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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5254fjev1 20/8/1215    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Flourakis, M. Articles by Prevarskaya, N. Search for Related Content PubMed PubMed Citation Articles by Flourakis, M. Articles by Prevarskaya, N.

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@yahoo.fr; fabien.vancoppenolle@univ-lille1.fr; natacha.prevarskaya{at}univ-lille1.fr

SPECIFIC AIM

The aim of this study was to explore the role of the translocon complex [present on the endoplasmic reticulum (ER) membrane and involved in protein translocation during translation] in the activation of SOCE (store operated calcium entry). We addressed the hypothesis about a possible functional link between the passive leakage via the translocon and SOCE stimulation.

PRINCIPAL FINDINGS

1. Thapsigargin and EGTA deplete ER calcium stores via translocon
Thapsigargin and EGTA are the commonly used drugs that induce calcium release from the ER. Nevertheless, the mechanism and the molecular counterpart of this calcium leak are still ill-defined.

Using confocal calcium imaging, we have demonstrated that by lowering the number of opened translocons by anisomycin (an inhibitor of peptidyl-transferase, leaving the translocon closed) neither thapsigargin nor calcium chelator EGTA could induce calcium release from the ER. However, the inhibition of the calcium leak via the translocon by anisomycin did not reduce the IP₃-evoked ER store depletion.

These results suggest that the translocon is the main ER channel, which mediates passive Ca²⁺ leak induced by thapsigargin and calcium chelators.

2. 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).

Previous studies undertaken by our laboratory have put forward the hypothesis of the coexistence of two functionally distinct types of SOC, depending on the mode of store depletion: (i) SOC_CC (conformational coupling), involving "active" dynamic protein–protein interaction between IP₃ receptors and SOC, and (ii) SOC_CIF (calcium influx factor) activated by iPLA₂-regulated signaling stimulated following passive leakage from intracellular stores.

In this study, we tried to determine whether ER store depletion via translocon could activate SOCE and what would be the mechanism of "translocon-to-SOC" coupling.

In our experiments, lowering the number of opened translocon by anisomycin resulted in a decrease of the SOC current density evaluated during whole-cell recording. Anisomycin pretreatment reduced both EGTA-evoked I_SOC (1.05±0.18 pA/pF under control condition (n=13) and 0.36±0.2 pA/pF after anisomycin pretreatment (n=11)) and thapsigargin-evoked I_SOC [0.73±0.08 pA/pF (n=4) under control conditions and 0.22±0.182 pA/pF after anisomycin pretreatment (n=6)] (Fig. 1 A, B, and D). The same pretreatment was unable to reduce the IP₃-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), (Fig. 1C and D ).

View larger version (35K): [in this window] [in a new window]   Figure 1. 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.

3. iPLA₂ activity is required for SOCE stimulation by passive Ca²⁺ leakage via the translocon
We also investigated whether Ca²⁺ passive leakage occurring specifically through the translocon could activate the SOC current via iPLA₂ activation. We measured the SOCE using barium imaging with Fluo 4 (5 µM) as a Ca²⁺ cytoplasmic dye. As shown in Fig. 2 , puromycin induced a rise of [Ba²⁺]_in, which was absent in a barium-free solution (Fig. 2A ). In anisomycin-pretreated cells, this increase was inhibited by 76.7 ± 5.3% (n=43), however, in BEL-pretreated cells (a specific iPLA₂ inhibitor) the same increase was reduced by 90.3±1.4% (n=47) (Fig. 2B ). Thapsigargin induced an increase similar to that evoked from puromycin in [Ba²⁺]_in (Fig. 2C ). This thapsigargin-evoked SOCE was also reduced in anisomycin-pretreated and BEL-pretreated cells (respectively, 45.1 ± 5.3% (n=74) and 88.3 ± 1.4% (n=49), Fig. 2D ). A summary of this data is presented in Fig. 2E . The same pretreatment could not reduce the IP₃(100 µM)-evoked SOCE. These results were confirmed by patch-clamp experiments (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 2. 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.

The results of this study show that calcium leakage occurring via the translocon could activate SOC_CIF current (but not SOCcc current), thus highlighting our hypothesis that calcium leakage occurring via the translocon is a first step for SOC_CIF activation.

CONCLUSION AND SIGNIFICANCE

In summary, for the first time, 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. Figure 3 demonstrates a "translocon–SOC activation" hypothesis consistent with our data. 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 calmodulin from iPLA2ßbeta; and so leads to the production of lysophospholipids and to SOC_CIF activation.

View larger version (59K): [in this window] [in a new window]   Figure 3. 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.

FOOTNOTES

² These authors equally contributed to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5254fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5254fjev1 20/8/1215    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Flourakis, M. Articles by Prevarskaya, N. Search for Related Content PubMed PubMed Citation Articles by Flourakis, M. Articles by Prevarskaya, N.