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J. Biol. Chem., Vol. 277, Issue 48, 46696-46705, November 29, 2002

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Calreticulin Differentially Modulates Calcium Uptake and Release in the Endoplasmic Reticulum and Mitochondria^*

Serge Arnaudeau, Maud Frieden, Kimitoshi Nakamura^§, Cyril Castelbou, Marek Michalak^§¶, and Nicolas Demaurex

From the  Department of Physiology, University of Geneva, 1211 Geneva 4, Switzerland and ^§ Canadian Institutes of Health Research Membrane Proteins Research Group and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Received for publication, March 12, 2002, and in revised form, September 24, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To study the role of calreticulin in Ca²⁺ homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca²⁺ signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca²⁺ concentration within the ER lumen, [Ca²⁺]_ER, from 306 ± 31 to 595 ± 53 µM, and doubled the rate of ER refilling. [Ca²⁺]_ER remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca²⁺ ATPases. Under these conditions, store-operated Ca²⁺ influx appeared inhibited but could be reactivated by decreasing [Ca²⁺]_ER with the low affinity Ca²⁺ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca²⁺]_ER decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca²⁺ response and, surprisingly, with a shorter mitochondrial Ca²⁺ response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca²⁺ responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca²⁺ fluxes across the ER but decreases mitochondrial Ca²⁺ and membrane potential. The increased Ca²⁺ turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ signals control key biological functions, including fertilization, development, cardiac contraction, and secretion of neurotransmitters and hormones (1). At the cellular level, Ca²⁺ can be either a life and death signal, as changes in cytosolic free Ca²⁺ concentration can control cell growth and proliferation or induce apoptosis, the programmed cell death (2). These diverging effects reflect the precise spatial and temporal encoding of Ca²⁺ signals, which depends largely on the controlled release of Ca²⁺ from intracellular organelles. The main intracellular Ca²⁺ store is the endoplasmic reticulum (ER),¹ but mitochondria also take up and release Ca²⁺ very efficiently and are often strategically located close to Ca²⁺ sources (see Refs. 3-6 and reviewed in Ref. 7). This intimate connection allows mitochondria to shape Ca²⁺ signals (8) by modulating the release of Ca²⁺ from the ER (9) and the influx of Ca²⁺ across the plasma membrane (10), or by providing a local source of Ca²⁺ for ER refilling (11). To achieve such precise control over Ca²⁺ fluxes, the ER and mitochondria are equipped with a variety of Ca²⁺ transport and storage proteins and exert a tight control of the Ca²⁺ concentration within their lumen. Ca²⁺ fluxes across the ER membrane are stringently dependent on the free Ca²⁺ concentration within the ER, [Ca²⁺]_ER, as Ca²⁺ allosterically modulates the activity of the InsP₃ receptor, the main Ca²⁺-release channel of the ER. In addition, changes in [Ca²⁺]_ER regulate the Ca²⁺ permeability of store-operated channels (SOC) at the plasma membrane (12). The mechanism of this "capacitative" coupling is still elusive and has been proposed to involve the diffusion of a soluble messenger (13), direct interaction between InsP₃ receptors and SOC channels (14), or a secretion-like docking mechanism (15).

In addition to these Ca²⁺ signaling functions, the Ca²⁺ concentration within the ER lumen and the mitochondrial matrix also affects many functions of these organelles. The activity of several ER resident chaperone proteins is modulated by changes in [Ca²⁺]_ER, which thereby indirectly regulates the processing, sorting, and secretion of cargo proteins (16). In mitochondria, Ca²⁺ directly controls the activity of several dehydrogenases, thereby coupling the cell metabolism to the Ca²⁺ signal (17, 18). The mitochondrial "decoding" of Ca²⁺ signals allows cells to quickly respond to an increased energy demand but can be turned into a death signal during concomitant exposure to apoptotic stimuli (reviewed in Ref. 19). In the presence of ceramide, even physiological Ca²⁺ responses of mitochondria to InsP₃-generating agonists are sufficient to induce apoptosis, possibly via Ca²⁺-dependent opening of the permeability transition pore (20). The Ca²⁺ content of the ER also affects the cell sensitivity to apoptotic stimuli. A decreased [Ca²⁺]_ER was observed in cells overexpressing the antiapoptotic protein Bcl-2 (21, 22), and a variety of conditions that decreased [Ca²⁺]_ER has been shown to protect cells from ceramide-induced cell death (23). The opposite effect was observed in cells overexpressing the Ca²⁺-ATPases (SERCA2b) or the ER-resident Ca²⁺-binding chaperone calreticulin, which increased the Ca²⁺ content of the ER (23-25). Conversely, cells lacking the calreticulin had a decreased ER Ca²⁺ content and were more resistant to apoptotic stimuli (26). Calreticulin-deficient cells, however, had normal [Ca²⁺]_ER levels, suggesting that the ability of calreticulin to modulate the cell sensitivity to apoptotic stimuli might be linked to changes in the total Ca²⁺ content of the ER rather than to changes in [Ca²⁺]_ER.

Calreticulin is a 46-kDa Ca²⁺-binding chaperone that interacts in a Ca²⁺-dependent fashion with several ER resident proteins, with unfolded glycoproteins, and with Ca²⁺ transporters at the ER membrane (27, 28). Calreticulin is composed of three structural and functional domains as follows: a highly conserved N-terminal domain, involved in chaperone function and in the interactions with other ER chaperones; a proline-rich P-domain, which shares significant amino acid sequence identity with calnexin, calmegin, and CALNUC and is involved in the chaperone function of calreticulin; and a C-terminal domain that binds Ca²⁺ ions with low affinity and high capacity (29). The Ca²⁺-binding C-domain has been postulated to be the "Ca²⁺ sensor" that regulates calreticulin interactions with other proteins (25, 29). Because of the central role of the ER in Ca²⁺ signaling, both the chaperoning functions of calreticulin as well as its interactions with ER Ca²⁺ transporters can interfere with Ca²⁺ signals. For example, calreticulin inhibits repetitive Ca²⁺ waves by interacting selectively with distinct isoforms of SERCA2 (30, 31). On the other hand, conflicting results have been reported regarding the role of calreticulin in the modulation of store-operated Ca²⁺ influx (SOC). Stable up-regulation of calreticulin in HEK-293 cells inhibits thapsigargin-induced Ca²⁺ or Mn²⁺ influx (32), whereas transient expression in RBL-1 cells only delays the activation of the I_CRAC current, to an extent that correlated with the extent of store depletion (33). Similarly, in Xenopus oocytes overexpressing calreticulin and stimulated with InsP₃-generating agonists, SOC inhibition correlated with increased [Ca²⁺]_ER levels as expected from the capacitative mechanism (34).

