Interaction of STIM1 with endogenously expressed human canonical TRP1 upon depletion of intracellular Ca2+ stores

STIM1 (stromal interaction molecule 1) has recently been proposed to communicate the intracellular Ca(2+) stores with the plasma membrane to mediate store-operated Ca(2+) entry. Here we describe for the first time that Ca(2+) store depletion stimulates rapid STIM1 surface expression and association with endogenously expressed human canonical TRP1 (hTRPC1) independently of rises in cytosolic free Ca(2+) concentration. These events require the support of the actin cytoskeleton in human platelets, as reported for the coupling between type II inositol 1,4,5-trisphosphate receptor in the Ca(2+) stores and hTRPC1 in the plasma membrane, which has been suggested to underlie the activation of store-operated Ca(2+) entry in these cells. Electrotransjection of cells with anti-STIM1 antibody, directed toward the N-terminal sequence that includes the Ca(2+)-binding region, prevented the migration of STIM1 toward the plasma membrane, the interaction between STIM1 and hTRPC1, the coupling between hTRPC1 and type II inositol 1,4,5-trisphosphate receptor, and reduced store-operated Ca(2+) entry. These findings provide evidence for a role of STIM1 in the activation of store-operated Ca(2+) entry probably acting as a Ca(2+) sensor.     

Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+ channels

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca(2+) selective I(CRAC) and relatively Ca(2+) selective I(SOC), respectively. STIM1 has been proposed to communicate the Ca(2+) content of the intracellular Ca(2+) stores to the plasma membrane store-operated Ca(2+) channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca(2+) stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca(2+). In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca(2+) permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca(2+) entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca(2+) entry.     

Temperature-dependent STIM1 activation induces Ca²+ influx and modulates gene expression

Intracellular Ca(2+) is essential for diverse cellular functions. Ca(2+) entry into many cell types including immune cells is triggered by depleting endoplasmic reticulum (ER) Ca(2+), a process termed store-operated Ca(2+) entry (SOCE). STIM1 is an ER Ca(2+) sensor. Upon Ca(2+) store depletion, STIM1 clusters at ER-plasma membrane junctions where it interacts with and gates Ca(2+)-permeable Orai1 ion channels. Here we show that STIM1 is also activated by temperature. Heating cells caused clustering of STIM1 at temperatures above 35 °C without depleting Ca(2+) stores and led to Orai1-mediated Ca(2+) influx as a heat off-response (response after cooling). Notably, the functional coupling of STIM1 and Orai1 is prevented at high temperatures, potentially explaining the heat off-response. Additionally, physiologically relevant temperature shifts modulate STIM1-dependent gene expression in Jurkat T cells. Therefore, temperature is an important regulator of STIM1 function.     

The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions

The activation of store-operated Ca(2+) entry by Ca(2+) store depletion has long been hypothesized to occur via local interactions of the endoplasmic reticulum (ER) and plasma membrane, but the structure involved has never been identified. Store depletion causes the ER Ca(2+) sensor stromal interacting molecule 1 (STIM1) to form puncta by accumulating in junctional ER located 10-25 nm from the plasma membrane (see Wu et al. on p. 803 of this issue). We have combined total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording to localize STIM1 and sites of Ca(2+) influx through open Ca(2+) release-activated Ca(2+) (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca(2+) entry to discrete sites comprising a small fraction of the cell surface. Orai1, an essential component of the CRAC channel, colocalizes with STIM1 after store depletion, providing a physical basis for the local activation of Ca(2+) influx. These studies reveal for the first time that STIM1 and Orai1 move in a coordinated fashion to form closely apposed clusters in the ER and plasma membranes, thereby creating the elementary unit of store-operated Ca(2+) entry.     

