scholarly journals STIM1 regulates Ca2+entry via arachidonate-regulated Ca2+-selective (ARC) channels without store depletion or translocation to the plasma membrane

2007 ◽  
Vol 579 (3) ◽  
pp. 703-715 ◽  
Author(s):  
Olivier Mignen ◽  
Jill L. Thompson ◽  
Trevor J. Shuttleworth
Keyword(s):  
Plasma Membrane ◽  
Store Depletion

Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+entry in mouse pancreatic acinar cells

2005 ◽  
Vol 288 (1) ◽  
pp. C214-C221 ◽  
Author(s):  
Juan A. Rosado ◽  
Pedro C. Redondo ◽  
Ginés M. Salido ◽  
Stewart O. Sage ◽  
Jose A. Pariente
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Cell ◽  
Botulinum Toxin A ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Amylase Secretion ◽  
Cell Type ◽  
Store Depletion

We recently reported that store-operated Ca2+entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as “secretion-like coupling.” As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

scholarly journals Microdomains of High Calcium Are Not Required for Exocytosis in Rbl-2h3 Mucosal Mast Cells

2001 ◽  
Vol 153 (2) ◽  
pp. 339-350 ◽  
Author(s):  
Sahar F. Mahmoud ◽  
Clare Fewtrell
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Crac Channels ◽  
Store Depletion ◽  
Mucosal Mast Cells ◽  
High Calcium ◽  
Constant Potential

We have previously shown that store-associated microdomains of high Ca2+ are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca2+ microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca2+ influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca2+ release–activated Ca2+ (CRAC) current (ICRAC) through CRAC channels. In the second, a Ca2+ ionophore was used to transport Ca2+ randomly across the plasma membrane. Since store depletion by Ca2+ ionophore will also activate ICRAC, different means of inhibiting ICRAC before ionophore addition were used. Ca2+ responses and secretion in individual cells were compared using simultaneous indo-1 microfluorometry and constant potential amperometry. Secretion still takes place when the increase in intracellular Ca2+ occurs diffusely via the Ca2+ ionophore, and at an average intracellular Ca2+ concentration that is no greater than that observed when Ca2+ entry via CRAC channels triggers secretion. Our results suggest that microdomains of high Ca2+ near the plasma membrane, or associated with mitochondria or Ca2+ stores, are not required for secretion. Therefore, we conclude that modest global increases in intracellular Ca2+ are sufficient for exocytosis in these nonexcitable cells.

scholarly journals The store-operated Ca2+ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel

2021 ◽  
Vol 118 (10) ◽  
pp. e2010789118
Author(s):  
Yihan Shen ◽  
Nagendra Babu Thillaiappan ◽  
Colin W. Taylor
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fluorescence Intensity ◽  
Small Cluster ◽  
Store Depletion ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cytosolic Domains ◽  
Intracellular Ca ◽  
The Many ◽  
Orai Channels

Increases in cytosolic Ca2+ concentration regulate diverse cellular activities and are usually evoked by opening of Ca2+ channels in intracellular Ca2+ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP3), IP3 receptors coordinate the contributions of these two Ca2+ sources by mediating Ca2+ release from the endoplasmic reticulum (ER). Loss of Ca2+ from the ER then activates store-operated Ca2+ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca2+ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca2+ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.

Mechanisms of capacitative calcium entry

2001 ◽  
Vol 114 (12) ◽  
pp. 2223-2229 ◽  
Author(s):  
James W. Putney ◽  
Lisa M. Broad ◽  
Franz-Josef Braun ◽  
Jean-Philippe Lievremont ◽  
Gary St J. Bird
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Binding Sites ◽  
Cell Types ◽  
Store Depletion ◽  
Blocking Effects ◽  
Conformational Coupling ◽  
Phosphatidylinositol 4 ◽  
Filling State ◽  
Ca2 Channels

Capacitative Ca2+ entry involves the regulation of plasma membrane Ca2+ channels by the filling state of intracellular Ca2+ stores in the endoplasmic reticulum (ER). Several theories have been advanced regarding the mechanism by which the stores communicate with the plasma membrane. One such mechanism, supported by recent findings, is conformational coupling: inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptors in the ER may sense the fall in Ca2+ levels through Ca2+-binding sites on their lumenal domains, and convey this conformational information directly by physically interacting with Ca2+ channels in the plasma membrane. In support of this idea, in some cell types, store-operated channels in excised membrane patches appear to depend on the presence of both Ins(1,4,5)P3 and Ins(1,4,5)P3 receptors for activity; in addition, inhibitors of Ins(1,4,5)P3 production that either block phospholipase C or inhibit phosphatidylinositol 4-kinase can block capacitative Ca2+ entry. However, the electrophysiological current underlying capacitative Ca2+ entry is not blocked by an Ins(1,4,5)P3 receptor antagonist, and the blocking effects of a phospholipase C inhibitor are not reversed by the intracellular application of Ins(1,4,5)P3. Furthermore, cells whose Ins(1,4,5)P3 receptor genes have been disrupted can nevertheless maintain their capability to activate capacitative Ca2+ entry channels in response to store depletion. A tentative conclusion is that multiple mechanisms for signaling capacitative Ca2+ entry may exist, and involve conformational coupling in some cell types and perhaps a diffusible signal in others.