Because of the plethoric effects of the protein and the different expression system used, the role of calreticulin in Ca²⁺ signaling remains controversial. To clarify the role of calreticulin in Ca²⁺ homeostasis and in apoptosis, we generated cell lines inducible for either the full-length calreticulin, an N-truncated version lacking the chaperoning N-domain, or its chaperone homologue calnexin. The effects of a controlled increase in protein levels on cytosolic, ER, and mitochondrial Ca²⁺ signals were measured using genetically encoded Ca²⁺-sensitive "cameleon" indicators. The bright fluorescence and molecular targeting of the probes allowed precise quantification of the changes in free [Ca²⁺] occurring within the different cell compartments at different times after the induction of protein expression.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Dulbecco's modified Eagle's culture medium, fetal calf serum, penicillin, streptomycin, and geneticin were obtained from Invitrogen. Thapsigargin, nigericin, monensin, ATP, and HEPES were purchased from Sigma. Ionomycin was obtained from Calbiochem. Hygromycin B, doxycycline, EGTA, and HEDTA were from Fluka (Buchs, Switzerland). JC-1 and TMRM were from Molecular Probes (Eugene, OR). Transfast transfection reagent was purchased from Promega (Catalys AG, Switzerland). All other chemicals were of analytic grade and were obtained from Fluka or Sigma. The "Ca²⁺ medium" contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose, and 20 mM Hepes, pH 7.4. For the "Ca²⁺-free medium" CaCl₂ was omitted, and 0.5 mM EGTA was included. Drugs were dissolved in dimethyl sulfoxide (Me₂SO) or ethanol and diluted in the recording medium on the day of use, at a final solvent concentration <0.1%.

Constructs-- Plasmids YC2, YC2.1, and YC4_ER were kindly provided by Dr. R. Y. Tsien. Plasmid YC2_mit and YC4.1_mit were generated as described previously (11). cDNA encoding full-length or truncated (P + C-domain HA-tagged) rabbit calreticulin and canine calnexin were subcloned into the pTRE plasmid to generate pTRE-CRT, pTRE-P + C and pTRE-CNX expression vectors, respectively. These vectors were used to generate Tet-On inducible cell lines. Plasmid DNAs were purified using a Qiagen column by the Maxi-prep purification protocol recommended by the manufacturer.

Generation of the Tet-On Cell Lines-- The Tet-On cell lines were generated by co-transfecting pTRE-CRT, pTRE-P + C, or pTRE-CNX with pTK-Hyg at a ratio 20:1 into HEK-293 cells (HeLa cells) by the Ca²⁺-phosphate protocol. Transfected cells were selected for growth in the presence of 200 µg of hygromycin B/ml of culture medium. Single colonies of the hygromycin B-resistant cells were tested for doxycycline (Dox)-dependent expression of calreticulin, P + C-domain, and calnexin by Western blotting with anti-calreticulin, anti-HA, and anti-calnexin antibodies. Three cell lines with the highest inducible expression of calreticulin, P + C-domain, and calnexin were selected for this study.

Cell Culture-- HEK-293 or Tet-On cell lines were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin and were maintained in a humidified incubator at 37 °C in the presence of 5% CO₂, 95% air. Cells (~200,000) were plated on 25-mm glass coverslips. With HEK-293 at 60% of confluency, the cells were transiently transfected with cDNAs encoding the yellow cameleon probes. Cells were imaged 3-5 days after transfection. Stable HEK-293 transfectants were grown in the presence of geneticin (100 µg/ml) for 3 weeks, and ~20 clones were expanded for each condition and tested for expression of the probes. 2 µg of Dox/ml was added into the culture medium to induce expression of calreticulin, its P + C-domain, or calnexin in Tet-On cell lines.

Immunoblotting and Immunocytochemistry-- Western blot analysis with the use of goat anti-calreticulin, anti-HA, and rabbit anti-calnexin antibodies was carried out as described (25). For indirect immunofluorescence of calreticulin expressing HEK Tet-On cells were plated on coverslips pretreated with polylysine and cultured in the presence or absence of 2 µg of Dox/ml for 72 h. Cells were washed 3 times with PBS, fixed with 3.7% paraformaldehyde for 20 min, and permeabilized with 0.3% Triton X-100 for 20 min. Calreticulin was detected by incubation with a goat anti-calreticulin antibody followed by staining with a rabbit anti-goat antibody conjugated to Texas Red (Jackson ImmunoResearch).

[Ca²⁺] Measurements-- Cells plated on 25-mm coverslips were superfused at 37 °C in a thermostatic chamber (Harvard Apparatus, Holliston, MA) equipped with gravity feed inlets and vacuum outlet for solution changes. Dual-emission ratio imaging of [Ca²⁺] with cameleon probes was performed as described previously (11). Cameleon fluorescence from cells was imaged on a Axiovert S100 TV using a 100×, 1.3 NA oil-immersion objective (Carl Zeiss AG, Feldbach, Switzerland). Cells were excited by the 430 ± 10 nm line from a monochromator (DeltaRam, Photon Technology International Inc., Monmouth Junction, NJ) through a 455DRLP dichroic mirror. Fluorescence emission from the cameleons was imaged using a cooled, 16-bits CCD back-illuminated frame transfer MicroMax camera (Princeton Instruments, Roper Scientific, Trenton, NJ) at two emission wavelengths, using a filter wheel (Ludl Electronic Products, Hawthorn, NY) to alternately change the two emission filters (475DF15 and 535DF25, Omega Optical, Brattleboro, VT). Image acquisition and analysis was performed with the Metamorph/Metafluor 4.1.2 software (Universal Imaging, West Chester, PA). Changes in fluorescence ratio, R = (fluorescence intensity at 535 nm  background intensity at 535 nm)/(fluorescence intensity at 475 nm  background intensity at 475 nm), were calibrated in [Ca²⁺] using Equation 1,

(Eq. 1)

where R_max and R_min are the ratios obtained, respectively, in the absence of Ca²⁺ and at saturating Ca²⁺. K is the apparent dissociation constant, and n is the Hill coefficient of the Ca²⁺ calibrations curves obtained in situ for each cameleon.

