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Passive calcium leak via translocon is a first step for iPLA₂-pathway regulated store operated channels activation

INSERM U800, Villeneuve d’Ascq, France; Université Lille I, Villeneuve d’Ascq, France; and Equipe Labellisee Ligue Nationale Contre le Cancer, Villeneuve d’Ascq, France

¹Correspondence: Laboratoire de Physiologie Cellulaire, INSERM U800, Bâtiment SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France. E-mail: flourakismatt{at}yahoo.fr; fabien.vancoppenolle{at}univ-lille1.fr; natacha.prevarskaya{at}univ-lille1.fr

ABSTRACT

Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiopathology. One of the most enigmatic mechanisms responsible for Ca²⁺ concentration in the ER is passive calcium leak. Previous studies have shown that the translocon complex is permeable to calcium. However, the involvement of the translocon in the passive calcium leak has not been directly demonstrated. Furthermore, the question whether the passive store depletion via the translocon could activate SOC (store operated channels) replenishing the ER, remains still unresolved. In this study, for the first time, we show that thapsigargin and calcium chelators deplete ER via translocon. Indeed, using confocal imaging, we demonstrate that when the number of opened translocons was lowered neither thapsigargin nor calcium chelators could induce ER store depletion. We also demonstrate that calcium leakage occurring via the translocon activates store-operated current, which is, by its kinetic and pharmacology, similar to that evoked by thapsigargin and EGTA (but not IP3), thus highlighting our hypothesis that calcium leakage via the translocon is a first step for activation of the specific iPLA₂-regulated SOC. As the translocon is present in yeast and mammalian cells, our findings suggest that translocon-related calcium signaling is a common phenomenon.—Flourakis, M., Van Coppenolle, F., Lehen’kyi, V., Beck, B., Skryma, R., Prevarskaya, N. Passive calcium leak via translocon is a first step for iPLA₂-pathway regulated store operated channels activation.

Key Words: calcium leak • store-operated channels • translocon • iPLA₂ • calcium homeostasis

THE ENDOPLASMIC RETICULUM (ER) is the largest calcium store in all types of cells, playing a major role in Ca²⁺ signaling and cell physiopathology (1 , 2) . Tight regulation of ER lumen Ca²⁺ concentration is essential for protein folding and maturation (3 , 4) .

Under resting conditions, ER Ca²⁺ concentration results from a balance between the activity of SERCA pumps (sarcoplasmic and ER calcium ATP-ases), which import calcium into the ER lumen and calcium leak that balances the influx created by the pumps. This balance between Ca²⁺ uptake and Ca²⁺ leakage appears to be a common property of Ca²⁺-stores. However, if the SERCA pumps are now extensively studied, the passive calcium leak remains the most enigmatic of the processes involved in regulation of calcium homeostasis.

The main experimental way to study this passive calcium leak consists in pharmacological artificial shifting the ER calcium balance toward calcium leakage due to either suppression of ER Ca²⁺ uptake (classical SERCA pump inhibitor thapsigargin), or to excessive cytosolic Ca²⁺ buffering (calcium chelators as EGTA). Indeed, these commonly used drugs induce calcium release from the ER. Nevertheless, the mechanism responsible for this type of calcium leak and its molecular counterpart are still ill-defined and remain for a long time one of the most intriguing question of cell biology.

The studies of Wonderlin’s group have shown that the permeability of the ER is dynamically coupled to protein synthesis and that polarized molecules could cross the ER membrane through the translocon, the complex implicated in protein translocation during translation (5 , 6) . Indeed, two previous studies of Lomax et al. (7) and of our group (8) have demonstrated that the translocon complex (opened by puromycin) is permeable to calcium and, therefore, the authors suggested that translocon could potentially play a role in calcium leakage. However, the involvement of the translocon in the passive calcium leak due to thapsigargin and calcium chelators has never been directly demonstrated.

