©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Overexpression of Calreticulin Increases Intracellular Ca Storage and Decreases Store-operated Ca Influx (*)

(Received for publication, August 10, 1995; and in revised form, November 30, 1995)

Laurence Mery (1)(§) Nasrin Mesaeli (2)(¶) Marek Michalak (2)(**) Michal Opas (3) Daniel P. Lew (1) Karl-Heinz Krause (1)

From the  (1)Division of Infectious Diseases, University Hospital, 1211 Geneva 14, Switzerland, the (2)Medical Research Council Group in Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada, and the (3)Department of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The widely distributed and highly conserved Ca-binding protein calreticulin has been suggested to play a role as a Ca storage protein of intracellular Ca stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin expression vector. The calreticulin content of the overexpressers was increased by 1.6 ± 0.2-fold compared with mock-transfected cells. The total cellular Ca content of calreticulin-overexpressing and control cells, as assessed by equilibrium Ca uptake, was 141 ± 8 and 67 ± 6 pmol of Ca/10^6 cells, respectively (i.e. a 2.1 ± 0.2-fold increase in the Ca content of calreticulin-overexpressing cells). Over 80% of the increased Ca content was found within thapsigargin-sensitive Ca stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca] elevations due to Ca release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin-induced Ca and Mn influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca influx.


INTRODUCTION

Ca stores are intracellular compartments that are characterized by (i) their high intraluminal Ca content and (ii) their participation in the regulation of the cytosolic free Ca concentration ([Ca]) (^1)through rapid Ca accumulation and Ca release (1, 2, 3) . Intracellular Ca stores are faced with the dilemma to store large amounts of Ca within a restricted fraction of the cellular volume. For this reason, it is widely assumed that intracellular Ca stores contain intraluminal Ca buffers that allow the accumulation of large amounts of Ca without an excessive increase in the free intraluminal Ca concentration. The protein that has been suggested to make the physiologically most relevant contribution to Ca storage in nonmuscle Ca stores is calreticulin(2, 4, 5, 6, 7) . Calreticulin is able to bind Ca with high capacity and low affinity (8) and was found in many (but not all) studies to colocalize with other markers of intracellular Ca stores. However, while circumstantial evidence suggested a role for calreticulin in Ca storage, it turned out to be extremely difficult to provide scientific proof for this hypothesis.

Besides playing a crucial role in Ca uptake and Ca release, intracellular Ca stores appear to regulate, in a large variety of cell types, the divalent cation permeability of the plasma membrane: a decrease in the store Ca concentration activates the so-called store-operated Ca influx(9, 10) . The intraluminal Ca sensor that detects the drop in the Ca concentration within intracellular stores is not known, nor is the biochemical machinery that mediates the signaling from Ca stores to the plasma membrane.

In this study, we demonstrate that overexpression of calreticulin in L fibroblasts leads to an increase in the Ca content of thapsigargin-sensitive Ca stores and to a decrease in store-operated Ca influx.


EXPERIMENTAL PROCEDURES

Materials

Thapsigargin and Geneticin were obtained from Life Technologies (Basel, Switzerland); monensin, ionomycin, and DTPA were obtained from Sigma. Ca was purchased from Du Pont de Nemours/New England Nuclear Inc. (Dreieich, Germany), and fura-2/AM was from Molecular Probes, Inc. (Eugene, OR). Dulbecco's modified Eagle's culture medium and fetal calf serum were obtained from Gibco (Paisley, Scotland). Restriction endonucleases and DNA-modifying enzymes were purchased from Boehringer Mannheim, Life Technologies, Inc., and Bio/Can Scientific. Mouse L fibroblasts were a generous gift of D. Kobasa (Department of Biochemistry, University of Alberta). All other chemicals were of the highest grade available and were obtained from Fluka (Buchs, Switzerland) or Sigma. The medium referred to as ``Ca-free medium'' contained 138 mM NaCl, 6 mM KCl, 1 mM MgCl(2), 20 mM glucose, and 20 mM HEPES, pH 7.4. The medium referred to as ``Ca medium'' contained, in addition, 1.0 mM CaCl(2). When drugs were added as dimethyl sulfoxide solutions, final concentrations of dimethyl sulfoxide in the recording medium did not exceed 0.25%.

