scholarly journals Separation and Characterization of Currents through Store-operated CRAC Channels and Mg2+-inhibited Cation (MIC) Channels

2002 ◽  
Vol 119 (5) ◽  
pp. 487-508 ◽  
Author(s):  
Murali Prakriya ◽  
Richard S. Lewis
Keyword(s):  
Calcium Release ◽  
Divalent Cations ◽  
Noise Analysis ◽  
Crac Channels ◽  
Unitary Conductance ◽  
New Information ◽  
Crac Channel ◽  
High Doses ◽  
Whole Cell Recordings

Although store-operated calcium release–activated Ca2+(CRAC) channels are highly Ca2+-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na+and Cs+with a unitary conductance of ∼40 pS, and that intracellular Mg2+modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg2+-inhibited cation (MIC) channels that open as Mg2+is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8–10 mM intracellular Mg2+to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca2+to divalent-free extracellular solution evokes Na+current through open CRAC channels (Na+-ICRAC) that is initially eightfold larger than the preceding Ca2+current and declines by ∼80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs+(PCs/PNa= 0.13 vs. 1.2 for MIC). Neither the decline in Na+-ICRACnor its low Cs+permeability are affected by intracellular Mg2+(90 μM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca2+channels.

scholarly journals A Store-operated Calcium Channel in Drosophila S2 Cells

2004 ◽  
Vol 123 (2) ◽  
pp. 167-182 ◽  
Author(s):  
Andriy V. Yeromin ◽  
Jack Roos ◽  
Kenneth A. Stauderman ◽  
Michael D. Cahalan
Keyword(s):  
Mammalian Cells ◽  
Divalent Cations ◽  
Noise Analysis ◽  
Reversal Potential ◽  
S2 Cells ◽  
Pipette Solution ◽  
Crac Channels ◽  
Drosophila S2 Cells ◽  
Cell Recording ◽  
Inward Currents

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

scholarly journals Orai1 pore residues control CRAC channel inactivation independently of calmodulin

2016 ◽  
Vol 147 (2) ◽  
pp. 137-152 ◽  
Author(s):  
Franklin M. Mullins ◽  
Michelle Yen ◽  
Richard S. Lewis
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Negative Charge ◽  
Noise Analysis ◽  
Dominant Negative ◽  
Side Chain ◽  
Open Probability ◽  
Crac Channels ◽  
Channel Inactivation ◽  
Crac Channel

Ca2+ entry through CRAC channels causes fast Ca2+-dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca2+–CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl− modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.

scholarly journals Characterization of the Calcium Release-Activated Calcium (CRAC) Channel From the Human Pathogen Schistosoma mansoni

2018 ◽  
Vol 114 (3) ◽  
pp. 286a
Author(s):  
Ana Eliza Zeraik ◽  
Aparna Gudlur ◽  
Ricardo DeMarco ◽  
Ana Paula U. Araujo ◽  
Patrick Hogan
Keyword(s):  
Schistosoma Mansoni ◽  
Calcium Release ◽  
Human Pathogen ◽  
Crac Channel

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.

Abstract TP272: Calcium Release-activated Calcium (CRAC) Channel Inhibition Protects Against Brain Injury and Neuronal Death Induced by Activated Microglia

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Rachid Kacimi ◽  
Jong Youl Kim ◽  
Ken Stauderman ◽  
Michael Dunn ◽  
Sudarshan Hebbar ◽  
...  
Keyword(s):  
Brain Injury ◽  
Calcium Signaling ◽  
Calcium Release ◽  
Neuronal Cell ◽  
Western Blots ◽  
Activated Microglia ◽  
Crac Channels ◽  
Channel Inhibition ◽  
Bv2 Cells ◽  
Crac Channel