STIM1 and the noncapacitative ARC channels

Our understanding of the nature and regulation of receptor-activated Ca(2+) entry in nonexcitable cells has recently undergone a radical change that began with the identification of the stromal interacting molecule proteins (e.g., STIM1) as playing a critical role in the regulation of the capacitative, or store-operated, Ca(2+) entry. As such, current models emphasize the role of STIM1 located in the endoplasmic reticulum membrane, where it senses the status of the intracellular Ca(2+) stores via a luminal N-terminal Ca(2+)-binding EF-hand domain. Dissociation of Ca(2+) from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca(2+) channels (e.g., CRAC channels). Thus, the specific dependence on store-depletion, and the role of the Ca(2+)-binding EF-hand domain in this process, are critical to all current models of the action of STIM1 on Ca(2+) entry. However, until recently, the effects of STIM1 on other modes of receptor-activated Ca(2+) entry have not been examined. Surprisingly, we found that STIM1 exerts similar, although not identical, actions on the arachidonic acid-regulated Ca(2+)-selective (ARC) channels-a widely expressed mode of agonist-activated Ca(2+) entry whose activation is completely independent of Ca(2+) store depletion. Regulation of the ARC channels by STIM1 is not only independent of store depletion, but also of the Ca(2+)-binding function of the EF-hand, and translocation of STIM1 to the plasma membrane. Instead, it is the pool of STIM1 that constitutively resides in the plasma membrane that is critical for the regulation of the ARC channels. Thus, ARC channel activity is selectively inhibited by exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1. Similarly, introducing mutations in STIM1 that prevent the N-linked glycosylation-dependent constitutive expression of the protein in the plasma membrane specifically inhibits the activity of the ARC channels without affecting the CRAC channels. These studies demonstrate that STIM1 is a far more universal regulator of Ca(2+) entry pathways than previously assumed, and has multiple, and entirely distinct, modes of action. Precisely how this same protein can act in such separate and specific ways on these different pathways of agonist-activated Ca(2+)entry remains an intriguing, yet currently unresolved, question.     

STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release

Cytosolic Ca(2+) signals encoded by repetitive Ca(2+) releases rely on two processes to refill Ca(2+) stores: Ca(2+) reuptake from the cytosol and activation of a Ca(2+) influx via store-operated Ca(2+) entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca(2+) sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca(2+) channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca(2+) channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca(2+) signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca(2+) sensor for their Ca(2+) homeostasis and intracellular signaling.     

Molecular basis of early epithelial response to streptococcal exotoxin: role of STIM1 and Orai1 proteins

Streptolysin O (SLO) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pyogenes. SLO induces diverse types of Ca(2+) signalling in host cells which play a key role in membrane repair and cell fate determination. The mechanisms behind SLO-induced Ca(2+) signalling remain poorly understood. Here, we show that in NCI-H441 cells, wild-type SLO as well as non-pore-forming mutant induces long-lasting intracellular Ca(2+) oscillations via IP(3) -mediated depletion of intracellular stores and activation of store-operated Ca(2+) (SOC) entry. SLO-induced activation of SOC entry was confirmed by Ca(2+) add-back experiments, pharmacologically and by overexpression as well as silencing of STIM1 and Orai1 expression. SLO also activated SOC entry in primary cultivated alveolar type II (ATII) cells but Ca(2+) oscillations were comparatively short-lived in nature. Comparison of STIM1 and Orai1 revealed a differential expression pattern in H441 and ATII cells. Overexpression of STIM1 and Orai1 proteins in ATII cells changed the short-lived oscillatory response into a long-lived one. Thus, we conclude that SLO-mediated Ca(2+) signalling involves Ca(2+) release from intracellular stores and STIM1/Orai1-dependent SOC entry. The phenotype of Ca(2+) signalling depends on STIM1 and Orai1 expression levels. Our findings suggest a new role for SOC entry-associated proteins in S. pyogenes-induced lung infection and pneumonia.     

Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and degranulation in a rat mast cell line

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.     