For better three-dimensional rendering wide field or confocal image stacks were deconvoluted after acquisition on a Silicon Graphics Octane work station using the Huygens 2 software, and shadow projections were constructed using the Imaris software (Bitplane AG, Zurich, Switzerland).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To generate cells inducible for calreticulin, we stably transfected HEK-293 cells with a rabbit calreticulin cDNA construct driven by the tetracycline promoter (Tet-ON). The activation of calreticulin gene transcription by doxycycline (Dox), added to the culture medium, was confirmed by immunoblotting with a goat polyclonal CRT antibody (Fig. 1A). Quantification of the immunoblot indicated that the cellular calreticulin content increased by 2.5-fold within 24 h and remained at this level for up to 5 days in culture. The induction was specific for calreticulin, as addition of Dox had no effect on the expression of other ER luminal chaperones such as ERp57 or Bip (not shown). An immunostaining with a calreticulin-specific antibody confirmed that protein expression was much stronger in Dox-induced cells and still displayed the reticular pattern typical of the ER (Fig. 1B, left). No immunoreactivity was observed in the cytosol or at the plasma membrane, confirming that, after induction, calreticulin remained localized within the ER lumen. The ER structure was not noticeably altered, because Dox induction did not affect the intracellular distribution of the ER-targeted Ca²⁺ indicator YC4_ER (Fig. 1B, right). This indicated that the increase in calreticulin did not interfere with the import, ER retention, or folding efficiency of the GFP-based indicator. Moreover, the Ca²⁺ affinity of both the ER-targeted probe YC4_ER and of the cytosolic probe YC2, measured in situ in cells permeabilized with ionomycin or digitonin, was not affected by the increased expression of calreticulin (Fig. 1C). Thus, Dox induction increased the amount of calreticulin within the ER lumen in a controlled manner, without interfering with the targeting specificity or Ca²⁺ dependence of the cameleon Ca²⁺ indicators.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Time course and specificity of calreticulin induction by doxycycline. A, Western blot of HEK-293 cells stably transfected with the Tet-ON CRT construct. Cells were treated with 2 µM doxycyline for the indicated times. B, effects of doxycycline induction on calreticulin immunostaining and on YC4ER fluorescence. Tet-ON cells were cultured for 3 days in the absence (top) or presence (bottom) of 2 µM doxycycline. The cells were then fixed and stained with a goat anti-CRT antibody and observed by confocal microscopy using identical acquisition settings (left). YC4ER fluorescence was assessed in live cells by confocal microscopy (right). Images are shadow projections of six adjacent, 400-nm wide z sections deconvoluted with the iterative constrained Tikhonov-Miller restoration algorithm. The induced calreticulin retained its reticular staining pattern and did not affect the subcellular distribution of YC4ER. cont, control. C, in situ Ca2+ calibration curves of cells expressing YC2cyt and YC4ER, cultured in the absence (control, black circles) or presence of doxycycline for 3 days (Dox, green circles). The induction of calreticulin had no effect on the Ca2+ affinity of the probes. Size bar indicates 5 µm.

Effect of Calreticulin Induction on ER [Ca²⁺] Homeostasis-- To assess whether the sustained increase in calreticulin levels interfered with ER Ca²⁺ homeostasis, we measured the changes in the free Ca²⁺ concentration within the ER lumen, [Ca²⁺]_ER, using the low affinity ER-targeted ratiometric "cameleon" indicator YC4_ER (K_D = 290 µM (11)). YC4_ER measurements revealed that the induction of calreticulin markedly increased the resting [Ca²⁺]_ER levels (Fig. 2) with the basal [Ca²⁺]_ER values averaging 306 ± 31 µM in the absence and 595 ± 53 µM 72 h after Dox-dependent induction of calreticulin expression. The increase could not be attributed to a specific ER region, as higher [Ca²⁺]_ER levels were observed throughout the ER network in the ratio images (Fig. 2A). Thus, the 2.5-fold increase in calreticulin levels caused, after 3 days of induction, a doubling in the free Ca²⁺ concentration within the ER lumen.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of calreticulin induction on ER [Ca2+] homeostasis. A, YC4ER emission ratio images (535/475 nm) of control and calreticulin-induced cells, measured before and after stimulation with the SERCA inhibitor thapsigargin (Tg). B, [Ca2+]ER changes in control (cont) and Dox-treated cells in response to Tg (1 µM) followed by ionomycin (Iono) to maximally deplete ER stores. The spatially averaged 535/475 ratio values were converted into [Ca2+] using the calibration curve of Fig 1. C, [Ca2+]ER changes induced by 100 µM carbachol (CCh). Stimuli were applied in the absence of external Ca2+, and Ca2+ was re-added when indicated. Inset, rate of ER Ca2+ pumping, measured at different [Ca2+]ER values in control () and Dox-induced cells (). Data are pooled from 5 and 4 independent experiments. D, average [Ca2+]ER values measured in control and Dox-treated cells under resting and depleted conditions. Data are means ± S.E. (*, p < 0.01). The number of experiments for each condition is indicated. Size bar indicates 5 µm.