Plasmid Construction

To construct pRCR calreticulin expression vector, the DraI/SmaI restriction DNA fragment (nucleotides 20-1653) of pcDx-CRT (GenBank accession number J05138) (4) was first inserted into SmaI-digested pSVL vector (Pharmacia Biotech Inc.) to generate pSVL-CRT (pSCR-1) calreticulin expression vector. Next, the XhoI/SstI DNA restriction fragment, containing cDNA encoding full-length calreticulin, was excised from pSCR-1 vector and inserted into XhoI/SstI restriction sites of pRc/CMV vector (Invitrogen) to generate pRCR. pRCR vector was used for generation of stably transfected mouse L fibroblasts overexpressing calreticulin.

Stable Transfection

Mouse L fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C with 5% CO(2) in a humidified incubator. For transfection experiments, plasmid DNA was purified using a QIAGEN column by the Meg-Plasmid purification protocol as recommended by the manufacturer. Mouse L fibroblasts were electroporated (1500 V cm, 25 microfarads) with 20 µg of pRCR plasmid containing calreticulin cDNA in sense orientation or mock-transfected with 20 µg of pRc/CMV vector. Cells were selected for resistance to Geneticin (G418) (200 µg/ml). After 14 days of growth in the presence of G418, 50 clones were obtained and tested for the expression of calreticulin(11) . Cells transfected with the calreticulin expression vector are referred to as overexpressers; mock-transfected controls are referred to as control cells.

Culture of Stably Transfected Fibroblasts

Cells were grown in Dulbecco's modified Eagle's medium (Nut mix F-12, Gibco) supplemented with 10% heat-inactivated fetal calf serum, 100 µg/ml Geneticin, 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mML-glutamine. Cells were used for experiments during a period of 5 weeks to 3 months after thawing. In the first 5 weeks after thawing, no thapsigargin-induced Ca influx was observed in control cells. After 3 months, a decrease in calreticulin overexpression was observed despite the presence of Geneticin in the culture medium.

Immunocytochemistry

For intracellular localization of calreticulin, polyclonal antibodies against calreticulin were used at 1:50 dilution in phosphate-buffered saline (PBS)(12) . Fluorescein isothiocyanate-conjugated secondary antibodies were used at 1:30 dilution in PBS. For immunofluorescence confocal microscopy, cells on coverslips were fixed in 3.8% formaldehyde in PBS for 10 min; extracted with 0.1% Triton X-100 in buffer containing 100 mM PIPES, 1 mM EGTA, and 4% (w/v) polyethylene glycol 8000, pH 6.9, for 3 min; washed in PBS for 10 min; and then processed for labeling with primary antibodies followed by fluorescein isothiocyanate-conjugated secondary antibodies.

Ca Measurements

Fibroblast monolayers were cultured (under the above-described standard conditions) for 54 h in the presence of 10 µCi/ml Ca. Monolayers were then washed three times with the culture medium (without fetal calf serum), detached by trypsinization (0.25% trypsin and 0.02% EDTA in Ca/Mg-free PBS), and finally resuspended in Ca-free medium. For the measurement of total cell-associated Ca, aliquots of 10^6 cells were centrifuged; the radioactivity in the supernatant was counted and subtracted from the radioactivity measured for the same volume of cell suspension. For the measurement of Ca release in response to thapsigargin, ionomycin, and monensin, aliquots of 10^6 cells were removed immediately before and at the indicated times after thapsigargin addition and centrifuged, and the radioactivity was counted in the supernatant. Supernatants were placed in a vial containing a liquid scintillation mixture (Ultima Gold, Packard Instrument Co.), and the radioactivity was measured using a Packard 1900 TR scintillation counter.