Furthermore, it is well established that ER depletion could activate store-operated Ca²⁺ entry (SOCE) (9) . This process triggers an increase of cytoplasmic Ca²⁺ concentration, in order to replenish internal Ca²⁺ stores. Indeed, previous studies undertaken by our laboratory have put forward the hypothesis of the coexistence in the single cell type of two functionally distinct types of store-operated channels (SOC), depending on the mode of store depletion (10) : (i) SOC_CC (conformational coupling), activating by IP₃ and involving "active" dynamic protein–protein interaction between IP₃ receptors and SOC, and (ii) SOC_CIF (calcium influx factor) activating by iPLA₂-regulated signaling stimulated following passive leakage from intracellular stores (with thapsigargin and BAPTA) (11 , 12) . The alternative terminology for these distinct classes of Ca²⁺-conducting channels has later been proposed by Bolotina (13) : SOCs, which are activated by depletion of Ca²⁺ stores through CIF-iPLA₂ pathway, and IP₃ROCs, which are activated by IP₃R through a direct coupling mechanism. However, it is important to note that the preferential mode of SOC activation may also depend on the degree of ER compartmentalization due to existence of specialized ER subregions involved in different SOC-controlling signaling pathways (14 , 15) . Considering this as well as a potential physiological role of translocon in ER depletion, in the present study we asked whether or not the store depletion via the translocon could activate SOCE and, if so, what would be the type of SOC activated and the mechanism of "translocon-to-SOC" coupling.

Hence, for the first time, we demonstrate directly that thapsigargin and EGTA deplete ER via open translocon complex. Furthermore, by electrophysiological and calcium imaging techniques, we demonstrate that calcium leakage occurring via the translocon can activate SOC_CIF current (but not SOCcc current!), which, by its kinetic and pharmacology is similar to SOC current induced by thapsigargin and EGTA (but not by IP₃), thus highlighting our hypothesis that the translocon-mediated calcium leakage is a first step for SOC_CIF activation. Moreover, since our results show that SOC current mediated by translocon is inhibited by specific iPLA₂ inhibitors, this work also hints to a new Ca²⁺ pathway with a crucial role in Ca²⁺ homeostasis.

MATERIALS AND METHODS

Cell Culture
LNCaP cells from the American Type Culture Collection (Manassas, VA) were cultured in RPMI 1640 medium (Life Technologies, Inc., Fontenay sous Bois, France) supplemented with 5 mM L-glutamine (Life Technologies, Inc., L’Isle d’Abeau, France), 10% FBS (Seromed, Poly-Labo, Strasbourg, France) and 1% kanamycin (Life Technologies, Inc., L’Isle d’Abeau, France). Cells were routinely grown in 50 ml flasks (Nunc, Poly-labo) and kept at 37°C in a humidified incubator in an air/CO₂ (95/5%) atmosphere. For Ca²⁺ imaging experiments, the cells were subcultured in Petri dishes (Nunc) and used after 3–6 d.

Calcium imaging
[Ca²⁺]_in was measured by using ratiometric dye Fura-2 and quantified according to Grynkiewicz and Tsien formula (25) . The extracellular solution contained: NaCl-120, KCl-6, CaCl₂-2, MgCl₂-2, HEPES-10, and glucose (Glc)-12. For Ca²⁺-free HBSS, CaCl₂ was removed and EGTA (0.5 mM) was added.

Calcium imaging within the ER
LNCaP cells were grown on glass coverslips and loaded with 5 µM of Mag-Fluo 4 acetoxymethyl ester (AM) (Molecular Probes, Leiden, The Netherlands), for 45 min at 37°C. After incubation with the dye, the plasma membrane was selectively permeabilized: cells were rinsed briefly in a high K⁺ solution of the following composition (in mM): KCl-125, NaCl-25, HEPES-10, EGTA-1, CaCl₂-0.5, and MgCl₂-0.1 (free Ca²⁺ clamped to 170 nM, pH 7.2) and exposed for 1 min to the same solution at 37°C in presence of digitonin (0.5 mg/ml). Permeabilized cells were then continuously perfused with the high-K⁺ solution supplemented with 0.2 mM Mg-ATP. Ratio imaging measurements of Mag-Fluo 4 were made using a confocal microscope (LSM 510, Zeiss, Le Pecq, France).