The doubling in resting [Ca²⁺]_ER could reflect either an increased Ca²⁺ pumping activity, or a decrease in the passive Ca²⁺ permeability, or "leak" of the ER. To distinguish between these possibilities, we studied the effect of the SERCA inhibitor thapsigargin (Tg) on calreticulin-dependent changes in free ER Ca²⁺. Tg induced a slow decrease in [Ca²⁺]_ER in both control and calreticulin-induced cells (Fig. 2B). A linear fit of the initial [Ca²⁺]_ER decay revealed that the kinetics of Ca²⁺ release were nearly identical (Ca²⁺ = 5.2 ± 0.3 versus 6.0 ± 0.3 µM/s), despite the higher [Ca²⁺]_ER in the calreticulin overexpressers. Consequently, calreticulin-overexpressing cells retained a higher [Ca²⁺]_ER level throughout the course of Tg stimulation. A further decline was observed upon addition of the ionophore ionomycin (Fig. 2, B and D), indicating that the ER Ca²⁺ store was not fully depleted by Tg. Thus, a block of SERCA ATPases unmasked a nearly identical passive Ca²⁺ permeability in the ER, regardless of the increase in calreticulin levels.

In contrast, upon stimulation with the InsP₃-generating agonist carbachol (CCh), [Ca²⁺]_ER decreased much faster in CRT-induced cells, and similar depleted levels were achieved within 100 s of agonist stimulation (Fig. 2, C and D). The faster kinetics of Ca²⁺ release (Ca²⁺ = 4.5 ± 0.6 versus 11.4 ± 1.4 µM/min) suggested that calreticulin overexpression increased the InsP₃-stimulated Ca²⁺ permeability of the ER. Importantly, re-addition of Ca²⁺ to the external medium resulted in a rapid increase of the [Ca²⁺]_ER in calreticulin-overexpressing cells (Fig. 2C). The recovery rates were 1.9-fold higher in calreticulin overexpressers than in control, non-induced cells, at any given [Ca²⁺]_ER (Fig. 2C, inset). Because this assay measures the net flow of Ca²⁺ from the external space to the ER, this indicates that both the influx of Ca²⁺ across the plasma membrane and the ER Ca²⁺ pumping activity were increased in cells expressing high levels of calreticulin. In the absence of agonist stimulation, the increased rates of ER refilling were not balanced by a parallel increase in the endogenous ER Ca²⁺ permeability, resulting in higher [Ca²⁺]_ER levels at rest. However, induction of calreticulin expression markedly increased the agonist-induced ER Ca²⁺ permeability, and therefore, upon stimulation, more Ca²⁺ was released from the ER lumen.

Effect of CRT Induction on Cytosolic Ca²⁺ Signals-- To assess how these changes in ER luminal Ca²⁺ homeostasis influenced Ca²⁺ signals in the cytosol, we monitored changes in cytoplasmic Ca²⁺, [Ca²⁺]_cyt, with the cytosolic YC2 probe (K_D = 1.24 µM). Ca²⁺ release from ER stores was measured in the absence of external Ca²⁺, and Ca²⁺ influx was subsequently measured by re-adding Ca²⁺ to the external medium. Fig. 3 shows that both CCh and Tg elicited a much larger increase in [Ca²⁺]_cyt in calreticulin overexpressing cells, indicating that substantially more Ca²⁺ was released from the intracellular Ca²⁺ stores. Compared with previous studies using fura-2 (32), the differences between control and calreticulin overexpresser cells were striking, reflecting the better adequacy of the YC2 probe to quantify [Ca²⁺]_cyt changes in the micromolar range. Subsequent addition of Ca²⁺ to assess the activity of store-operated Ca²⁺ channels at the plasma membrane revealed that, as previously reported (32), Ca²⁺ influx was severely blunted in Tg-stimulated calreticulin-overexpressing cells (Fig. 3). This decreased influx correlated well with the increased [Ca²⁺]_ER levels measured with YC4_ER (Fig. 2) and indicated that, consistent with the capacitative hypothesis, the activity of SOC channels is determined by changes in [Ca²⁺]_ER levels. Accordingly, Ca²⁺ influx was similar in control and Dox-induced cells stimulated with CCh, which had comparable [Ca²⁺]_ER levels (Figs. 2 and 3). However, in this case the activity of SOC channels could not be readily inferred from the changes in [Ca²⁺]_cyt, because of the concomitant ER Ca²⁺ pumping activity. Although Ca²⁺ re-addition produced similar [Ca²⁺]_cyt changes, larger [Ca²⁺]_ER increases were observed in calreticulin-overexpressing cells, indicating that substantially more Ca²⁺ was taken up by the ER (Fig. 2A). This suggested that the net flux of Ca²⁺ ions across the plasma membrane was, in fact, larger in calreticulin-induced cells but that the Ca²⁺ entering the cell was rapidly taken up by the ER. Thus, the increased [Ca²⁺]_ER levels observed in the presence of Tg correlated with decreased SOC activity. In contrast, SOC activity was high in calreticulin overexpresser cells stimulated with CCh but did not translate into a larger cytosolic Ca²⁺ signal because of the high concomitant ER Ca²⁺ pumping activity.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of calreticulin induction on cytosolic Ca2+ signals. A, [Ca2+]cyt changes induced by Tg. Cells were stimulated in the absence of external Ca2+, which was re-added when indicated. B, average [Ca2+]cyt responses elicited by Tg in control (cont) and calreticulin-induced cells. C, [Ca2+]cyt responses elicited by CCh. D, average [Ca2+]cyt responses measured with CCh. Data are mean ± S.E. of the indicated number of experiment (*, p < 0.02; **, p < 0.0001).