Fluorescence Measurements

Cells (2 times 10^7/ml) suspended in Ca medium containing 0.1% bovine serum albumin were loaded for 40 min with 2 µM fura-2/AM, taking the following precautions to minimize compartmentalization of the dye. (i) Loading was performed at room temperature; (ii) 0.02% pluronic acid was included to prevent precipitation of solid fura-2/AM; and (iii) sulfinpyrazone (at a final concentration of 250 µM), an inhibitor of organic ion transport, was added to avoid intracellular transport or extrusion from the cells of fura-2-free acid(13) . To measure the amount of cytosolic versus compartmentalized fura-2, two types of protocols were used. (i) Cells were suspended in Ca-free medium containing 1 mM EGTA in the fluorometer cuvette, and fura-2 fluorescence was monitored at = 340 nm. After 3 min, 20 µM digitonin was added to permeabilize the plasma membrane. Calibration was performed by adding 5 mM Tris and 0.1% Triton (minimum) and 10 mM HCl and 3 mM Ca (maximum). (ii) Digitonin (20 µM) was added to 10^6 cells suspended in 2.4 ml of Ca-free medium. After 5 min of incubation at 37 °C, cells were centrifuged. The supernatant was transferred to a fluorometer cuvette, and 2 mM Ca was added. The pellet was resuspended in 2.4 ml of Ca medium, and cells were lysed in 0.1% Triton X-100. Fluorescence was measured at = 340 nm in the supernatant and in the cell lysate. Results are expressed as percent of the sum of the total fluorescence in the pellet and in the supernatant. All further steps of the fluorescence measurements were performed as described previously(14) . To measure Mn influx, 10 µM Mn was added to the extracellular medium, and the Mn-dependent decrease in intracellular fura-2 fluorescence (quenching) was measured at the Ca-independent wavelength of fura-2 ( = 360 nm). The decay of fura-2 fluorescence upon Mn addition is expressed as percent decrease in fura-2 fluorescence per time unit (initial fluorescence = 100%). The immediate fluorescence increase after addition of the heavy metal chelator DTPA reflects the ``unquenching'' of the extracellular fura-2 fluorescence and therefore gives an estimate of the amount of extracellular fura-2. For details concerning the Mn influx measurements, see (15) .

Protein Gel Electrophoresis and Quantitative Immunoblots

Cells were directly lysed in Laemmli sample buffer (containing 0.5% SDS and a mixture of protease inhibitors), followed by sonication to decrease viscosity of the samples. The proteins were separated by SDS-polyacrylamide gel electrophoresis using minigel systems. Quantitative immunoblotting with polyclonal anti-calreticulin antibodies was performed and analyzed as described previously(16) ; however, chemiluminescence (ECL Western blotting analysis system, Amersham International, Buckinghamshire, United Kingdom) rather than alkaline phosphatase was used for detection of secondary antibody binding.


RESULTS

Mouse L fibroblasts were stably transfected with a calreticulin expression vector or a control vector as described under ``Experimental Procedures.'' To assess the level of calreticulin overexpression, we performed quantitative immunoblotting of calreticulin-transfected and control cells (Fig. 1A). Immunoblots were scanned, and the slope of the correlation between cell number and optical density was determined. The slope of calreticulin-transfected cells divided by the slope of control cells yielded directly the increase in calreticulin content due to expression of calreticulin in transfected cells (see also (16) ). Fig. 1B shows that cells transfected with the calreticulin expression vector had a 1.6 ± 0.2-fold increase in the level of immunoreactive calreticulin.


Figure 1: Calreticulin levels and total cellular Ca content of calreticulin overexpressers and of control cells. Mouse L fibroblasts were stably transfected with a calreticulin expression vector or a control vector containing only the Geneticin resistance gene. A, to quantitate calreticulin, calreticulin overexpressers and control cells were analyzed by Western blotting with polyclonal anti-calreticulin antibodies. For each condition, three different amounts of cells (0.06, 0.08, and 0.11 times 10^6 cells/well) were analyzed. The immunblots were developed by a chemiluminescence assay. B, the immunoblots were scanned by a densitometer. The slope of the cell number versus optical density plot was obtained by a linear fit; it is a direct measure of the relative cellular calreticulin content. The calreticulin content of transfected cells is shown as percent of the calreticulin content of control cells. C, the total cellular Ca content was determined using equilibrium incubation with Ca. The total cellular Ca content is given as picomoles of Ca/10^6 cells. Data shown in B and C are means ± S.E. of three separate experiments performed in duplicate.



We next investigated whether calreticulin overexpression leads to a change in the pattern of calreticulin localization. Both control cells (Fig. 2A) and overexpressers (Fig. 2B) were heavily stained by anti-calreticulin antibodies. In both cells types, calreticulin appears to be located in the perinuclear system of the membrane corresponding in localization to the endoplasmic reticulum. More important, neither in control cells nor in overexpressers was nuclear, cytoplasmic, or surface staining observed.