Inflammatory responses following ischemia can worsen neurological outcome, and represent a potential target for therapeutic intervention. Store-operated Ca 2+ entry (SOCE) mediated by CRAC channels contribute to calcium signaling in immune cells. CRAC channels consist of the endoplasmic reticulum resident Ca 2+ -sensing protein stromal interaction molecule 1 (STIM1) and the calcium channel protein ORAI1 located in the plasma membrane. Prolonged Ca 2+ entry through CRAC channels activates, via calcineurin, nuclear factor of activated T cells (NFAT), involved in T cell proliferation and cytokine expression. Microglia mediate inflammation in the injured brain, but little is known about the role of CRAC channels in this process. We studied novel CRAC channel inhibitors to explore their therapeutic potential in microglia-mediated injury. A neuron cell line (Neuro-2A, N-2A) was cultured alone or with microglial BV2 cells then exposed to 2h oxygen glucose deprivation (OGD). Some cultures were treated with a novel CRAC channel inhibitor. Toll-like receptor (TLR) -3, -4 agonists or IFNγ were also used to activate microglia. Western blots revealed the presence of CRAC channel proteins STIM1 and ORAI1 in microglia. CRAC channel inhibition decreased NO release and inflammatory proteins iNOS and COX-2 expression in activated microglia, but did not affect STIM1 or ORAI1 expression. CRAC channel inhibitors also reduced agonist induced intracellular calcium accumulation in BV2 cells. Agonists also activated JNK1/2 kinase, NFAT, NF-κB, CREB & STAT1 in microglia, but only JNK1/2 kinase & NFAT were attenuated by inhibitor. OGD decreased N2A neuronal cell viability, further exacerbated by BV2 cells, but neuronal cells were protected by CRAC channel inhibition (n=5, *p<0.05). We then treated male C57/BL6 mice exposed to experimental brain trauma (TBI) and found that CRAC channel inhibition led to decreased lesion size, brain hemorrhage and improved neurological deficits (n=6-7/grp, *p<0.05). We suggest a novel anti-inflammatory approach for treating acute brain injury. Our observations also shed light on new calcium signaling pathways, not previously described in brain injury models.

Bidirectional regulation of calcium release–activated calcium (CRAC) channel by SARAF

2021 ◽  
Vol 220 (12) ◽  
Author(s):  
Elia Zomot ◽  
Hadas Achildiev Cohen ◽  
Inbal Dagan ◽  
Ruslana Militsin ◽  
Raz Palty
Keyword(s):  
Gene Expression ◽  
Calcium Release ◽  
Cell Receptor ◽  
Crac Channels ◽  
Bidirectional Regulation ◽  
Dependent Inactivation ◽  
Store Operated Calcium Entry ◽  
Crac Channel ◽  
Initial Activation ◽  
Downstream Gene Expression

Store-operated calcium entry (SOCE) through the Ca2+ release–activated Ca2+ (CRAC) channel is a central mechanism by which cells generate Ca2+ signals and mediate Ca2+-dependent gene expression. The molecular basis for CRAC channel regulation by the SOCE-associated regulatory factor (SARAF) remained insufficiently understood. Here we found that following ER Ca2+ depletion, SARAF facilitates a conformational change in the ER Ca2+ sensor STIM1 that relieves an activation constraint enforced by the STIM1 inactivation domain (ID; aa 475–483) and promotes initial activation of STIM1, its translocation to ER–plasma membrane junctions, and coupling to Orai1 channels. Following intracellular Ca2+ rise, cooperation between SARAF and the STIM1 ID controls CRAC channel slow Ca2+-dependent inactivation. We further show that in T lymphocytes, SARAF is required for proper T cell receptor evoked transcription. Taking all these data together, we uncover a dual regulatory role for SARAF during both activation and inactivation of CRAC channels and show that SARAF fine-tunes intracellular Ca2+ responses and downstream gene expression in cells.

scholarly journals Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

2020 ◽  
Author(s):  
Adam G Grieve ◽  
Yi-Chun Yeh ◽  
Lucrezia Zarcone ◽  
Johannes Breuning ◽  
Nicholas Johnson ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Calcium Release ◽  
Calcium Influx ◽  
Cell Mediated Immunity ◽  
Crac Channels ◽  
Rhomboid Proteases ◽  
Store Operated Calcium Entry ◽  
Crac Channel

SummaryCalcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease, RHBDL2. We show that RHBDL2 prevents stochastic signalling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved, disease-linked proline residue is responsible for RHBDL2 recognising only the active conformation of Orai1, and cleavage by RHBDL2 is required to sharpen switch-like signalling triggered by store-operated calcium entry. Loss of RHBDL2 control of Orai1 causes severe dysregulation of CRAC channel effectors including transcription factor activation, inflammatory cytokine expression and T cell activation. We propose that this seek-and-destroy function may represent an ancient activity of rhomboid proteases in degrading unwanted signalling proteins.