Time Course of the CRT Effects on ER and Cytosolic [Ca²⁺]-- To better assess the effects of high expression of calreticulin on Ca²⁺ handling, we measured the [Ca²⁺]_ER and [Ca²⁺]_cyt responses at different times following the induction of protein expression. Fig. 4A shows that the resting [Ca²⁺]_ER levels were increased 24 h after Dox-dependent induction of calreticulin expression and remained elevated thereafter. In contrast, Ca²⁺ influx, taken as the peak [Ca²⁺]_cyt upon Ca²⁺ re-addition to Tg-treated cells, was inhibited only 3 days after the induction with Dox (Fig. 4B, circles). The amount of releasable Ca²⁺ followed a similar delayed time course; the peak of Tg-induced [Ca²⁺]_cyt release was only marginally increased 24 h post-induction and became significantly increased only 2 or 3 days after Dox induction of calreticulin expression (Fig. 4B, squares). The strong correlation between SOC activation and the total stored Ca²⁺ likely reflected the higher residual [Ca²⁺]_ER levels achieved at the end of the Tg stimulation in calreticulin-overexpressing cells. Although an ~5-min stimulation with Tg is routinely used to deplete Ca²⁺ stores, YC4_ER measurements indicated that [Ca²⁺]_ER did not reach fully depleted levels within the first 5 min in cells induced to express calreticulin for 3 days (Fig. 2). Thus, induction of calreticulin expression had two temporally distinct effects on Ca²⁺ homeostasis as follows: acute induction caused an immediate increase in [Ca²⁺]_ER, whereas more sustained expression of high levels of calreticulin was required to increase the total amount of stored Ca²⁺ and to inhibit store-operated Ca²⁺ influx.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Time course of calreticulin effects on ER and cytosolic [Ca2+]. A, [Ca2+]cyt responses measured at different times after Dox induction. B, the average [Ca2+]cyt responses elicited by Tg in Ca2+-free medium (release, squares) or during subsequent readdition of Ca2+ (influx, circles) are plotted against the duration of calreticulin induction. C, corresponding [Ca2+]ER levels measured at different times during Dox induction. Data are mean ± S.E. (*, p < 0.02; **, p < 0.001).

To show that the [Ca²⁺]_ER levels were indeed the prime determinant of SOC activity, we acutely modulated [Ca²⁺]_ER using the low affinity Ca²⁺ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). This cell membrane-permeant Ca²⁺ chelator has a K_D that matches the free Ca²⁺ levels in the ER lumen, providing an excellent tool to clamp [Ca²⁺]_ER without affecting the [Ca²⁺]_cyt responses (35). Fig. 5 shows that addition of TPEN produced a rapid decrease in [Ca²⁺]_ER but had only minor effect on [Ca²⁺]_cyt. The effects of TPEN were reversible (not shown). The chelator did not prevent the [Ca²⁺]_ER changes induced by Tg. Therefore, it was possible to artificially impose normal resting and depleted [Ca²⁺]_ER levels in calreticulin-overexpressing cells (Fig. 5A). Cytosolic Ca²⁺ measurements revealed that a robust Ca²⁺ influx could be elicited with Tg in the presence of the ER luminal Ca²⁺ chelator, as expected from the capacitative mechanism (Fig. 5B). This indicates that SOC channels were fully functional in calreticulin overexpresser cells when [Ca²⁺]_ER was artificially clamped to the level found in non-induced cells. Therefore, the high expression of calreticulin had no effect per se on the activity of SOC channels, which is determined primarily by the [Ca²⁺]_ER level in the ER lumen.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effects of TPEN on ER and cytosolic [Ca2+]. A, effect of the cell-permeant low affinity Ca2+ chelator TPEN on [Ca2+]ER, measured in the absence of external Ca2+ in Dox-induced cells. Iono, ionomycin. B, averaged [Ca 2+]ER levels measured before and after the addition of TPEN, which reduced the free ER Ca2+ by 2.2 times. C, effect of TPEN on the Tg-induced [Ca2+]cyt responses. D, averaged [Ca2+]cyt responses in control cells and in calreticulin-induced cells treated with TPEN. Data are mean ± S.E. (* p < 0.05).

Effect of Calreticulin Induction on Mitochondrial Ca²⁺ Signals-- In addition to communicating with the plasma membrane, the ER is also involved in a cross-talk with mitochondria, which are strategically located close to the sites of Ca²⁺ release and can capture part of the Ca²⁺ released by the ER (6). To assess whether the increased [Ca²⁺]_ER levels and InsP₃-induced ER permeability also affected mitochondria, we measured Ca²⁺ changes within the mitochondrial matrix, [Ca²⁺]_mit. Two mitochondrial probes of different affinities were used as follows: the high affinity YC2_mit probe (K_D = 1.24 µM), to allow accurate measurements within the low micromolar range, and the low affinity YC4.1_mit probe (K_D = 105 µM), to better resolve the high levels achieved during peak [Ca²⁺]_mit responses (11). The basal [Ca²⁺]_mit levels reported by the YC2_mit probe were not affected by the induction of calreticulin. Surprisingly, however, YC4.1_mit measurement revealed that the [Ca²⁺]_mit responses were blunted in calreticulin-induced cells (Fig. 6A). The peak [Ca²⁺]_mit levels measured with YC4.1_mit were close to 100 µM, consistent with earlier findings in chromaffin and HeLa cells (11, 36) and were only slightly reduced in calreticulin cells (120 ± 16 (n = 10) versus 77 ± 12 µM (n = 9), p = 0.05). However, a marked difference in the decay kinetics of the [Ca²⁺]_mit signal was observed, as [Ca²⁺]_mit returned much faster to basal levels despite the continuous presence of the InsP₃-generating agonist (Fig. 6B). This suggested that mitochondria were still able to take up the Ca²⁺ released by the ER but that Ca²⁺ extrusion from mitochondria was facilitated. As a result, the average [Ca²⁺]_mit level measured during CCh application was significantly reduced in calreticulin-induced cells (Fig. 6B). This finding was unexpected, as calreticulin induction increased both the amount of releasable Ca²⁺, the driving force for ER-to-cytosol Ca²⁺ release, and the InsP₃-induced Ca²⁺ permeability of the ER (Figs. 2 and 3). The shorter duration of the [Ca²⁺]_mit signal suggested that the ability of mitochondria to retain Ca²⁺ loads was impaired.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of calreticulin induction on mitochondrial Ca2+ signals. A, [Ca2+]mit changes measured with the low affinity YC4mit probe, whose KD matches the peak [Ca2+]mit values. B, mean [Ca2+]mit levels recorded during the time of CCh application (~120 s). Data are mean ± S.E. (* p < 0.01). cont, control.