Figure 2: Localization of calreticulin in overexpressers and in control cells by immunofluorescence confocal microscopy. Control cells (A) and overexpressers (B) were stained with goat anti-calreticulin antibodies. In both cell types, calreticulin localized predominantly to an endoplasmic reticulum-like intracellular network. Calreticulin also delineates the nuclear envelope in these cells. We did not observe nuclear, cytoplasmic, or cell surface staining. When the preimmune serum was substituted for the calreticulin antibody, no labeling was observed (data not shown).



To assess whether the Ca content of intracellular Ca stores is modified by the overexpression of calreticulin, we performed equilibrium loading experiments with Ca. Calreticulin overexpressers and control cells were cultured for 54 h in the regular culture medium containing 10 µCi/ml Ca. The time required to obtain isotopic equilibrium was within 36-48 h and was not significantly different between overexpressers and control cells (data not shown). The total cellular Ca content was then calculated based on the cell-associated radioactivity and on the specific activity of Ca in the culture medium. Control cells contained 67 ± 6 pmol of Ca/10^6 cells, whereas the overexpressers contained 141 ± 8 pmol of Ca/10^6 cells (Fig. 1C). Thus, the 1.6 ± 0.2-fold increase in cellular content of calreticulin protein led to a 2.1 ± 0.2-fold increase in cellular Ca content.

We next wanted to know whether the increased cellular Ca content reflected an increased Ca load of agonist-sensitive, rapidly exchangeable intracellular Ca stores. Agonist-sensitive, rapidly exchangeable intracellular Ca stores accumulate Ca through sarcoplasmic-endoplasmic reticulum Ca-ATPases. We therefore used thapsigargin, an inhibitor of sarcoplasmic-endoplasmic reticulum Ca-ATPases(17) , to measure the amount of Ca associated with rapidly exchangeable intracellular Ca stores. To assess the residual amount of Ca contained within thapsigargin-insensitive luminal Ca stores, we added the Ca ionophore ionomycin. Finally, as ionomycin has been shown to be inactive in releasing Ca from acidic intracellular compartments, we added the sodium proton ionophore monensin. For these experiments, cells were equilibrium-loaded with Ca isotope as described above and resuspended in a nonradioactive Ca-free buffer. Unidirectional fluxes to the extracellular medium after addition of the respective compounds were then measured. For more details concerning this approach, see (18) . Fig. 3shows that in both calreticulin overexpressers and control cells, 60% of the total cellular Ca could be released by thapsigargin. The remaining Ca was almost completely released by ionomycin. The increase in cellular Ca content of calreticulin overexpressers was mostly due to an increase in the size of thapsigargin-sensitive Ca stores (Fig. 3B). The monensin-induced Ca release was very small, suggesting that neither controls nor calreticulin-transfected cells contained relevant quantities of Ca stored within acidic compartments.


Figure 3: Characterization of Ca pools in calreticulin overexpressers and in control cells. Cells were cultured for 54 h with Ca to reach isotopic equilibrium, detached from the culture flask, and resuspended in Ca-free medium. Cell suspensions were preincubated for 3 min at 37 °C and sequentially stimulated with thapsigargin (100 nM), ionomycin (2 µM), and monensin (2 µM). Aliquots of the suspensions (corresponding to 10^6 cells) were collected at the indicated times and centrifuged. The radioactivity in the supernatant (i.e. the amount of Ca released from the cells) was measured. Background values (i.e. counts/minute in the supernatant before addition of thapsigargin) were substracted. A, typical experiment; B, mean ± S.E. of three separate experiments performed in duplicate. Ionomycin- and monensin-induced Ca release were defined as the additional release caused by the application of the respective compound.



To study the effect of the increased Ca content of intracellular Ca stores on the regulation of [Ca](i), we next performed experiments with the Ca-sensitive fluorescent dye fura-2. Cells were loaded with fura-2/AM under conditions preventing sequestration of the dye into subcellular organelles (e.g. endoplasmic reticulum, lysosomes). (i) Indicator loading was performed at room temperature; (ii) pluronic acid was added during incubation with fura-2/AM; (iii) sulfinpyrazone, an inhibitor of organic anion transport, was included in the buffer for dye loading and [Ca](i) measurements(19) . To document the efficacy of these precautions, we measured the amount of fura-2 that was released by digitonin (20 µM) or Triton X-100 (0.1%); >90% of the fura-2 from both control and calreticulin-overexpressing cells was released by digitonin, demonstrating the cytosolic localization of the dye (Fig. 4, A and B). Note also that the maximal fluorescence in control cells and overexpressers was comparable (Fig. 4A), suggesting that both cell lines contained the same amount of cytosolic fura-2.


