set at 40 mV.
Detection of peptide pull downs by flow cytometry
Streptavidin was covalently conjugated to 3.2-µm-diam carboxylate-modified polystyrene latex beads. The beads were then incubated in 150 mM NaCl, 10 mM Hepes, pH 7.4, for 30 min at RT with 100 µg/ml biotinylated peptide corresponding either to the NH2 terminus of junctate or an irrelevant peptide (biotin-SHIPGLRPSQQQQL). After washing, the beads were incubated with postnuclear lysates (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 6 mM EDTA, 1% NP-40, 0.5% deoxycholate; Boulay et al., 1999) from 2 x 105 HEK293 or T3-HEK293 cells for 60 min at RT. Beads were washed and stained with goat anti-InsP3R type 3 and subsequently with antigoat IgG-FITC. Bead fluorescence was acquired on a FACScalibur cytometer (Becton Dickinson Immunocytometry Systems); geometric mean fluorescence intensity calculated using CellQuest software.
LM and EM analysis of GFP-junctate expressing cells
Transfected COS-7 cells were examined 48 h after transfection using an inverted microscope (model Diaphot 300; Nikon) equipped with a PlanApo x 100/1.4 oil-immersion objective under fluorescent light (excitation 480 nm; emission 510 nm) as described previously (Treves et al., 2000). FACS-sorted GFP-positive T3-HEK293 cells were fixed in 3% glutaraldehyde in PBS and pelleted. The cells were maintained in the fixative for several days. The pellets were fixed after in osmium tetraoxide, en-bloc "stained" in uranyl acetate, and embedded. Thin sections showed 1200 profiles of cells/section cut in random orientations. The section were stained in uranyl acetate and lead containing solutions and examined in an electron microscope (model 410; Philips).
Statistical analysis
Statistical analysis was performed using the t test for unpaired samples; means were considered statistically significant when the P value was <0.05.
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Acknowledgments |
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This work was supported by grants from Ministero Universita' e Ricerca scientifica e Tecnologica 40%, F.I.R.B. RBAUO01ERMX, HPRN-CT-2002-00331 from the European Union, by the Dept. of Anaesthesia, Kantonsspital Basel and by National Institute of Health grants AR PO144650 to CFA and RO1 NS42183 to M.X. Zhu.
Submitted: 13 April 2004
Accepted: 6 July 2004
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