Effects of Calreticulin on Mitochondria Morphology and Membrane Potential-- The abnormal mitochondrial response of calreticulin cells suggested that calreticulin induction might cause structural or functional damages to mitochondria. Because mitochondria are tightly coupled to Ca²⁺ release sites at the ER membrane (6, 37), subtle change in the architecture of the mitochondrial network might be sufficient to cause dramatic effects on [Ca²⁺]_mit signals. On the other hand, changes in mitochondrial membrane potential, _m, which determines the driving force for Ca²⁺, also directly impact on [Ca²⁺]_mit. To distinguish between these two possibilities, we measured _m and assessed the morphology of mitochondria as well as their interactions with the ER. To assess the morphology of mitochondria without relying on the extent of their negative membrane potential, we used the genetically targeted indicator DsRed_mit. Fig. 7A shows that the staining pattern of DsRed_mit was not markedly altered in calreticulin-induced cells. Upon Dox induction, mitochondria retained their "worm-like" appearance and did not appear swollen or condensed (Fig. 7A). Although a variety of mitochondria morphologies was observed both in control and Dox-induced cells, no systematic alterations could be observed in association with the induction of calreticulin expression. More importantly, the overlap between the mitochondrial and the ER signal was similar in control and calreticulin cells, as assessed by co-labeling cells with YC4_ER and Mitotracker Red (Fig. 7B). In both conditions, mitochondria appeared embedded into the ER, suggesting that the induction of calreticulin did not disrupt the interactions between the ER and mitochondria. Thus, although the resolution of the confocal microscope did not allow us to resolve the contact points between the ER and mitochondria, the structural integrity as well as the relationship between the two organelles appeared to be preserved.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Effect of calreticulin induction on mitochondria morphology and membrane potential. A, staining patterns of a mitochondrial targeted DsRedmit (kindly provided by Dr. T. Pozzan, Padua). Images are shadow projections of z stacks of confocal images deconvoluted and processed using the Huygens and Imaris software. B, YC4ER-expressing cells were co-labeled with Mitotracker Red to assess the interaction between the ER and mitochondria. Images were acquired and processed as in A. C, TMRM (left) and JC-1 (right) measurements of the mitochondrial membrane potential. m is expressed as the ratio of the mitochondrial over cytosolic TMRM fluorescence (left) or as the percentage of the JC-1 stained area containing red fluorescent aggregates (right). Data are mean ± S.E. (*, p < 0.005). Size bar indicates 5 µm.

We next measured the mitochondrial membrane potential, _m, using the rhodamine-based dye TMRM, which accumulates into polarized mitochondria. The _m-driven accumulation of TMRM into mitochondria was quantified as the ratio of the mitochondrial over cytosolic fluorescence intensity (38). The TMRM ratio was significantly lower in Dox-induced cells (Fig. 7C, left panel), indicating that _m was reduced by long term overexpression of calreticulin. The decrease in _m was not due to TMRM photoactivation and subsequent local generation of reactive oxygen species (ROS) (39), as determined by time lapse imaging. The TMRM ratio was already lower in Dox-induced cells illuminated for the first time, and did not change subsequently over the 20 min recording period (data not shown). The decrease in _m was confirmed by measurements with JC-1, a potentiometric dye that forms red-emitting aggregates at negative _m (38). As shown in Fig. 7C, the proportion of red-emitting JC-1 aggregates was markedly reduced in Dox-induced cells (Fig. 7C, right panel). Thus, the abnormal [Ca²⁺]_mit response of calreticulin-overexpressing cells correlated with a decreased mitochondrial membrane potential, with no visible alteration in the mitochondrial architecture.

Role of the Ca²⁺ Binding C-domain of Calreticulin-- Calreticulin is a multifunctional protein, and different regions of the protein perform different functions (29). For example, the N- + P-domains of calreticulin are involved in chaperone function, whereas the C-domain of the proteins plays a role of Ca²⁺ storage and "Ca²⁺ sensing" in the ER lumen (29). To identify the region of calreticulin involved in Ca²⁺ and organelle homeostasis, we generated Tet-ON cells inducible for a truncated calreticulin, encoding the P + C-domain, which contains a critical Ca²⁺-binding region in calreticulin. Dox-induced expression of the P + C-domain was at similar levels as the wild-type protein, as assessed by immunofluorescence and Western blotting (not shown). Fig. 8 shows that the P + C-domain mimicked the effects of the full-length calreticulin. The Dox-induced cells overexpressing the P + C-domain had a higher resting [Ca²⁺]_ER, increased residual [Ca²⁺]_ER levels after Tg stimulation, and a lower [Ca²⁺]_mit signal (Fig. 8, C and D). In addition the reduction of TMRM fluorescence was also measured in P + C-induced cells (Fig. 8E). This suggested that the Ca²⁺ sensing and Ca²⁺ storage C-domain of calreticulin was responsible for the deleterious effects. Despite repeated attempts we were unable to generate cells overexpressing either the N- or C-domain alone. However, it is unlikely that the chaperone P-domains of calreticulin play a role because Dox-inducible expression of ER chaperone calnexin, which contains a similar P-domain, did not reproduce the effect on [Ca²⁺]_ER (Fig. 8B). In summary, these data suggest that the low affinity, high capacity Ca²⁺-binding C-domain, rather that the chaperone interacting regions of calreticulin, mediate the effects on [Ca²⁺]_ER leading to modulation of SOC and mitochondrial Ca²⁺ homeostasis.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effect of calreticulin domains. A, schematic representation of calreticulin and calnexin domains (27). B, average [Ca2+]ER values in cells induced to express the P-domain containing chaperone calnexin. [Ca2+]ER was measured in the absence of external Ca2+, before and 5 min after application of Tg. C, [Ca2+]ER values in cells induced to express the P + C-domain of calreticulin (P + C-domain). D, [Ca2+]mit responses measured with the YC4.1mit probe in the P + C-domain Tet-ON cells. The average values recorded during the CCh application are shown. E, TMRM fluorescence intensity ratio between mitochondria and the cytosol, measured in control and P + C-domain-induced cells. Data are mean ± S.E. (*, p < 0.05; **, p < 0.005).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we report that differential expression of calreticulin in the lumen of ER affects the Ca²⁺ homeostasis of distinct cellular compartments. Altered Ca²⁺ signals were observed in the ER, in the cytosol, at the plasma membrane, and in the mitochondria. The most predominant effects of increased expression of calreticulin occurred at the level of ER, where the protein resides. Consistent with all previous studies (23, 32, 34, 40), we found that calreticulin overexpression increased the total amount of Ca²⁺ stored in the ER, an effect that occurred within days after the induction of protein expression. In addition, we found that the increased expression of calreticulin has a significant effect on the free intraluminal ER Ca²⁺. The free Ca²⁺ concentration within the ER lumen, [Ca²⁺]_ER, nearly doubled within 24 h of induction of calreticulin expression and remained at these elevated levels for several days. This is in contrast to an earlier report wherein oocyte [Ca²⁺]_ER levels were either not affected (34) or slightly decreased (31) when calreticulin was overexpressed. Although different expression systems were used, these diverging effects of calreticulin relate to cellular systems expressing the same SERCA isoform. In this study, increased [Ca²⁺]_ER levels and Ca²⁺ pumping activity were observed in calreticulin-overexpressing HEK-293 cells, which contain predominantly the SERCA2b isoform (Fig. 2). In contrast, in oocytes co-injected with calreticulin and the SERCA2b expression vectors, decreased [Ca²⁺]_ER and Ca²⁺ pumping activities were observed (31). In both cases, Ca²⁺ pumping activity directly correlated with the [Ca²⁺]_ER levels, consistent with recent results (24) showing that overexpression of SERCA2b increases [Ca²⁺]_ER by 25% in Chinese hamster ovary cells. In the present study, calreticulin levels were increased by 2.5-fold, Ca²⁺ pumping activity by 1.9-fold, and [Ca²⁺]_ER by 1.8-fold. This excellent correlation reflected the imbalance between the increased Ca²⁺ pumping activity and the endogenous Ca²⁺ permeability of the ER, which was unaffected by calreticulin.