Patch clamp recording
Membrane currents in LNCaP cells were recorded in the whole-cell configuration using the patch-clamp technique and also by using a computer-controlled EPC-9 amplifier (HEKA Electronic, Germany) as described previously (10) Patch pipettes were made on a P-97 puller (Sutter, Novato, CA) from borosilicate glass capillaries (WPI, Sarasota, FL). Extracellular solution used to record Ca²⁺-carried I_SOC contained (in mM): 120 NaCl, 5 KCl, 10 CaCl₂, 2 MgCl₂, 5 Glc, 10 HEPES (pH adjusted to 7.3 with TEA OH). The pipettes were filled with the basic intracellular pipette solution (in mM): 120 Cs Methane sulfonate, 10 CsCl, 10 HEPES, 10 BAPTA, 6 MgCl₂ (pH adjusted to 7.2 with CsOH).

Barium imaging
The rate of Ba²⁺ increases was measured using Ca²⁺ dye Fluo-4 AM. Ratio imaging measurements of Fluo 4 were made using a confocal microscope (LSM 510, Zeiss, Le Pecq, France). The extracellular solution contained: NaCl-120, KCl-6, CaCl₂-2, MgCl₂-2, HEPES-10, and Glc-12 and BaCl-2 was added when necessary.

Western blot analysis
Subconfluent LNCaP cells were treated with an ice-cold lysis buffer containing: 10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 10 mM MgCl; 1 mM PMSF; 1% Nonidet P-40; and protease inhibitor cocktail from Sigma (l’Isle d’Abeau, France). The lysates were centrifuged 15,000 g at 4°C for 20 min, mixed with a sample buffer containing: 125 mM Tris-HCl, pH 6.8; 4% SDS; 5% ß-mercaptoethanol; 20% glycerol; 0.01% bromphenol blue. Total protein samples were subjected to 8–10% SDS-PAGE and transferred to a nitrocellulose membrane by semidry Western blotting (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked in a 5% milk containing TNT buffer (Tris-HCl, pH 7.5; 140 mM NaCl; and 0.05% Tween 20) overnight then probed using specific rabbit polyclonal anti-ß iPLA2 (Calbiochem) and anti-ß-actin (Lab Vision Co., Fremont, CA) antibodies. The bands on the membrane were visualized using enhanced chemiluminescence method (Pierce Biotechnologies Inc., San Francisco, CA).

Reagents and chemicals
All chemicals were purchased from Sigma except Fura-2-acetoxymethyl ester (Euromedex, France). rBEL and sBEL were generously provided by Dr. Victoria Bolotina (Boston University School of Medicine).

Data analysis and statistics
Each experiment was repeated several times. Data were analyzed by using PulseFit (HEKA Electronics, Germany) and Origin 7.0 (Microcal Software Inc., Northampton, MA). Results were expressed as mean ± SEM.

RESULTS

Involvement of the translocon in calcium leak from the ER evoked by thapsigargin and EGTA
To study the possible role of translocon in calcium leak, we first used thapsigargin, a classic SERCA pump inhibitor. We measured, in a Ca²⁺-free medium, the passive calcium leak using Fura-2 (2 µM) as a calcium cytoplasmic dye. Figure 1 A shows the time course of a representative thapsigargin (1 µM) response in a calcium-free medium. Under control conditions, the cytoplasmic calcium concentration ([Ca²⁺]_c) peak was 235.5 +/– 8.85 nM (n=150). The cells were treated with anisomycin, a peptidyl-transferase inhibitor (16) , to reduce the number of opened translocons (8) . In anisomycin-treated cells (200 µM, 1 h), the [Ca²⁺]_c peak decreased by 36%, as compared with control conditions (n=150). Figure 1B illustrates the peak values of [Ca²⁺]_c increase evoked by thapsigargin (100 nM, 1 and 10 µM) in anisomycin-pretreated cells and in control cells. Figure 1C shows quantification of Ca²⁺ leakage, taken from Fura-2 measurements. Anisomycin pretreatment resulted in a significant decrease of the rate of calcium leakage from the ER (4.05±0.4 nM/s in control conditions (n=150) and 1.03 ± 0.1 nM/s in anisomycin treated cells (n=150)). Peak values of the calcium leakage rate induced by TG (1 µM and 100 nM) under control conditions and in anisomycin-treated cells are shown in Figure 1D .