scholarly journals Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions, regulates Ca2+ dynamics in T cells

2016 ◽  
Vol 113 (10) ◽  
pp. 2762-2767 ◽  
Author(s):  
Jin Seok Woo ◽  
Sonal Srikanth ◽  
Miyuki Nishi ◽  
Peipei Ping ◽  
Hiroshi Takeshima ◽  
...  
Keyword(s):  
T Cells ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Genetic Manipulation ◽  
Erk Signaling ◽  
Excitable Cells ◽  
Activated T Cells ◽  
Activation Markers ◽  
Extracellular Signaling ◽  
Store Depletion

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca2+ entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca2+ sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER–PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER–PM junctions to regulate Ca2+ signaling. Silencing or genetic manipulation of JP4 decreased ER Ca2+ content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca2+-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate–JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca2+ homeostasis and mediate SOCE in T cells.

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

2011 ◽  
Vol 194 (2) ◽  
pp. 335-346 ◽  
Author(s):  
Basile Darbellay ◽  
Serge Arnaudeau ◽  
Charles R. Bader ◽  
Stephane Konig ◽  
Laurent Bernheim
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Cell Types ◽  
Actin Binding ◽  
Slow Process ◽  
Excitable Cells ◽  
Store Depletion ◽  
Mammalian Tissues ◽  
New Protein ◽  
Ca2 Channels

Cytosolic Ca2+ signals encoded by repetitive Ca2+ releases rely on two processes to refill Ca2+ stores: Ca2+ reuptake from the cytosol and activation of a Ca2+ influx via store-operated Ca2+ 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 Ca2+ sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca2+ channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca2+ channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca2+ 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 Ca2+ sensor for their Ca2+ homeostasis and intracellular signaling.

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

2008 ◽  
Vol 294 (6) ◽  
pp. C1323-C1331 ◽  
Author(s):  
Geoffrey E. Woodard ◽  
Ginés M. Salido ◽  
Juan A. Rosado
Keyword(s):  
Plasma Membrane ◽  
Botulinum Neurotoxin ◽  
Surface Expression ◽  
Membrane Expression ◽  
Botulinum Neurotoxin A ◽  
Crac Channels ◽  
Store Depletion ◽  
Protein Receptor ◽  
Intracellular Ca ◽  
Crac Channel

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.

scholarly journals State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation

2014 ◽  
Vol 143 (5) ◽  
pp. 621-631 ◽  
Author(s):  
Anna Amcheslavsky ◽  
Olga Safrina ◽  
Michael D. Cahalan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cadmium Ions ◽  
Store Depletion ◽  
State Dependent ◽  
Conducting Pathway ◽  
Cysteine Scanning ◽  
Crac Channel ◽  
High Concentrations ◽  
Cysteine Scanning Mutagenesis

After endoplasmic reticulum (ER) Ca2+ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca2+-selective Ca2+ release-activated Ca2+ (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1–Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB–activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd2+). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB–activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd2+-induced block that is state dependent: Cd2+ only blocks 2-APB–activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB–activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.

scholarly journals A Cytosolic STIM2 Preprotein Created by Signal Peptide Inefficiency Activates ORAI1 in a Store-independent Manner

2011 ◽  
Vol 286 (18) ◽  
pp. 16174-16185 ◽  
Author(s):  
Sarah J. L. Graham ◽  
Marie A. Dziadek ◽  
Lorna S. Johnstone
Keyword(s):  
Plasma Membrane ◽  
Gene Transcription ◽  
Signal Peptide ◽  
Independent Manner ◽  
Methionine Residue ◽  
Store Depletion ◽  
Amino Acid Signal Peptide ◽  
Er Targeting ◽  
Amino Acid Signal ◽  
Er Membrane

Calcium (Ca2+) influx through the plasma membrane store-operated Ca2+ channel ORAI1 is controlled by Ca2+ sensors of the stromal interaction molecule (STIM) family. STIM1 responds to endoplasmic reticulum (ER) Ca2+ store depletion by redistributing and activating ORAI1 from regions of the ER juxtaposed to the plasma membrane. Unlike STIM1, STIM2 can regulate ORAI1 in a store-dependent and store-independent manner, but the mechanism by which this is achieved is unknown. Here we find that STIM2 is translated from a highly conserved methionine residue and is directed to the ER by an incredibly long 101-amino acid signal peptide. We find that although the majority of the total STIM2 population resides on the ER membrane, a second population escapes ER targeting to accumulate as a full-length preprotein in the cytosol, signal peptide intact. Unlike STIM2, preSTIM2 localizes to the inner leaflet of the plasma membrane where it interacts with ORAI1 to regulate basal Ca2+ concentration and Ca2+-dependent gene transcription in a store-independent manner. Furthermore, a third protein comprising a fragment of the STIM2 signal peptide is released from the ER membrane into the cytosol where it regulates gene transcription in a Ca2+-independent manner. This study establishes a new model for STIM2-mediated regulation of ORAI1 in which two distinct proteins, STIM2 and preSTIM2, control store-dependent and store-independent modes of ORAI1 activation.

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