The increased Ca²⁺ pumping activity, however, was not mediated by SERCA isoforms, as inferred from the effects of thapsigargin. Thapsigargin, added at concentrations that fully inhibit SERCA, unmasked a nearly identical passive ER Ca²⁺ permeability in control and calreticulin overexpressers (Fig. 2B). Because at steady state the Ca²⁺ pumping activity is equal to the ER Ca²⁺ leak, this indicates that, under resting conditions, the activity of SERCA was not altered in the calreticulin overexpressers. Thus, thapsigargin-insensitive Ca²⁺ pumps mediate the increased ER refilling observed during Ca²⁺ re-addition to Ca²⁺-depleted cells (Fig. 2C). A likely candidate is the Pmr1 family of Ca²⁺ transport ATPases, which has been shown recently (41) to be expressed and functional in mammalian cell lines. The thapsigargin-insensitive Pmr1 pump is localized mainly to the Golgi complex, but a substantial fraction is present and functional in the ER. The Pmr1 store had a reduced Ca²⁺ leak and weak InsP₃ responses, and COS-7 cells overexpressing the Pmr1 pump had delayed Ca²⁺ influx (42). It is tempting to speculate that calreticulin, by interacting with the Golgi-targeted Pmr1 pump, might promote its retention in the ER, thereby accounting for the increased Ca²⁺ pumping activity observed in calreticulin overexpressers. In any case, the existing evidence strongly suggests that calreticulin interacts differentially with distinct Ca²⁺ pump isoforms and modulates the rates of Ca²⁺ uptake into the ER, thereby directly altering [Ca²⁺]_ER. The physiological relevance of these interactions is not clear, but a decreased [Ca²⁺]_ER has been shown to activate the transcription of the calreticulin gene (43). Therefore, an increase in calreticulin level in the ER would rapidly restore normal [Ca²⁺]_ER levels, thereby abrogating its transcriptional activation. Consistent with such a feedback mechanism, the [Ca²⁺]_ER increase was the first perturbation observed upon the induction of calreticulin.

In addition to increasing the total and free Ca²⁺ of the ER, calreticulin also increased the rates of agonist-induced Ca²⁺ release. Increased release was observed over a wide range of [Ca²⁺]_ER, indicating that it did not simply reflect the increased driving force for Ca²⁺ but increased fluxes through InsP₃-gated channels. This was unexpected, because it was reported recently (24) that the rates of ATP-induced Ca²⁺ release were decreased in cells with increased [Ca²⁺]_ER due to overexpression of SERCA. This effect was attributed to the Ca²⁺-dependent inhibition of InsP₃-gated channels. Because in our calreticulin-induced cells the InsP₃ channels were also exposed to higher amounts of Ca²⁺ ions, both on the ER and on the cytosolic side, the increased release might reflect a direct action of calreticulin on InsP₃-gated Ca²⁺ channels.

Because of the increased ER Ca²⁺ load and the increased driving force for Ca²⁺, more Ca²⁺ was released into the cytosol during stimulation with agonists and/or thapsigargin, and store-operated Ca²⁺ influx was reduced when measured with the Ca²⁺ re-addition protocol (Fig. 3). However, analysis of the cytosolic and ER responses at different times after induction indicated that calreticulin levels had no direct effects on store-operated Ca²⁺ influx. Decreased SOC activity was only observed in cells induced to express CRT for 3 days and correlated with an increase in total stored Ca²⁺, rather than with the resting [Ca²⁺]_ER levels (Fig. 4). In previous studies, decreased Ca²⁺ influx was observed in stable calreticulin overexpressers (32) but not in cells transiently transfected with calreticulin (33). Our observations reconcile these apparently discrepant findings and caution against the Ca²⁺ re-addition protocol to assess store-operated Ca²⁺ influx, because 1) the degree of store depletion cannot be readily estimated from the cytosolic Ca²⁺ responses, and 2) the concomitant activity of SERCA greatly affects the dynamics of the [Ca²⁺]_cyt signal, precluding accurate estimates of the influx component.