View larger version (22K): [in this window] [in a new window]   Figure 1. Anisomycin treatment reduces thapsigargin-induced calcium release from the ER. A) Typical [Ca2+]c traces in response to 1 µM thapsigargin under control conditions and after 1-h incubation with 200 µM anisomycin. All measurements were made at room temperature in a Ca2+-free HBSS. B) Cumulative data (mean±SEM) of peak [Ca2+]c increases evoked by thapsigargin (100 nM, 1 µM, 10 µM) responses under control conditions (n=150) and with anisomycin 200 µM (n=150). C) The apparent Ca2+ leak rate is plotted as a function of time after application of thapsigargin (1 µM). D) Cumulative data (mean±SEM) of the peak Ca2+ leak rate evoked by thapsigargin (100 nM, 1 µM, 10 µM) responses under control conditions (n=150) and with anisomycin 200 µM (n=150).

To study translocon-related variations in the ER specifically, we performed further investigations using the compartmentalized fluorescent Ca²⁺ indicator Mag fluo 4 on cells that had been permeabilized using digitonin treatment. In this series of experiments, we used various drugs that are known to induce passive Ca²⁺ leakage such as thapsigargin, EGTA, ionomycin, and also IP₃, which induces active calcium release specifically via IP₃R. Puromycin (200 µM), thapsigargin (1 µM), and EGTA (1 mM) induced a decrease in [Ca²⁺]_ER [respectively 30±2.3% (n=35), 29.6±3.1% (n=32), 52.65±3.2% (n=68)] (see Fig. 2 A–C). After an anisomycin pretreatment, the release was only 5 ± 2.5% (n=53), 12.3 ± 0.766% (n=40), and 28.88 ± 2.11% (n=43) for puromycin-evoked, thapsigargin-evoked, and EGTA-evoked Ca²⁺ release, respectively. Futhermore, we used ionomycin (1 µM), which induces a nonspecific calcium store depletion of 72.5 ± 3.53%, a decrease in [Ca²⁺]_ER (n=30). This Ca²⁺ calcium release was not inhibited by anisomycin pretreatment (74.366±1.458%) (n=62) (see Fig. 2D ). Finally, IP₃, which induces a calcium-store depletion independent of passive calcium leakage, induced a 12.4 ± 1.4% decrease in [Ca²⁺]_ER (n=17). This release was not inhibited by anisomycin pretreatment (12.8±2.1%) (n=34) (figure 2E) . Moreover, puromycin had a cumulative effect after IP₃-induced calcium release, which was not observed in anisomycin-pretreated cells. Figure 2F illustrates the peak values of [Ca²⁺]_ER depletion evoked by puromycin 200 µM, thapsigargin 1 µM, EGTA 1 mM, ionomycin 1µM, and IP₃ 100 µM under control conditions and in anisomycin-treated cells. Taken together, these results suggest that the translocon is the main channel through which Ca²⁺ passive leakage occurs in cancer prostate epithelial cells.

View larger version (21K): [in this window] [in a new window]   Figure 2. Anisomycin treatment reduces leak channel activity in the ER membrane. Typical traces of the estimate of passive Ca2+ leak in digitotin-permeabilized cells with the use of puromycin 200 µM (A), thapsigargin 1µM (B), EGTA 1 mM (C), ionomycin 1 µM (D), IP3 100µM (E), under control conditions (plotted as black squares), and after 1-h incubation with 200 µM anisomycin (plotted as open circles). F) Cumulative data (mean±SEM) for % release from internal Ca2+ stores.