Figure 4: Fura-2 loading of calreticulin overexpressers and control cells. Cells were loaded with the fluorescent Ca indicator fura-2, taking precautions to avoid dye sequestration (see ``Experimental Procedures'' for details). A, fura-2 fluorescence ( = 340 nm) was measured in unstimulated cells in Ca-free medium after addition of (i) 1 mM EGTA and 20 µM digitonin (EGTA+Dig.), (ii) 0.1% Triton X-100 and 5 mM Tris (min), and (iii) 10 mM HCl and 3 mM CaCl(2) (max). Data are means ± S.E. of four determinations of three independent experiments. B, cells were centrifuged after 5 min of incubation with 20 µM digitonin in Ca-free medium at 37 °C. The supernatant was transferred to a cuvette, and 1 mM Ca was added. The pellet was resuspended in an identical volume of Ca medium, and cells were lysed with 0.1% Triton X-100. Ca-dependent fura-2 fluorescence ( = 340 nm) was measured in the supernatant and in the resuspended pellet. Results are shown as percent of total fluorescence. Data are means ± S.D. of two independent experiments.



We next examined the effect of calreticulin overexpression on [Ca](i) elevations in response to Ca release from intracellular stores. We first investigated the effect of the receptor agonist ATP, which in mouse L fibroblasts has been shown to activate P2y purinergic receptors linked to phospholipase C(19) . ATP stimulation of cells suspended in Ca-free medium caused a rapid and transient [Ca](i) increase that was totally abolished by thapsigargin pretreatment of the cells (data not shown). The amplitude of the [Ca](i) elevation elicited by ATP was increased by a factor 1.5 in calreticulin overexpressers as compared with control cells (Fig. 5, A and E). Similarly, increased [Ca](i) elevations due to Ca release were observed when calreticulin overexpressers were concomitantly stimulated by thapsigargin and ionomycin (Fig. 5, D and E). In contrast, when cells were stimulated separately either by the sarcoendoplasmic reticulum Ca-ATPase inhibitor or by the ionophore, the peak and the duration of the [Ca](i) elevations due to Ca release were comparable in overexpressers and in control cells (Fig. 5, B, C, and E). Fig. 5F shows the amount of Ca that was released under the identical conditions in Ca experiments. Note that the increased amount of Ca that is mobilized by either thapsigargin or ionomycin alone is not reflected by increased [Ca](i) elevations (Fig. 5, compare E and F; see also ``Discussion''). No significant Ca release was detected after ATP stimulation of either control cells or overexpressers, suggesting that most of the released Ca was rapidly reaccumulated by intracellular Ca stores.


Figure 5: [Ca] elevations in response to Ca release from intracellular stores. Calreticulin overexpressers and control cells were loaded with the fluorescent Ca indicator fura-2. A-D, typical traces showing the stimulation of cells in Ca-free medium by the receptor agonist ATP (100 µM) followed by the Ca-ATPase inhibitor thapsigargin (100 nM) (A), by 100 nM thapsigargin only (B), by 2 µM ionomycin (C), or by a combination of 2 µM ionomycin and 50 nM thapsigargin (D). E, [Ca]([Ca]) elevations in response to stimulation by ATP, thapsigargin, ionomycin, and a combination of ionomycin and thapsigargin in calreticulin overexpressers, expressed as percent of values seen in control cells. Absolute values of [Ca] elevations in control cells were 123 ± 45 nM (ATP), 193 ± 16 nM (thapsigargin), 226 ± 36 nM (ionomycin), and 269 ± 33 nM (ionomycin + thapsigargin). Data are means ± S.E. of 8-12 independent experiments. F, Ca release from Ca-loaded control and calreticulin-overexpressing cells in response to stimulation by thapsigargin, ionomycin, and a combination of thapsigargin and ionomycin. Ca experiments were performed as described in the legend of Fig. 3. Values shown are counts/minute recovered in the supernatant 5 min after stimulation subtracted from the counts present in the medium before addition of thapsigargin (100 nM), ionomycin (2 µM), or a combination of thapsigargin (50 nM) and ionomycin (2 µM). Unstimulated Ca release was negligible in the period investigated. Data are means ± S.E. of three independent experiments.