STIM1 is required for Ca2+ signaling during mammalian fertilization

During fertilization in mammals, a series of oscillations in the oocyte's intracellular free Ca(2+) concentration is responsible for oocyte activation and stimulation of embryonic development. The oscillations are associated with influx of Ca(2+) across the plasma membrane that is probably triggered by the depletion of the intracellular stores, a mechanism known as store-operated Ca(2+) entry. Recently, STIM1 has been identified in oocytes as a key component of the machinery that generates the Ca(2+) influx after store depletion. In this study, the involvement of STIM1 in the sperm-induced Ca(2+) oscillations and its significance in supporting subsequent embryo development were investigated. Downregulation of STIM1 levels in pig oocytes by siRNA completely inhibited the repetitive Ca(2+) signal triggered by the fertilizing sperm. In addition, a significantly lower percentage of oocytes cleaved or formed blastocysts when STIM1 was downregulated prior to fertilization compared to the control groups. Restoring STIM1 levels after fertilization in such oocytes by means of mRNA injection could not rescue embryonic development that in most cases was arrested at the 2-cell stage. On the other hand, STIM1 overexpression prior to fertilization did not alter the pattern of sperm-induced Ca(2+) oscillations and development of these fertilized oocytes up to the blastocyst stage was also similar to that registered in the control group. Finally, downregulation of STIM1 had no effect on oocyte activation when activation was stimulated artificially by inducing a single large elevation in the oocyte's intracellular free Ca(2+) concentration. These findings suggest that STIM1 is essential for normal fertilization as it is involved in the maintenance of the long-lasting repetitive Ca(2+) signal.     

Capacitative and non-capacitative signaling complexes in human platelets

Discharge of the intracellular Ca(2+) stores activates Ca(2+) entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca(2+) signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca(2+) stores with thapsigargin, independently of changes in cytosolic Ca(2+) concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca(2+) entry. These results indicate that Ca(2+) store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca(2+) influx in human platelets.     

STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.     

Visualisation and identification of the interaction between STIM1s in resting cells

Store-operated Ca(2+) channels are a major Ca(2+) entry pathway in nonexcitable cells, which drive various essential cellular functions. Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca(2+) release-activated Ca(2+) (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca(2+) sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca(2+) stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca(2+) stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.     

Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.     

Ca2+ depletion and inositol 1,4,5-trisphosphate-evoked activation of Ca2+ entry in single guinea pig hepatocytes

Store-operated Ca(2+) entry was investigated by monitoring the Ca(2+)-dependent K(+) permeability in voltage-clamped guinea pig hepatocytes. In physiological conditions, intracellular Ca(2+) stores are discharged following agonist stimulation, but depletion of this stores can be achieved using Ca(2+)-Mg(2+)-ATPase inhibitors such as 2,5-di(tert-butyl)-1,4-benzohydroquinone and thapsigargin. The effect of internal Ca(2+) store depletion on Ca(2+) influx was tested in single cells using inositol 1,4,5-trisphosphate (InsP(3)) release from caged InsP(3) after treatment of the cells with 2, 5-di(tert-butyl)-1,4-benzohydroquinone or thapsigargin in Ca(2+)-free solutions. We show that the photolytic release of 1-d-myo-inositol 1,4-bisphosphate 5-phosphorothioate, a stable analog of InsP(3), and Ca(2+) store depletion have additive effects to activate a high level of Ca(2+) entry in single guinea pig hepatocytes. These results suggest that there is a direct functional interaction between InsP(3) receptors and Ca(2+) channels in the plasma membrane, although the nature of these Ca(2+) channels in hepatocytes is unclear.     