Ca²⁺ leakage through translocon activates SOCE
To further explore the putative role of translocon in Ca²⁺ homeostasis, it was also potentially rewarding to examine whether passive store depletion via the translocon could activate SOCE through SOC (store-operated channels). In complete agreement with our previous studies (10) store-operated current (I_SOC) could be activated either passively (by TG-mediated store depletion in response to extracellular application of 0.1 µM TG or by excessive cytosolic Ca²⁺ buffering with EGTA) or actively by inclusion of 100 µM IP₃ in the intracellular pipette solution. In our experiments, lowering the number of opened translocon by anisomycin resulted in a decrease of the SOC current (induced by TG and EGTA) density evaluated during whole-cell recording. The typical time courses of the I_SOC and the corresponding current-potential (I-V) relations recorded from control cells and cells treated with anisomycin 200 µM for 1 h are shown in Fig. 3A-C and a summary of the data gathered is laid out in Fig. 3 D. Anisomycin pretreatment reduced both EGTA-evoked and thapsigargin-evoked I_SOC. For EGTA: 1.05 ± 0.18 pA/pF under control condition (n=13) and 0.36 ± 0.2 pA/pF after anisomycin pretreatment (n=11). For thapsigargin, 0.73 ± 0.08 pA/pF (n=4) under control conditions and 0.22 ± 0.182 pA/pF after anisomycin pretreatment (n=6), see Fig. 3A, B, and D . The same pretreatment was unable to reduce the IP₃(100 µM)-evoked I_SOC (0.93±0.178 pA/pF under control conditions (n=14) and 0.8±0.27 pA/pF after anisomycin pretreatment (n=15)), see Fig. 3C and D . Puromycin was also able to activate a small I_SOC (0.2 pA/pF), however, this trend was hardly monitored (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 3. Ca2+ passive leak via translocon activates ISOC. Representative time courses of the whole cell ISOC in LNCaP cells evoked by EGTA 10 mM (A), thapsigargin 1µM (B), and IP3 100 µM (C) under control conditions (plotted in black squares) and after anisomycin pretreatment (open circles). D) Cumulative data (mean±SEM) of maximal current density under control conditions, or with anisomycin 200 µM pretreatment.

iPLA₂ activity is required for SOC activation evoked by passive Ca²⁺ leakage occurring through the translocon
Recent studies have shown the involvement of the Ca²⁺-independent phospholipase A2 (iPLA₂ ß) (11) in the mechanism of SOC activation by passive Ca²⁺ leakage. Therefore, using a specific inhibitor of iPLA₂ ß (sBEL), we have studied further the potential involvement of this enzyme in the I_SOC activated following the store depletion via translocon. We first detected iPLA₂ ß by Western blotting in epithelial cancer cell line (LNCaP cells) (see Fig. 4 A). Figure 4B represents the typical time course of EGTA (10 mM)-evoked I_SOC (1.32±0.19 pA/pF; n=12). I_SOC was inhibited by BEL (25 µM) and sBEL (10 µM), the active enantiomere on iPLA₂ ß (respectively, 0.32±0.11 pA/pF; n=7 and 0.34±0.17 pA/pF; n=6). rBEL (10µM) and the inactive enantiomere on iPLA₂ ß (1.43±0.10 pA/pF; n=7) was without effect. We also checked whether BEL inhibits the IP₃-evoked I_SOC. Figure 4C represents the typical time course of the I_SOC evoked by IP₃ (1.54±0.4 pA/pF; n=7). BEL treatment resulted in a small decrease of IP₃-induced I_SOC (1.1±0.2 pA/pF; n=9). Nevertheless, the observed decrease was statistically nonsignificant. A summary of the data gathered is presented in Fig. 4D . These results hint to the involvement of iPLA₂ ß in SOC activation by Ca²⁺ passive leakage.

View larger version (21K): [in this window] [in a new window]   Figure 4. ISOC evoked by passive calcium leak via translocon is iPLA2-dependent. A) Western blot detection of iPLA2ß in LNCaP cells. B, C) Representative time courses of the whole cell ISOC in LNCaP cells evoked by EGTA 10 mM or IP3 100 µM under control conditions and after BEL treatment. D) Cumulative data (mean±SEM) for maximal current density.

We also investigated whether Ca²⁺ passive leakage occurring specifically through the translocon could activate the SOC current via iPLA₂ activation. As previously noted, puromycin activates a very small I_SOC, which was hardly monitored. To avoid this problem, we measured the SOCE using barium imaging with Fluo 4 (5 µM) as a Ca²⁺ cytoplasmic dye. As shown in Fig. 5 A, puromycin induced a rise of [Ba²⁺]_in, which was absent in a barium free solution. In anisomycin-pretreated cells, this increase was inhibited by 76.7 ± 5.3% (n=43), in BEL-pretreated cells the same increase was reduced by 90.3 ± 1.4% (n=47). Thapsigargin induced a similar rise of [Ba²⁺]_in, see Fig. 5C . This increase was reduced in a barium-free solution (this was due to thapsigargin-evoked ER depletion). The thapsigargin-evoked SOCE was reduced in anisomycin-pretreated and BEL-pretreated cells [respectively, 45.1 ± 5.3% (n=74) and 88.3 ± 1.4% (n=49)]. A summary of this data is presented in Fig. 5E . These results clearly indicate that, even if puromycin-evoked I_SOC is very small, calcium leakage occurring specifically via the translocon activates iPLA2-regulated SOCE.

