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

2006 ◽  
Vol 174 (6) ◽  
pp. 803-813 ◽  
Author(s):  
Minnie M. Wu ◽  
JoAnn Buchanan ◽  
Riina M. Luik ◽  
Richard S. Lewis
Keyword(s):  
Plasma Membrane ◽  
Jurkat Cells ◽  
Causal Role ◽  
Cell Periphery ◽  
Patch Clamp Recording ◽  
Channel Activation ◽  
Crac Channels ◽  
Store Depletion ◽  
Electron Microscopy Analysis ◽  
Crac Channel

Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca2+ sensor controlling store-operated Ca2+ entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca2+ release–activated Ca2+ (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 Ca2+ entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.

scholarly journals S-acylation of Orai1 regulates store-operated Ca2+ entry

2021 ◽  
Vol 135 (5) ◽  
Author(s):  
Savannah J. West ◽  
Goutham Kodakandla ◽  
Qioachu Wang ◽  
Ritika Tewari ◽  
Michael X. Zhu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Types ◽  
Central Component ◽  
Channel Activation ◽  
Crac Channels ◽  
Store Depletion ◽  
Crac Channel ◽  
Underlying Mechanisms ◽  
Different Cell Types

ABSTRACT Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER–PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER–PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER–PM junctions, subsequent binding to STIM1 and channel activation.

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

2006 ◽  
Vol 174 (6) ◽  
pp. 815-825 ◽  
Author(s):  
Riina M. Luik ◽  
Minnie M. Wu ◽  
JoAnn Buchanan ◽  
Richard S. Lewis
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Plasma Membranes ◽  
Patch Clamp Recording ◽  
Elementary Unit ◽  
Local Activation ◽  
Crac Channels ◽  
Store Depletion ◽  
Crac Channel ◽  
First Time

The activation of store-operated Ca2+ entry by Ca2+ 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 Ca2+ 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 Ca2+ influx through open Ca2+ release–activated Ca2+ (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca2+ 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 Ca2+ 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 Ca2+ entry.

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 Complex role of STIM1 in the activation of store-independent Orai1/3 channels

2014 ◽  
Vol 143 (3) ◽  
pp. 345-359 ◽  
Author(s):  
Xuexin Zhang ◽  
Wei Zhang ◽  
José C. González-Cobos ◽  
Isaac Jardin ◽  
Christoph Romanin ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cell Types ◽  
Hek293 Cells ◽  
Patch Clamp Recording ◽  
Channel Activation ◽  
Whole Cell ◽  
Perforated Patch ◽  
Crac Channels ◽  
Crac Channel

Orai proteins contribute to Ca2+ entry into cells through both store-dependent, Ca2+ release–activated Ca2+ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) and leukotriene C4 (LTC4)-regulated Ca2+ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels.

scholarly journals Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.

1995 ◽  
Vol 105 (2) ◽  
pp. 209-226 ◽  
Author(s):  
A Zweifach ◽  
R S Lewis
Keyword(s):  
Time Course ◽  
Measurement Techniques ◽  
Fast Inactivation ◽  
Patch Clamp Recording ◽  
Crac Channels ◽  
Whole Cell Patch Clamp ◽  
Close Proximity ◽  
Rapid Inactivation ◽  
Voltage Dependent ◽  
Crac Channel

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

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 An essential and NSF independent role for α-SNAP in store-operated calcium entry

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Yong Miao ◽  
Cathrine Miner ◽  
Lei Zhang ◽  
Phyllis I Hanson ◽  
Adish Dani ◽  
...  
Keyword(s):  
Essential Role ◽  
Calcium Release ◽  
Calcium Entry ◽  
Snare Complex ◽  
Functional Coupling ◽  
Crac Channels ◽  
Store Depletion ◽  
Store Operated Calcium Entry ◽  
Crac Channel

Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca2+ sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE.

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 Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.

1996 ◽  
Vol 107 (5) ◽  
pp. 597-610 ◽  
Author(s):  
A Zweifach ◽  
R S Lewis
Keyword(s):  
T Lymphocytes ◽  
Voltage Dependence ◽  
Single Channel ◽  
Current Amplitude ◽  
Second Phase ◽  
Channel Activation ◽  
Calcium Dependent ◽  
Crac Channels ◽  
Crac Channel ◽  
Two Phases

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.

scholarly journals Physiological CRAC channel activation and pore properties require STIM1 binding to all six Orai1 subunits

2018 ◽  
Vol 150 (10) ◽  
pp. 1373-1385 ◽  
Author(s):  
Michelle Yen ◽  
Richard S. Lewis
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
Ion Selectivity ◽  
Functional Coupling ◽  
Open Probability ◽  
Channel Activation ◽  
Channel Current ◽  
Crac Channels ◽  
Crac Channel ◽  
Pore Properties

The binding of STIM1 to Orai1 controls the opening of store-operated CRAC channels as well as their extremely high Ca2+ selectivity. Although STIM1 dimers are known to bind directly to the cytosolic C termini of the six Orai1 subunits (SUs) that form the channel hexamer, the dependence of channel activation and selectivity on the number of occupied binding sites is not well understood. Here we address these questions using dimeric and hexameric Orai1 concatemers in which L273D mutations were introduced to inhibit STIM1 binding to specific Orai1 SUs. By measuring FRET between fluorescently labeled STIM1 and Orai1, we find that homomeric L273D mutant channels fail to bind STIM1 appreciably; however, the L273D SU does bind STIM1 and contribute to channel activation when located adjacent to a WT SU. These results suggest that STIM1 dimers can interact with pairs of neighboring Orai1 SUs. Surprisingly, a single L273D mutation within the Orai1 hexamer reduces channel open probability by ∼90%, triples the size of the single-channel current, weakens the Ca2+ binding affinity of the selectivity filter, and lowers the selectivity for Na+ over Cs+ in the absence of divalent cations. These findings reveal a surprisingly strong functional coupling between STIM1 binding and CRAC channel gating and pore properties. We conclude that under physiological conditions, all six Orai1 SUs of the native CRAC channel bind STIM1 to effectively open the pore and generate the signature properties of extremely low conductance and high ion selectivity.

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