STIM2 is an inhibitor of STIM1-mediated store-operated Ca2+ Entry

The coupling mechanism between endoplasmic reticulum (ER) Ca(2+) stores and plasma membrane (PM) store-operated channels (SOCs) remains elusive [1-3]. STIM1 was shown to play a crucial role in this coupling process [4-7]; however, the role of the closely related STIM2 protein remains undetermined. We reveal that STIM2 is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. This contrasts with gain of SOC function in STIM1-expressing cells. While STIM1 is expressed in both the ER and plasma membrane, STIM2 is expressed only intracellularly. Store depletion induces redistribution of STIM1 into distinct "puncta." STIM2 translocates into puncta upon store depletion only when coexpressed with STIM1. Double labeling shows coincidence of STIM1 and STIM2 within puncta, and immunoprecipitation reveals direct interactions between STIM1 and STIM2. Independent of store depletion, STIM2 colocalizes with and blocks the function of a STIM1 EF-hand mutant that preexists in puncta and is constitutively coupled to activate SOCs. Thus, whereas STIM1 is a required mediator of SOC activation, STIM2 is a powerful inhibitor of this process, interfering with STIM1-mediated SOC activation at a point downstream of puncta formation. The opposing functions of STIM1 and STIM2 suggest they may play a coordinated role in controlling SOC-mediated Ca(2+) entry signals.     

How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation?

Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orai1 expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orai1 expression, that STIM1 accumulation is not sufficient for Orai1 accumulation in the same areas, and that normal Ca(2+) release-activated Ca(2+) current (I(CRAC)) can be activated in STIM1-deficient cells. These data challenge the idea of direct conformational coupling between STIM1 and Orai1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements.     

Local cytosolic Ca2+ elevations are required for stromal interaction molecule 1 (STIM1) de-oligomerization and termination of store-operated Ca2+ entry

The Ca(2+) depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca(2+) entry (SOCE) pathway that sustains long-term Ca(2+) signals critical for cellular functions. ER Ca(2+) depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca(2+) refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca(2+) concentrations ([Ca(2+)](ER)) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca(2+)](ER) levels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca(2+) using a membrane-permeable Ca(2+) chelator to provide a large reservoir of buffered Ca(2+). This procedure rapidly restored pre-stimulatory [Ca(2+)](ER) levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca(2+)] remained low. STIM1 dissociation evoked by Ca(2+) readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca(2+) elevations restricted to STIM1 puncta, indicating that Ca(2+) acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca(2+) stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca(2+) elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE.     

Relation between phosphatidylserine exposure and store-operated Ca(2+) entry in stimulated cells

A significant increase in intracellular Ca(2+) is required to trigger the remodeling of the cell plasma membrane. Scott syndrome is an extremely rare inherited disorder of the transmembrane migration of phosphatidylserine toward the exoplasmic leaflet in blood cells. We have recently reported a reduced capacitative Ca(2+) entry in Scott cells [Martínez et al. (1999) Biochemistry 38, 10092-10098]. We have investigated here the links between defective phosphatidylserine exposure and Ca(2+) signaling in Scott cells by focusing on the Ca(2+) entry following the emptying of intracellular stores. After depletion of caffeine- or thapsigargin-sensitive stores, Ca(2+) entry was lower in Scott compared to control lymphoblasts. However, the simultaneous depletion of both types of stores restored a normal Ca(2+) influx across the plasma membrane in Scott cells and phosphatidylserine externalization ability was improved concomitantly with capacitative Ca(2+) entry. These observations point to the essential role of capacitative Ca(2+) entry in the control of phosphatidylserine exposure of stimulated cells.     

Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane

Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca(2+) release-activated Ca(2+) (CRAC) channel activation, but it is unclear whether redistribution is rapid enough to play a causal role. Furthermore, the location of STIM1 puncta is uncertain, with recent reports supporting retention in the ER as well as insertion into the plasma membrane (PM). Using total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording from single Jurkat cells, we show that STIM1 puncta form several seconds before CRAC channels open, supporting a causal role in channel activation. Fluorescence quenching and electron microscopy analysis reveal that puncta correspond to STIM1 accumulation in discrete subregions of junctional ER located 10-25 nm from the PM, without detectable insertion of STIM1 into the PM. Roughly one third of these ER-PM contacts form in response to store depletion. These studies identify an ER structure underlying store-operated Ca(2+) entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.