View larger version (24K): [in this window] [in a new window]   Figure 5. Puromycin- and thapsigargin-evoked calcium leakage activates SOCE. Typical [Ba2+]c traces in response to puromycin 200 µM (A) and thapsigargin 1 µM (B) under control conditions and after pretreatment with 200 µM anisomycin or BEL 25 µM (C, D). E) Cumulative data (mean±SEM) for the normalized rate of barium increase.

DISCUSSION

In the present work, we have demonstrated that the translocon is the main Ca²⁺ leak channel in epithelial prostate cancer cells and that physiological passive store depletion through the translocon could activate SOC via an iPLA₂ ß-dependent mechanism.

The mammalian translocon complex contains several subunits (Sec61, Sec61ß, Sec61, TRAM, and Bip) and numerous associated proteins such as SP, calnexin, and SRP (17) . The stoechiometry of the translocon (especially the pore structure) is still unknown (18) . In physiological conditions, the pore diameter of the ribosome-free translocon is 0.9 to 1.5 nM (19) . During translation, in ribosome-bound conditions, the pore aperture has a theoretical diameter of between 4 and 6 nM (20) . This pore is the largest one in the ER’s membrane, and it has been suggested that it is involved in Ca²⁺ leakage (21) .

To study the passive calcium leak through the translocon, our experimental approach was based on the pharmacological modulation of the translocon’s open state using two antibiotics: puromycin (a translation inhibitor, which specifically releases polypeptide chains, leaving the translocon open) and anisomycin (an inhibitor of peptidyl-transferase, leaving the translocon closed) (8) . It is important to note that, given the crucial role of the translocon in translation and the number of translocon-constitutive subunits, such classical approaches as the employment of siRNA (or antisens) to abolish the expression of at least one subunit of the translocon, such as Sec61 or overexpressing the whole translocon complex in lipid bilayers, were not deemed appropriate. Therefore, our experimental approach is currently the only one that enables decreases in [Ca²⁺]_ER, which occur through the translocon to be accurately measured in living cells.

One of the protocols we used to check the involvement of the translocon in passive store depletion consisted in applying thapsigargin to inhibit Ca²⁺ reuptake in the ER. This drug is commonly used to inhibit SERCA pumps. During thapsigargin application, calcium leaks from the ER to the cytoplasm. However, the exact mechanism by which ER depletion occurs after thapsigargin has been applied remains unclear.

Thus, in the present study, we measured the effects of exposure to anisomycin over a long time period (200 µM, 1 h) on thapsigargin-evoked Ca²⁺ release from the ER. Anisomycin pretreatment of LNCaP cells (loaded with Fura-2) induced a significant decrease in the amplitude and rate of thapsigargin response, compared with control (Fig. 1) . We obtained similar results with human prostate primary cultures (data not shown in this paper). In a previous study we showed that anisomycin treatment reduced the coefficient of colocalization between Sec61 and the ribosome (8) . This implies that the number of opened translocon and consequently of Ca²⁺ leak channels is lowered after anisomycin treatment. Thus, the effect of anisomycin on thapsigargin-induced Ca²⁺ release is due to a decrease in the number of ribosome-bound translocon complexes, which are involved in Ca²⁺ leakage.

Another protocol we used to observe Ca²⁺ leakage from the ER was to use mag-fluo 4 to measure directly the changes in luminal Ca²⁺ release induced by thapsigargin (Fig. 2) . Under the experimental conditions we put in place, permeabilized LNCaP cells were perfused with an "internal medium" with the same ATP concentration throughout the experiment (100 µM), in order to avoid Ca²⁺ leakage evoked by ATP (22) . Puromycin, thapsigargin, and EGTA induced a decrease in [Ca²⁺]_ER, but this decrease was greatly reduced after anisomycin pretreatment. Anisomycin treatment did not fully abolish the thapsigargin- and EGTA-evoked Ca²⁺ release. This may indicate that other proteins could be involved in association with the translocon in Ca²⁺ leak. Taken together, these results hint to the considerable role of the translocon in ER passive store depletion but not in active store depletion.