In many cell types, the depletion of intracellular Ca stores leads to the activation of a Ca influx across the plasma membrane, a phenomenon referred to as store-operated Ca entry. We therefore investigated the effect of calreticulin overexpression on the activation of Ca influx in response to store depletion. [Ca](i) elevations after addition of Ca to cells stimulated by thapsigargin in Ca-free medium were markedly decreased in calreticulin overexpressers (Fig. 6). These results raise the possibility that calreticulin overexpression diminishes store-operated Ca influx. However, [Ca](i) elevations in response to Ca readdition experiments are the complex result of the activity of a variety of Ca transport pathways and may, at least partially, be explained by an inhibition of the plasma membrane Ca-ATPase through extracellular Ca. To measure more specifically the activation of the Ca influx pathway, we studied thapsigargin-induced Mn influx. Mn is able to permeate through store-operated Ca channels, but is not transported by Ca transport ATPases. Mn influx is detected as a quenching of the fluorescence of cytosolic fura-2. In both control cells and calreticulin overexpressers, there was a relatively important Mn influx without thapsigargin stimulation (Fig. 7). This most likely reflects the previously observed presence of mechanisms that allow Mn entry independent of store-operated Ca influx(20) . However, in control cells, thapsigargin clearly increased the amplitude of the fura-2 quenching after addition of Mn, demonstrating the presence of store-operated Ca influx in control mouse L fibroblasts. In contrast, no thapsigargin activation of Mn influx was observed in the overexpressers (Fig. 7). The suppression of thapsigargin-induced Mn influx was even observed when Mn was added 10 min after stimulation (Fig. 7D), i.e. at times when there was an almost complete emptying of thapsigargin-sensitive Ca stores (see Fig. 3A).


Figure 6: [Ca] changes in response to Ca influx across the plasma membrane. Fura-2-loaded cells were suspended in Ca-free medium at 37 °C and exposed to either 100 nM thapsigargin or dimethyl sulfoxide. Five minutes after the stimulation, 3 mM Ca was added. A and B, typical traces showing [Ca]([Ca]) increases in both control cells (solid lines) and calreticulin overexpressers (dotted lines) in response to the Ca readdition. C and D, [Ca] values immediately before and at different times after the Ca readdition in control and calreticulin-overexpressing cells. Data are means ± S.E. of five independent experiments.




Figure 7: Thapsigargin-induced Mn influx. Cells were loaded with the fluorescent Ca indicator fura-2. Recordings were performed at the Ca-independent excitation wavelength of fura-2 (360 nm). A and B, typical traces showing spontaneous and thapsigargin-induced fura-2 quenching after addition of Mn to the extracellular medium in control cells (A) and in overexpressers (B). C and D, thapsigargin-induced Mn influx (Mn-induced fura-2 quenching after thapsigargin stimulation minus Mn-induced fura-2 quenching without thapsigargin stimulation) in calreticulin overexpressers and in control cells (mean ± S.E.). Mn was added either 5 min (C; n = 6) or 10 min (D; n = 3) after thapsigargin stimulation. The small amplitude of the fluorescence increase after DTPA addition witnesses the low amount of extracellular fura-2. Unstimulated fura-2 quenching (as percent fluorescence decrease/80 s) was 15.3 ± 0.8 (5 min after dimethyl sulfoxide (DMSO)) and 18.7 ± 1.7 (10 min after dimethyl sulfoxide) in control cells and 15.8 ± 0.4 (5 min after dimethyl sulfoxide) and 18.8 ± 1.1 (10 min after dimethyl sulfoxide) in overexpressers.




DISCUSSION

Calreticulin is thought to act as a high capacity Ca-binding protein within intracellular Ca stores(2, 5) . So far, however, this notion is based on circumstantial evidence(6, 7, 21, 22) . Here we demonstrate for the first time that the Ca content of cells that stably overexpress calreticulin is augmented. (^2)This elevation in stored Ca is mostly due to an increase in the size of thapsigargin-sensitive Ca stores. Thus, our results add novel arguments in favor of a role for calreticulin as a Ca storage protein of agonist-sensitive, rapidly exchangeable Ca stores. The precise mechanism through which calreticulin increases the Ca content of intracellular Ca stores is not known. Our results would be compatible with the concept that the increased intraluminal Ca buffering in overexpressers is compensated by an increased total Ca store content, resulting in an unchanged intraluminal free Ca concentration. However, alternatively, one might consider the possibility that the effect of calreticulin on cellular Ca storage could include a regulatory role for this protein, rather than being a simple function of its Ca buffering properties. The latter possibility is supported by a recent study that demonstrates that in Xenopus laevis oocytes, calreticulin overexpression inhibits repetitive intracellular Ca waves and that this inhibition is independent of the Ca storage domain of calreticulin (23) .