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.

scholarly journals Monovalent Permeability, Rectification, and Ionic Block of Store-operated Calcium Channels in Jurkat T Lymphocytes

1998 ◽  
Vol 111 (4) ◽  
pp. 521-537 ◽  
Author(s):  
Hubert H. Kerschbaum ◽  
Michael D. Cahalan
Keyword(s):  
T Lymphocytes ◽  
Intracellular Ph ◽  
Calcium Release ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Pipette Solution ◽  
Crac Channels ◽  
Cell Recording ◽  
Voltage Dependent ◽  
Crac Channel

We used whole-cell recording to characterize ion permeation, rectification, and block of monovalent current through calcium release-activated calcium (CRAC) channels in Jurkat T lymphocytes. Under physiological conditions, CRAC channels exhibit a high degree of selectivity for Ca2+, but can be induced to carry a slowly declining Na+ current when external divalent ions are reduced to micromolar levels. Using a series of organic cations as probes of varying size, we measured reversal potentials and calculated permeability ratios relative to Na+, PX/PNa, in order to estimate the diameter of the conducting pore. Ammonium (NH4+) exhibited the highest relative permeability (PNH4/PNa = 1.37). The largest permeant ion, tetramethylammonium with a diameter of 0.55 nm, had PTMA/PNa of 0.09. N-methyl-d-glucamine (0.50 × 0.64 × 1.20 nm) was not measurably permeant. In addition to carrying monovalent current, NH4+ reduced the slow decline of monovalent current (“inactivation”) upon lowering [Ca2+]o. This kinetic effect of extracellular NH4+ can be accounted for by an increase in intracellular pH (pHi), since raising intracellular pH above 8 reduced the extent of inactivation. In addition, decreasing pHi reduced monovalent and divalent current amplitudes through CRAC channels with a pKa of 6.8. In several channel types, Mg2+ has been shown to produce rectification by a voltage-dependent block mechanism. Mg2+ removal from the pipette solution permitted large outward monovalent currents to flow through CRAC channels while also increasing the channel's relative Cs+ conductance and eliminating the inactivation of monovalent current. Boltzmann fits indicate that intracellular Mg2+ contributes to inward rectification by blocking in a voltage-dependent manner, with a zδ product of 1.88. Ca2+ block from the outside was also found to be voltage dependent with zδ of 1.62. These experiments indicate that the CRAC channel, like voltage-gated Ca2+ channels, achieves selectivity for Ca2+ by selective binding in a large pore with current–voltage characteristics shaped by internal Mg2+.

Abstract 14278: Microglial Calcium Activity During Ischemic Stroke

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Kathryn N Kearns ◽  
Lei Liu ◽  
Khadijeh A Sharifi ◽  
Kenneth A Stauderman ◽  
Min S Park ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Calcium Release ◽  
Therapeutic Benefit ◽  
Artery Occlusion ◽  
Two Photon Microscopy ◽  
Crac Channels ◽  
Intracellular Ca ◽  
Crac Channel ◽  
Cerebral Artery Occlusion

Introduction: Ischemic stroke triggers waves of propagating action potentials followed by a loss of homeostatic ion gradients, known as cortical spreading depolarizations (SD). Our data indicate that microglia respond to SD by raising intracellular Ca 2+ , triggering a release of proinflammatory cytokines that may exacerbate post-stroke morbidity. Hypothesis: Inhibiting calcium release-activated calcium (CRAC) channels may block microglial Ca2+ influx and activation to potentially provide therapeutic benefit in ischemic stroke. Methods: We generated a mouse line expressing the Ca2+ indicator GCaMP5 in cortical microglia. Using two-photon microscopy, we imaged microglia in vivo following physical stroke via middle cerebral artery occlusion (MCAo) or chemical stroke via 1M KCl solution application. Controls were compared to mice treated with lipopolysaccharide (LPS) to mimic the inflammation of ischemic stroke. To study CRAC channels as a therapeutic target, we administered CM-EX-137, a CRAC channel blocker, and compared Ca2+ activity to controls. Results: We identified periodical Ca2+ waves in cortical microglia following MCAo (Fig. 1A), or localized KCl application (Fig. 1B-E). Further, when compared to controls, LPS-exposed mice expressed significantly greater microglial Ca2+ activity during KCl-triggered SD (Fig. 1C). Additionally, administration of CM-EX-137 effectively abolished the Ca2+ signals in the microglia and propagation of SD upon application of KCl (Fig. 1E). Conclusions: Blocking the Ca2+ influx into microglia after ischemic stroke may decrease the frequency of SD and reduce microglial activation, potentially leading to smaller stroke volumes and improved clinical outcomes. Figure 1: Microglial Ca 2+ after SD. (A) Ca 2+ transients in microglia after MCAo. (B) KCl-induced Ca 2+ activity in naïve microglia. (C) KCl-induced Ca 2+ activity after LPS. (D, E) KCl-induced Ca 2+ wave after (D) vehicle or (E) CM-EX-137 administration.

Sign in / Sign up

Export Citation Format

Share Document