It is well established that in physiological conditions ER permeability is coupled to protein synthesis (19) : the Ca²⁺ leaks through the pore of the translocon after release of the nascent protein (8) . Furthermore, an interesting study undertaken by Potter and Nicchitta (23) demonstrated that the ribosome stays on the translocon after translation. So, at this juncture, Ca²⁺-release through the translocon probably occurs and could be a way for the cell to regulate [Ca²⁺]_ER. It is also known that the translocon is involved in ER-associated degradation (ERAD) during ER stress (24) . Therefore, Ca²⁺ release via the translocon may also occur during ER stress.

To further explore the putative role of translocon in Ca²⁺ homeostasis, it was potentially rewarding to examine whether passive store depletion via the translocon could activate SOC. In our experiments, lowering the number of opened translocon using anisomycin led to a decrease in EGTA- and thapsigargin-evoked ER depletion. This was consistent with the decrease in SOCE density evaluated during whole-cell recording (see Fig. 3 ). The same pretreatment does not inhibit IP₃-evoked I_SOC. Intriguingly, in our experiments, puromycin induced very small I_SOC, which could be barely monitored. This could be due to the fact that the Ca²⁺, which is released by the translocon, may have been directly reuptaken into the ER by SERCA pumps or by mitochondria. To avoid such regulation, we used a classical approach for SOCE investigation with barium as carried ion (Fig. 5) . In these experiments, puromycin (like thapsigargin) could induce a capacitative barium entry due to SOC activation. Taken together, these results show that ER store depletion occurring through the translocon activates SOC in prostatic cancer epithelial cells.

Finally, we were interested in determining the mechanism of SOC activation after Ca²⁺ leak through the translocon. A recent work has put forward the iPLA₂ as an intermediate molecule between passive store depletion and SOC activation (11) . According to this hypothesis, dissociation of the complex calmodulin (CaM)-iPLA₂ by the CIF activates SOC. Indeed, the constitutive complex CaM-iPLA₂ inhibits iPLA₂ activity (11) , but when CIF dissociates CaM from iPLA₂, it triggers production of lysophospholipids, such as lysophosphatidycholine (LPC) and lysophosphatidylinositol (LPI), which directly activate SOC_CIF. In the present work, we have demonstrated, using a specific inhibitor of iPLA₂ (sBEL), that the activation of SOC_CIF evoked by Ca²⁺ leakage occurring via the translocon is iPLA₂ß dependent (Fig. 4) .

In summary, as shown in Fig. 6 , we have demonstrated that the passive calcium leak translocon channel mediates the thapsigargin- and EGTA-induced calcium release. We have also shown that calcium release that occurs via the translocon activates store-operated calcium current. This is the first characterization of SOCE triggered by a passive calcium leak channel with identified molecular structure in the ER. Our results also hint to a new Ca²⁺ pathway with a crucial role in Ca²⁺ homeostasis: Ca²⁺ leakage occurring through the translocon may induce CIF formation and/or CIF release from the ER, which dissociates CaM from iPLA2ß and so leads to the production of lysophospholipids and to SOC_CIF activation.

View larger version (71K): [in this window] [in a new window]   Figure 6. Speculative model for SOCCIF activation after calcium leak via the translocon. This activation occurs after passive ER depletion facilitated by thapsigargin or EGTA. On such depletion, CIF could be released from the ER and could stimulate membrane-bound Ca2+-independent phospholipase A2 (iPLA2), which in turn activates SOCCIF after production of lysophospholipids. In contrast, IP3R stimulation would directly activate SOCCC (IP3ROC) via protein–protein interaction. This latter pathway seems to be independent of Ca2+ leakage that occurs through the translocon. (ERM: ER membrane; PM: plasma membrane).

As the translocon complex is present in yeast and mammalian cells, our findings suggest that Ca²⁺ homeostasis using translocon pathways is a common phenomenon.

ACKNOWLEDGMENTS

This work was supported by the INSERM (INSERM), the Région Nord-Pas de Calais (regional government in the northern-most part of France), the Ligue Nationale contre le Cancer (the National Anti-Cancer League) and the Fondation pour la Recherche Médicale (FRM, the Foundation for Medical Research).

FOOTNOTES

² These authors equally contributed to this work.

Received for publication October 18, 2005. Accepted for publication January 17, 2006.

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