The amount of calreticulin overexpression found in this study is relatively modest (i.e. an 1.6 ± 0.2-fold increase over control), but we consider this relatively low overexpression as an advantage. Indeed, rather than studying a massive overexpression with its inherent uncertainties (correct protein targeting, nonspecific effects due to very high protein concentrations), we have studied an overexpression that is presumably still within the limits of the physiological range of calreticulin expression(24) . Our observation that the increased Ca storage occurred almost exclusively in thapsigargin-sensitive Ca stores (Fig. 3) adds weight to this argument.

The relationship between the increased Ca content of intracellular stores in calreticulin-transfected cells (as assessed by Ca measurements) and the [Ca](i) elevations in response to Ca release (as assessed by fura-2 measurements) is complex and depends on the applied stimulus. Increased [Ca](i) elevations in calreticulin overexpressers are observed with ATP or with a combination of thapsigargin and ionomycin, but not when thapsigargin or ionomycin is separately used as a stimulus. We think that, in the case of thapsigargin, the slowness of Ca release allows negative feedback mechanisms (in particular, Ca extrusion through the plasma membrane Ca-ATPase) to obscure the differences in Ca release in relation to the level of [Ca](i) measurements. In the case of ionomycin, a residual Ca accumulation through Ca stores might dampen [Ca](i) elevations. The latter explanation is supported by the observation that, in overexpressers, increased [Ca](i) elevations in response to Ca release are observed when cells are concomitantly stimulated with ionomycin and thapsigargin. Finally, the massive and rapid Ca release that occurs mainly through the inositol 1,4,5-trisphosphate receptor in cells stimulated with ATP appears to generate, at least for a short period (30 s; see Fig. 5A), [Ca](i) transients whose amplitude is not governed by negative feedback mechanisms, but rather by the amplitude of the Ca release itself.

The inhibition of the store-operated Ca influx pathway by calreticulin overexpression provides new and independent experimental evidences for the intimate connection between the Ca content of rapidly exchangeable intracellular Ca stores and the plasma membrane permeability for divalent cations. The calreticulin inhibition of Ca influx might be due to the increased intraluminal Ca buffering of intracellular Ca stores. However, we have measured thapsigargin-induced Mn influx even 10 min after thapsigargin addition. At this point, thapsigargin-sensitive Ca stores were almost completely depleted, as shown by Ca experiments (Fig. 3). Nevertheless, virtually no store-operated Ca influx was observed. Thus, our results suggest that high calreticulin concentrations do not simply slow down the appearance of store-operated Ca influx because of a delayed depletion of Ca stores through the increased intraluminal Ca buffering. High intraluminal calreticulin concentrations block store-operated Ca influx even after a complete depletion of Ca stores. This might be a first indication that calreticulin plays a direct regulatory role in the mechanism of store-operated Ca influx.


FOOTNOTES

*
This work was supported by Swiss National Foundation Grant 32 30161.90 and by grants from the Medical Research Council of Canada, the Carlos and Elsie de Reuter Foundation, the Sandoz Foundation, the Ernst and Lucie Schmidheiny Foundation, the Société Académique de Genève, the Helmut Horten Foundation, and the Zyma Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 41-22-3729818; Fax: 41-22-3729830; kkrause{at}cmu.unige.chandMery@dminov1.hcuge.ch.

Postdoctoral Fellow of the Heart and Stroke Foundation of Canada.

**
Alberta Heritage Foundation for Medical Research Senior Scholar and Medical Research Council Scientist.

(^1)
The abbreviations used are: [Ca], cytosolic free calcium concentration; DTPA, diethylenetriaminepentaacetic acid; fura-2/AM, fura-2 acetoxymethyl ester; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
Since submission of this manuscript, a publication by Bastianutto et al.(25) showed an increase in the capacity of rapidly exchanging Ca stores through transient overexpression of calreticulin in HeLa cells; the authors also observed an inhibition of Ca influx in calreticulin-transfected cells.


ACKNOWLEDGEMENTS

We thank Antoinette Monod and Elzbieta Huggler for skilled technical support.


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