The effects of calreticulin extended beyond the ER and affected another organelle, the mitochondria. However, the larger release of Ca²⁺ from the ER was not associated with an equally larger Ca²⁺ accumulation in mitochondria but with a reduced signal as [Ca²⁺]_mit rapidly returned to basal levels despite the presence of InsP₃-generating agonists (Fig. 6). The abnormal [Ca²⁺]_mit response did not reflect structural damage, because the shapes and numbers of mitochondria as well as their relationship to the ER appeared normal by confocal microscopy, but was associated with a mitochondrial depolarization (Fig. 7). The depolarization, by reducing the driving force for Ca²⁺, is expected to reduce mitochondrial Ca²⁺ uptake and might thus account for the blunted [Ca²⁺]_mit response. In addition, the activity of the mitochondrial Ca²⁺ uniporter might be further inhibited by the high Ca²⁺ concentrations found at the ER/mitochondria microdomain. Prolonged exposures to high Ca²⁺ concentrations might desensitize the uniporter, as exposures to low Ca²⁺ concentrations are needed to reset the uniporter into rapid uptake mode, its most efficient mode of Ca²⁺ uptake (44). Furthermore, the mitochondria Ca²⁺ uptake sites have been shown to be already close to saturation during physiological stimulations (6, 37), suggesting that exposure of mitochondria to higher Ca²⁺ microdomains might not translate into higher [Ca²⁺]_mit responses.

This mechanism might account for the preserved amplitude of the peak [Ca²⁺]_mit response in calreticulin overexpressers, despite the larger release of Ca²⁺ from the ER. The increased ER Ca²⁺ pumping activity of calreticulin overexpressers (Fig. 2) might further contribute to the abnormal [Ca²⁺]_mit response, by dissipating more efficiently the local Ca²⁺ microdomain surrounding mitochondria. Because of its slow kinetics, the increased ER Ca²⁺ pumping is not likely to affect the peak [Ca²⁺]_mit but might contribute to the faster decay of the [Ca²⁺]_mit response by removing more efficiently the Ca²⁺ released by mitochondria (11). Thus, several mechanisms might account for the abnormal [Ca²⁺]_mit response observed in calreticulin-induced cells, including a decrease in mitochondrial membrane potential, an inhibition of the Ca²⁺ uniporter, together with an increased Ca²⁺ uptake and release from the ER. A causal link between the increased ER Ca²⁺ release and mitochondrial depolarization might even be postulated, as mitochondria are likely to be damaged by the chronic exposure to high Ca²⁺ concentrations.

These perturbations of Ca²⁺ homeostasis are unlikely due to the chaperone function of calreticulin, as impaired ER and mitochondrial Ca²⁺ responses were observed in cells induced to express a truncated calreticulin lacking the chaperone N-domain of the protein (Fig. 8). Most importantly, the overexpression of calnexin, an ER chaperone similar to calreticulin and containing a chaperoning P-domain, did not affect cytosolic or ER Ca²⁺ homeostasis. This indicates that the effects do not require either the N- or the P-domain but are mediated by the unique C-domain of calreticulin. Thus, alterations in Ca²⁺ sensing, rather that in chaperone activity, are responsible for the increased Ca²⁺ pumping and release activity, which lead to higher Ca²⁺ turnover between the ER and mitochondria. These findings have important physiological implications because different levels of calreticulin are expressed in different tissues (28). Furthermore, expression of the protein is up-regulated under the conditions of stress and starvation (28). In the immune system, the CRT gene is activated in stimulated cytotoxic T-cells (45) where it may play a role in a Ca²⁺-dependent signaling and/or cytotoxic T-cell killing. In many cancer cells, including prostate cancer, calreticulin expression is increased or up-regulated by different steroids (46, 47). Expression of calreticulin is also differentially regulated during development (48). Because the Ca²⁺ signals of multiple cellular compartments are differentially modulated by calreticulin, changes in calreticulin expression levels might define specific patterns of cellular Ca²⁺ responses in these cell types. By allowing the ER to take up, store, and release more Ca²⁺, an increase in calreticulin might "arm" the cellular Ca²⁺ signaling machinery, thereby allowing previously "silent" cells to generate Ca²⁺ signals. In the long run, however, an increase in calreticulin appears to be deleterious for the cell, despite the reduction in Ca²⁺ influx that compensates for the increased Ca²⁺ release from the ER. The high Ca²⁺ microdomains around mitochondria, which might in the short term increase mitochondrial metabolism, might in the long run damage and depolarize mitochondria. This defective signaling might account for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.

	ACKNOWLEDGEMENTS

We thank Dr. Tullio Pozzan, Padua, for the kind gift of the mitochondria-targeted red fluorescent protein, and Drs. R. Y. Tsien and A. Miyawaki for providing the yellow cameleon constructs.

	FOOTNOTES

^* This work was supported by NSF Grants 31-46859.96 and 31-56802.99 from the Swiss National Science Foundation, the Canadian Institutes of Health Research, and the Heart and Stroke Foundation of Alberta.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Canadian Institutes of Health Research Senior Investigator and an Alberta Heritage Foundation for Medical Research Medical Scientist.

Fellow of the Dr. Max Cloetta Foundation. To whom correspondence should be addressed: Dept. of Physiology, University of Geneva Medical Center 1, Michel-Servet CH-1211 Geneva 4, Switzerland. Tel.: 4122-702-5399; Fax: 4122-702-5402; E-mail: Nicolas.Demaurex@medecine.unige.ch.

Published, JBC Papers in Press, September 24, 2002, DOI 10.1074/jbc.M202395200

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; Dox, doxycycline; InsP₃, inositol 1,4,5-trisphosphate; SOC, store-operated Ca²⁺ influx; SERCA, sarco(endo)plasmic reticulum Ca²⁺ transport ATPase, [Ca²⁺]_cyt, [Ca²⁺]_ER, and [Ca²⁺]_mit, cytosolic, ER, and mitochondria-free Ca²⁺ concentration, respectively; CCh, carbachol; YC, yellow cameleon; DsRed, Red fluorescent protein from Discosoma sp.; TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; Tg, thapsigargin; Tet, tetracycline; HA, hemagglutinin; TMRM, tetramethylrhodamine methyl ester; CRT, calreticulin; HEDTA, N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES