HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 294/6/C1323    most recent 00071.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map 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 Google Scholar Google Scholar Articles by Woodard, G. E. Articles by Rosado, J. A. Search for Related Content PubMed PubMed Citation Articles by Woodard, G. E. Articles by Rosado, J. A.

PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca²⁺ store depletion

¹National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and ²Department of Physiology (Cell Physiology Research Group), University of Extremadura, Cáceres, Spain

Submitted 11 February 2008 ; accepted in final form 8 April 2008

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Ca⁺ release-activated Ca²⁺ (CRAC) channels are activated when free Ca²⁺ concentration in the intracellular stores is substantially reduced and mediate sustained Ca²⁺ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca²⁺ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca²⁺-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca²⁺ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca²⁺ chelator that prevented TG-evoked cytosolic free Ca²⁺ concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca²⁺ entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca²⁺ entry.

Orai1; synaptosomal-associated protein-25; Ca²⁺ entry

Intracellular protein trafficking plays a relevant role in the activation of Ca²⁺ entry. Following store depletion, STIM1 has been reported to move from locations throughout the Ca²⁺ store membrane to accumulate in regions close to the plasma membrane (30). Aggregation of STIM1 underneath the plasma membrane induces clustering of Orai1, resulting in CRAC channel activation. Orai1 accumulates at discrete sites in the plasma membrane directly opposite the STIM1 clusters (31). Although the mechanism underlying this event is unknown, it should be finely regulated as for the exocytotic processes. The synaptosomal-associated protein-25 (SNAP-25) assembles into a multiprotein complex proposed to mediate vesicle docking and fusion with the plasma membrane (27). SNAP-25 has been reported to be important for the activation of CRAC entry by facilitating the interaction between elements in the membranes of the Ca²⁺ stores and the plasma membrane (17, 21, 32). Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins have been shown to be important for the insertion of TRPCs in the plasma membrane, whose surface expression is determined by a recycling type of trafficking mechanism in a number of cells (26). In the present study, we have investigated whether Orai1 surface expression is regulated by depletion of the intracellular Ca²⁺ stores and the possible involvement of the SNARE protein SNAP-25 in this process.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Materials. Thapsigargin (TG), carbachol, leupeptine, benzamidine, ionomycin, phenyl methyl sulphonyl fluoride, dimethyl BAPTA, botulinum neurotoxin A (BoNT A), and bovine serum albumin were from Sigma (St. Louis, MO). Rabbit polyclonal anti-Orai1 antibody was from ProSci (Poway, CA). Goat anti-β-tubulin antibody, goat anti-actin antibody, goat anti-SNAP-25 antibody (C-18), and horseradish peroxidase-conjugated goat anti-rabbit IgG and donkey anti-goat antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Fura 2-AM and Alexa Fluor 488, 568, and 647-conjugated secondary antibodies were from Invitrogen (Carlsbad, CA). Enhanced chemiluminescence detection reagents, immobilized streptavidin gel, and EZ-Link Sulpho-NHS-LC-Biotin were from Pierce (Rockford, IL). Hyperfilm ECL was from Amersham (Arlington Heights, IL). All other reagents were of analytic grade.

Cell culture. Human HeLa and human embryonic kidney 293T (HEK-293T) cells were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium, supplemented with 10% heat-inactivated fetal bovine serum, in a 37°C incubator with 5% CO₂. The experiments were performed in HEPES-buffered saline containing the following (in mM): 145 NaCl, 10 HEPES, 10 D-glucose, 5 KCl, 1 MgSO₄, pH 7.45. For experiments performed in the presence or absence of extracellular Ca²⁺, either 1 mM CaCl₂ or 100 µM EGTA were added to the medium, as indicated.

Biotinylation of cell-surface proteins. Aliquots of cell suspensions (1 ml) were stimulated with TG or left untreated. The reaction was terminated with ice-cold Söerscen's buffer (SB) containing 16 mM Na₂HPO₄ and 114 mM NaH₂PO₄, pH 8.0. Cell surface proteins were labeled by resuspending in EZ-Link Sulpho-NHS-LC-Biotin (2.5 mg/12 ml ice-cold SB) and incubated under mixing for 1 h at 4°C. The biotinylation reaction was terminated by addition of 100 µl of 1 M Tris base, and remaining biotinylating agent was removed by washing the cells in ice-cold SB. Labeled cells were resuspended in PBS and then lysed in RIPA buffer, pH 7.2, containing 316 mM NaCl, 20 mM Tris, 2 mM EGTA, 0.2% SDS, 2% sodium deoxycholate, 2% Triton X-100, 2 mM Na₃VO₄, 2 mM phenylmethylsulfonyl fluoride, 100 µg/ml leupeptin, and 10 mM benzamidine. Labeled proteins were collected using streptavidin-coated agarose beads. The beads were collected by centrifugation, resuspended in Laemmli's buffer, and subjected to Western blotting.

Western blotting. Western blotting was performed as described previously (19). Briefly, proteins were separated by 10 or 15% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (wt/vol) bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein binding sites. Immunodetection of Orai1 or SNAP-25 was achieved using the anti-Orai1 antibody or anti-SNAP-25 antibody, diluted 1:1,000 in TBST for 1 h. To detect the primary antibody, blots were incubated for 45 min with horseradish peroxidase-conjugated donkey anti-rabbit IgG or donkey anti-goat antibody diluted 1:10,000 in TBST and then exposed to enhanced chemiluminescence reagents for 4 min. Blots were then exposed to photographic films. The density of bands on the film was measured using a scanning densitometry.

Measurement of intracellular free calcium concentration. Cells were loaded with fura 2 by incubation with 2 µM fura 2-AM for 45 min at room temperature. Fluorescence was recorded from 1-ml aliquots of magnetically stirred cell suspensions at 37°C using a fluorescence spectrophotometer with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in intracellular free calcium concentration ([Ca²⁺]_c) were monitored using the fura 2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz and coworkers (3). TG-evoked Ca²⁺ influx was measured as the integral of the rise in [Ca²⁺]_c above basal for 3 min after the addition of TG in the presence of external Ca²⁺, corrected by subtraction of the integral over the same period for stimulation in the absence of external Ca²⁺ (with 100 µM EGTA). To compare the initial rate of Ca²⁺ entry between different treatments, traces were fitted to the equation y = 1 – e^–KT, where K is the rate constant and T is time (5). To compare the rate of decay of [Ca²⁺]_c to basal values after cell stimulation with TG in the absence and presence of BoNT A, we used the constant of the exponential decay. Traces were fitted to the equation y = A(1 – e^–K₁T)e^–K₂T, where A is the span; K₁ is the constant of the exponential increase, and K₂ is the constant of the exponential decay (22).

Immunofluorescence. Cultured cells were fixed using 3% paraformaldehyde (in PBS) for 10 min at room temperature. The cells were then permeabilized in PBS containing 0.025% (vol/vol) Nonidet P-40 detergent for 10 min at 4°C. Samples were incubated with rabbit anti-Orai1, goat anti-β-actin, and goat anti-β-tubulin antibodies overnight at room temperature in PBS containing 0.5% BSA as blocking agent, followed by incubation with Alexa Fluor 488, 568, and 647-conjugated secondary antibodies for 1 h. The samples were examined using a Zeiss LSM 510 confocal microscope. Staining intensity was quantified using Image J software.

Statistical analysis. Analysis of statistical significance was performed using Student's t-test. For multiple comparison, one-way ANOVA combined with the Dunnett tests was used.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Ca²⁺ store depletion enhances surface expression of Orai1. To investigate whether intracellular trafficking regulates the localization of Orai1, we assessed the surface expression of Orai1 in resting and Ca²⁺ store-depleted cells (treated with TG) by biotinylation of plasma membrane proteins and collection with streptavidin-coated agarose beads. SDS-PAGE and Western blotting were used to identify Orai1, and plasma membrane expression was quantified by scanning densitometry. As shown in Fig. 1, A and B, analysis of biotinylated proteins shows that Orai1 is present in the plasma membrane in resting HeLa and HEK-293 cells. Recent studies have reported that HEK cells express a number of proteins of the CRAC machinery, including Orai1, STIM1, and hTRPC3 and 6 (2, 4). Surface expression of Orai1 significantly increases in cells treated with 1 µM TG in the absence of extracellular Ca²⁺ (100 µM EGTA were added) to 246 ± 22 and 193 ± 20% of control (resting cells), respectively (Fig. 1, A and B, left; means ± SE; P < 0.05, ANOVA; n = 4). Interestingly, TG-induced Orai1 surface expression was impaired in HeLa and HEK cells loaded with dimethyl BAPTA, an intracellular Ca²⁺ chelator (20). Cells heavily loaded with the Ca²⁺ chelator dimethyl BAPTA were used for this study, so as to eliminate Ca²⁺-dependent, but not store depletion-dependent, surface expression of Orai1. For loading with dimethyl BAPTA, cells were incubated for 30 min at 37°C with 10 µM dimethyl BAPTA-AM (15). As shown in Fig. 1C, treatment of HEK cells with TG in a Ca²⁺-free medium (100 µM EGTA were added) resulted in a transient increase in [Ca²⁺]_c due to Ca²⁺ release from intracellular stores. TG-evoked elevation in [Ca²⁺]_c returned to basal levels within 3 min after stimulation, confirming that 3-min treatment with TG was sufficient to fully deplete the intracellular Ca²⁺ stores. In a Ca²⁺-free medium, dimethyl BAPTA loading prevented TG-evoked [Ca²⁺]_c elevations (Fig. 1C) and was able to significantly decrease TG-induced Orai1 plasma membrane expression, both in HeLa and HEK cells by 95 and 88%, respectively (Fig. 1, A and B, left). In contrast, cell loading with dimethyl BAPTA did not significantly modify the surface expression of Orai1 in resting cell (Fig. 1, A and B, left). To further investigate whether the effect of BAPTA loading was mediated by its effects on Ca²⁺ mobilization and not by a direct effect on cellular trafficking, we performed a series of experiments where dimethyl BAPTA-loaded cells were suspended in a medium containing 1 mM Ca²⁺ and then stimulated with 1 µM TG, 300 nM of the Ca²⁺ ionophore ionomycin (25), or a combination of TG + ionomycin. In the presence of 1 mM extracellular Ca²⁺, treatment of BAPTA-loaded HEK cells with TG, ionomycin, or both induced a detectable increase in [Ca²⁺]_c and enhanced Orai1 surface expression to 129 ± 6, 135 ± 8, and 170 ± 12% of control, respectively (unstimulated cells; Fig. 1D; n = 4). Overall, our data indicate that Ca²⁺ store depletion enhances surface expression of Orai1, which is dependent on rises in [Ca²⁺]_c.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Thapsigargin (TG) and carbachol (CCh) enhance Orai1 surface expression. A: HeLa cells loaded with dimethyl BAPTA or left untreated, as indicated, were suspended in a Ca2+-free medium (100 µM EGTA were added) and then stimulated in the absence or presence of 1 µM TG. B: human embryonic kidney (HEK) cells loaded with dimethyl BAPTA or left untreated were suspended in a Ca2+-free medium (100 µM EGTA were added) or in a medium containing 1 mM Ca2+ and then stimulated in the absence or presence of 1 µM TG or 15 µM CCh. Stimulation was terminated after 3 min in ice-cold Söercen's buffer, and cell surface proteins were labeled by biotinylation, extracted with streptavidin-coated agarose beads, and analyzed by SDS-PAGE and Western blotting using the anti-Orai1 antibody. Western blotting of the samples using anti-β-actin antibody was performed for sample protein controls. Positions of molecular mass markers are shown on the right. Histograms represent Orai1 surface expression as percentage of control (nontreated cells) and are expressed as average ± SE of four separate experiments. C: HEK cells, loaded with dimethyl BAPTA or left untreated, were stimulated with 1 µM TG in a Ca2+-free medium; extracellular free Ca2+ concentration ([Ca2+]o) = 0 mM; 100 µM EGTA were added. Traces are representative of 5 independent experiments. D: HEK cells, loaded with dimethyl BAPTA, were suspended in a medium containing 1 mM Ca2+ and then stimulated with either 1 µM TG, 300 nM ionomycin, or TG combined with ionomycin. Left: intracellular free Ca2+ concentration ([Ca2+]c) was determined as described in MATERIALS AND METHODS. Right: stimulation was terminated after 3 min in ice-cold Söercen's buffer, and cell surface proteins were labeled by biotinylation, extracted with streptavidin-coated agarose beads, and analyzed by SDS-PAGE and Western blotting using the anti-Orai1 antibody. Samples were reprobed using anti-β-actin antibody. n.s., Not significant.

Store depletion induces the recruitment of Orai1 in plasma membrane in close apposition to STIM1 aggregates to assemble functional units of CRAC channels in a stoichiometric manner (31). We have now investigated by immunofluorescence using anti-Orai1 antibody, followed by Alexa Fluor 647-conjugated secondary antibody, whether, in addition to stimulating Orai1 externalization, TG was able to induce Orai1 clustering in the plasma membrane in the absence of extracellular Ca²⁺ (100 µM EGTA were added). As shown in Fig. 2, Orai1 was distributed in the cell periphery (plasma membrane) but also in the cytoplasmic area in resting HeLa and HEK cells. Stimulation with TG for 3 min resulted in translocation of Orai1 to the plasma membrane with the formation of clusters that were more obvious in HeLa cells than in HEK cells, where we found that Orai1 was more evenly distributed in the plasma membrane after treatment with TG. In addition to Orai1, we visualized β-actin and β-tubulin in both cell types by confocal microscopy. Cells were incubated with anti-β-actin and anti-β-tubulin antibodies followed by Alexa Fluor 568- and 488-conjugated secondary antibodies, respectively. As an internal control for Orai1 staining, we visualized β-actin, which is a relatively stable cytoskeletal protein generally thought to be present at a constant level in cells, regardless of experimental treatment. The merged images show that the Orai1 located in the cell periphery is located externally to the actin and tubulin cytoskeleton, which confirms the surface expression of Orai1 (Fig. 2).

View larger version (60K): [in this window] [in a new window]   Fig. 2. Orai1 externalization and clustering after stimulation with TG. Confocal images of HeLa and HEK cells were immunostainded with anti-Orai1, anti-actin, and anti-β-tubulin antibodies, followed by Alexa Fluor 647, 568, and 488-conjugated secondary antibodies, respectively. An overlay of the three images is depicted on the right. Cells were stimulated in the absence (control) or presence of 1 µM TG, as indicated, in a Ca2+-free medium (100 µM EGTA were added). Scale bar: 10 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 3. BAPTA loading prevents TG-induced Orai1 externalization and clustering. Confocal images of dimethyl BAPTA-loaded HeLa and HEK cells were immunostainded with anti-Orai1, anti-actin, and anti-β-tubulin antibodies, followed by Alexa Fluor 647, 568, and 488-conjugated secondary antibodies, respectively. An overlay of the three images is depicted on the right. Cells were stimulated in the absence (control) or presence of 1 µM TG, as indicated, in a Ca2+-free medium (100 µM EGTA were added). Scale bar: 10 µm.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Botulinum neurotoxin A (BoNT A) impairs TG-induced surface expression of Orai1. A: HEK cells were treated for 24 h with 300 nM BoNT A or the vehicle (control) and lysed. Proteins were separated by 15% SDS-PAGE (50 µg total protein loaded/sample) and transferred onto a nitrocellulose membrane for Western blotting with anti-synaptosomal-associated protein-25 (SNAP-25) antibody. Results are representative of five independent experiments. B: HEK cells were incubated in the absence or presence of BoNT A (300 nM) for 24 h. In a Ca2+-free medium (100 µM EGTA were added), cells were stimulated with 1 µM TG or left untreated. Stimulation was terminated after 3 min in ice-cold Söercen's buffer, and cell surface proteins were labeled by biotinylation, extracted with streptavidin-coated agarose beads, and analyzed by SDS-PAGE and Western blotting using the anti-Orai1 antibody. Western blotting of the samples using anti-β-actin antibody was performed for sample protein controls. Positions of molecular mass markers are shown on the right. Histograms represent Orai1 surface expression as percentage of control (nontreated cells) and are expressed as means ± SE of four separate experiments. C: HEK cells were incubated in the absence or presence of BoNT A (300 nM) for 24 h. Top: cells were then stimulated with 1 µM TG in a medium containing 1 mM CaCl2. At the end of the experiment, extracellular Ca2+ was chelated by addition of 2 mM EGTA ([Ca2+]o = 0 mM). Bottom: cells were then stimulated with 1 µM TG in a Ca2+-free medium. Traces are representative of 5 separate experiments. D: confocal images of HEK cells immunostainded with anti-Orai1 antibody followed by Alexa Fluor 647-conjugated secondary antibody. HEK cells were treated for 24 h with 300 nM BoNT A or the vehicle (control) and then stimulated with 1 µM TG in a medium containing 1 mM Ca2+ or left untreated. Scale bar: 10 µm.

We have further explored by immunofluorescence using anti-Orai1 antibody, followed by Alexa Fluor 647-conjugated secondary antibody, whether treatment with BoNT A was able to impair TG-induced Orai1 clustering in the plasma membrane in the presence of 1 mM extracellular Ca²⁺. As shown in Fig. 4D, incubation for 24 h with 300 nM BoNT A prevented Orai1 externalization and clustering stimulated by TG in HEK cells. Since 40% of Ca²⁺ entry was still detectable in BoNT A-treated cells, our findings indicate that Orai1 clustering might not be essential for store-operated Ca²⁺ entry in these cells.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Recent studies have reported that Orai1 is required for CRAC channel function (1, 10, 28). The Orai1 protein has been shown to be expressed largely in the plasma membrane with cytoplasmic NH₂ and COOH termini of the tetra-spanning membrane protein (1, 13). Our results indicate that Ca²⁺ store depletion enhances surface expression of Orai1, a process that is likely dependent on rises in [Ca²⁺]_c. Stopped-flow fluorimetric studies have revealed that CRAC entry stimulated by agonists commences 500 ms after the initiation of Ca²⁺ release from the intracellular stores. At the time of initiation of Ca²⁺ entry, only a small increase in [Ca²⁺]_c has been detected as a result of the incipient release of Ca²⁺ from the stores (16). In fact, a requirement for the mechanism that initiates CRAC entry (or store-operated Ca²⁺ entry) has long been suggested to be the regulation of the putative mechanism by the Ca²⁺ content of the intracellular stores, independent of changes in [Ca²⁺]_c (24). The observation that the enhancement of Orai1 surface expression after store depletion requires a substantial elevation in [Ca²⁺]_c, either by Ca²⁺ released or entry, suggests that store depletion-induced Orai1 externalization is not involved in the initial stage of the activation of CRAC entry, but, instead, might be an essential event for the maintenance of Ca²⁺ influx.

CRAC current (I_CRAC) channels show a Ca²⁺-dependent inactivation mechanism that is alleviated by BAPTA (7), which is a Ca²⁺ buffer with fast association kinetics. Our findings, which indicate that BAPTA prevents externalization of Orai1, is not in contradiction with this observation and should be interpreted on the basis of the different currents reported to account for store-operated Ca²⁺ entry (I_SOC) (11). Although Orai1 has been presented as the prototype for I_CRAC, recent studies reporting the interaction of Orai1 with TRPC proteins (10, 18), which show different Ca²⁺ selectivity than I_CRAC, indicate that Orai1 is also involved in other I_SOC that might require Orai1 externalization. We suggest that a portion of Ca²⁺ entry occurs independently of the calcium-dependent insertion of Orai1. This portion, amplified by BAPTA removal of Ca²⁺ inhibition, could be that which is measured in electrophysiological studies of I_CRAC. In addition, store depletion induces Orai1 externalization, which might be involved in the conduction of I_SOC.

Store depletion-induced Orai1 externalization and clustering is strongly inhibited by BoNT A preincubation, which cleaves SNAP-25 and reduces the maintenance but not the initial stages of store-operated Ca²⁺ entry. The different effect of BoNT A on the initial and subsequent rates of increase in [Ca²⁺]_c induced by TG is consistent with the hypothesis that store depletion-induced Orai1 surface expression is not involved in the activation, but in the maintenance, of CRAC entry. If the insertion of Orai1 channels in the plasma membrane were involved in the activation of Ca²⁺ influx, the rate of Ca²⁺ entry would be expected to be reduced from the initial stages by a procedure that impairs Orai1 translocation, which was not the case. Our results indicate that there is a certain level of expression of Orai1 in the plasma membrane of resting cells, which might account, as well as other cation channels (4, 6, 10), for the initial Ca²⁺ current, which, in turn, would activate SNAP-25-mediated translocation of Orai1 to the plasma membrane to mediate further Ca²⁺ influx. Therefore, there is a functionally significant interaction between Orai1 and the member of the SNARE family of proteins, SNAP-25, that is involved in transport and fusion of intracellular vesicles with the plasma membrane. A key finding of this study is that surface expression of Orai1 in unstimulated and store-depleted cells is decreased when vesicular trafficking is inhibited by treatment of cells with BoNT A. It is widely accepted that the function of certain plasma membrane proteins, including ion channels (26), is regulated by modulation of their level of expression on the surface membrane (23). In some cases, there is a pool of the protein in endosomal compartments ready to be inserted into the plasma membrane. This pool depends on a specialized trafficking process (transport to, and retrieval from, the plasma membrane) called constitutive cycling, where SNARE proteins play a key role (23). Concerning Orai1 externalization, SNAP-25 might lead to the specific fusion of the channel-containing vesicle at the correct area of the target membrane opposite the STIM1 clusters in the Ca²⁺ stores. Proteins that show low surface expression under resting conditions undergo rapid constitutive internalization and subsequent slow recycling back to the plasma membrane. We show that store depletion with TG approximately doubles the surface expression of Orai1, although some Orai1 is still detected in intracellular pools. The remaining intracellular Orai1 pool suggests that cell stimulation with Ca²⁺-mobilizing agonists, which induce active Ca²⁺ release through inositol 1,4,5-trisphosphate generation, might stimulate greater Orai1 externalization than TG, thus adding a second level of regulation of Orai1 surface expression and function. We cannot rule out the possibility that increased Orai1 surface expression can also result from retention of the protein via interaction with scaffolding proteins into the plasma membrane, although the effect of BoNT A on Orai1 externalization indicates that an exocytotic-like mechanism is involved. Our findings that store depletion induces Orai1 surface expression involving SNAP-25 shed new light on the molecular basis of the involvement of Orai1 proteins in the formation of CRAC channels.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by Spanish Ministry of Education and Science Grant BFU2007-60104/BFI.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. A. Rosado, Dept. of Physiology, Univ. of Extremadura, Cáceres 10071, Spain (e-mail: jarosado{at}unex.es)

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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, Hogan PG, Lewis RS, Daly M, Rao A. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441: 179–185, 2006.[CrossRef][Medline]

2. Gross SA, Wissenbach U, Philipp SE, Freichel M, Cavalié A, Flockerzi V. Murine ORAI2 splice variants form functional Ca²⁺ release-activated Ca²⁺ (CRAC) channels. J Biol Chem 282: 19375–19384, 2007.[Abstract/Free Full Text]

3. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]

4. Liao Y, Erxleben C, Yildirim E, Abramowitz J, Armstrong DL, Birnbaumer L. Orai proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci USA 104: 4682–4687, 2007.[Abstract/Free Full Text]

5. López JJ, Camello-Almaraz C, Pariente JA, Salido GM, Rosado JA. Ca²⁺ accumulation into acidic organelles mediated by Ca²⁺- and vacuolar H⁺-ATPases in human platelets. Biochem J 390: 243–252, 2005.[CrossRef][Web of Science][Medline]

6. López JJ, Salido GM, Pariente JA, Rosado JA. Interaction of STIM1 with endogenously expressed human canonical TRP1 upon depletion of intracellular Ca²⁺ stores. J Biol Chem 281: 28254–28264, 2006.[Abstract/Free Full Text]

7. Louzao MC, Ribeiro CM, Bird GS, Putney JW Jr. Cell type-specific modes of feedback regulation of capacitative calcium entry. J Biol Chem 271: 14807–14813, 1996.[Abstract/Free Full Text]

8. Mignen O, Thompson JL, Shuttleworth TJ. Orai1 subunit stoichiometry of the mammalian CRAC channel pore. J Physiol 586: 419–425, 2008.[Abstract/Free Full Text]

9. Montecucco C, Schiavo G. Tetanus and botulism neurotoxins: a new group of zinc proteases. Trends Biochem Sci 18: 324–327, 1993.[CrossRef][Web of Science][Medline]

10. Ong HL, Cheng KT, Liu X, Bandyopadhyay BC, Paria BC, Soboloff J, Pani B, Gwack Y, Srikanth S, Singh BB, Gill DL, Ambudkar IS. Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components. J Biol Chem 282: 9105–9116, 2007.[Abstract/Free Full Text]

11. Parekh AB, Putney JW Jr. Store-operated calcium channels. Physiol Rev 85: 757–810, 2005.[Abstract/Free Full Text]

12. Peinelt C, Vig M, Koomoa DL, Beck A, Nadler MJ, Koblan-Huberson M, Lis A, Fleig A, Penner R, Kinet JP. Amplification of CRAC current by STIM1 and CRACM1 (Orai1). Nat Cell Biol 8: 771–773, 2006.[CrossRef][Web of Science][Medline]

13. Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A, Hogan PG. Orai1 is an essential pore subunit of the CRAC channel. Nature 443: 230–233, 2006.[CrossRef][Medline]

14. Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium 7: 1–12, 1986.[CrossRef][Web of Science][Medline]

15. Redondo PC, Ben-Amor N, Salido GM, Bartegi A, Pariente JA, Rosado JA. Ca²⁺-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca²⁺ entry in human platelets. Cell Signal 17: 1011–1021, 2005.[CrossRef][Web of Science][Medline]

16. Redondo PC, Harper MT, Rosado JA, Sage SO. A role for cofilin in the activation of store-operated calcium entry by de novo conformational coupling in human platelets. Blood 107: 973–979, 2006.[Abstract/Free Full Text]

17. Redondo PC, Harper AG, Salido GM, Pariente JA, Sage SO, Rosado JA. A role for SNAP-25 but not VAMPs in store-mediated Ca²⁺ entry in human platelets. J Physiol 558: 99–109, 2004.[Abstract/Free Full Text]

18. Redondo PC, Jardin I, Lopez JJ, Salido GM, Rosado JA. Intracellular Ca²⁺ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP₃ receptor and SERCA3 in human platelets. Biochem Biophys Acta. In press.

19. Redondo PC, Salido GM, Rosado JA, Pariente JA. Effect of hydrogen peroxide on Ca²⁺ mobilisation in human platelets through sulphydryl oxidation dependent and independent mechanisms. Biochem Pharmacol 67: 491–502, 2004.[CrossRef][Web of Science][Medline]

20. Rosado JA, Meijer EM, Hamulyak K, Novakova I, Heemskerk JW, Sage SO. Fibrinogen binding to the integrin _IIbβ₃ modulates store-mediated calcium entry in human platelets. Blood 97: 2648–2656, 2001.[Abstract/Free Full Text]

21. Rosado JA, Redondo PC, Salido GM, Sage SO, Pariente JA. Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca²⁺ entry in mouse pancreatic acinar cells. Am J Physiol Cell Physiol 288: C214–C221, 2005.[Abstract/Free Full Text]

22. Rosado JA, Sage SO. Regulation of plasma membrane Ca²⁺-ATPase by small GTPases and phosphoinositides in human platelets. J Biol Chem 275: 19529–19535, 2000.[Abstract/Free Full Text]

23. Royle SJ, Murrell-Lagnado RD. Constitutive cycling: a general mechanism to regulate cell surface proteins. Bioessays 25: 39–46, 2003.[CrossRef][Web of Science][Medline]

24. Sargeant P, Farndale RW, Sage SO. Calcium store depletion in dimethyl BAPTA-loaded human platelets increases protein tyrosine phosphorylation in the absence of a rise in cytosolic calcium. Exp Physiol 79: 269–272, 1994.[Abstract]

25. Schmidt K, Andrew P, Schrammel A, Groschner K, Schmitz V, Kojda G, Mayer B. Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells. Am J Physiol Heart Circ Physiol 281: H2053–H2061, 2001.[Abstract/Free Full Text]

26. Singh BB, Lockwich TP, Bandyopadhyay BC, Liu X, Bollimuntha S, Brazer SC, Combs C, Das S, Leenders AG, Sheng ZH, Knepper MA, Ambudkar SV, Ambudkar IS. VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca²⁺ influx. Mol Cell 15: 635–646, 2004.[CrossRef][Web of Science][Medline]

27. Söllner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman J. SNAP receptors implicated in vesicle targeting and fusion. Nature 362: 318–324, 1993.[CrossRef][Medline]

28. Vig M, DeHaven WI, Bird GS, Billingsley JM, Wang H, Rao PE, Hutchings AB, Jouvin MH, Putney JW, Kinet JP. CRACM1 is a plasma membrane protein essential for store-operated Ca²⁺ entry. Science 312: 1220–1223, 2006.[Abstract/Free Full Text]

29. Wu X, Babnigg G, Zagranichnaya T, Villereal ML. The role of endogenous human Trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells. J Biol Chem 277: 13597–13608, 2002.[Abstract/Free Full Text]

30. Wu MM, Buchanan J, Luik RM, Lewis RS. Ca²⁺ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J Cell Biol 174: 803–813, 2006.[Abstract/Free Full Text]

31. Xu P, Lu J, Li Z, Yu X, Chen L, Xu T. Aggregation of STIM1 underneath the plasma membrane induces clustering of Orai1. Biochem Biophys Res Commun 350: 969–976, 2006.[CrossRef][Web of Science][Medline]

32. Yao Y, Ferrer-Montiel AV, Montal M, Tsien RY. Activation of store-operated Ca²⁺ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell 98: 475–485, 1999.[CrossRef][Web of Science][Medline]

33. Yeromin AV, Zhang SL, Jiang W, Yu Y, Safrina O, Cahalan MD. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443: 226–229, 2006.[CrossRef][Medline]

34. Zhang SL, Yeromin AV, Zhang XH, Yu Y, Safrina O, Penna A, Roos J, Stauderman KA, Cahalan MD. Genome-wide RNAi screen of Ca²⁺ influx identifies genes that regulate Ca²⁺ release-activated Ca²⁺ channel activity. Proc Natl Acad Sci USA 103: 9357–9362, 2006.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: 294/6/C1323    most recent 00071.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map 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 Google Scholar Google Scholar Articles by Woodard, G. E. Articles by Rosado, J. A. Search for Related Content PubMed PubMed Citation Articles by Woodard, G. E. Articles by Rosado, J. A.