ORAI1 and ORAI2 modulate murine neutrophil calcium signaling, cellular activation, and host defense

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

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

10.1101/2020.04.04.025262 ◽

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.

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Large-conductance calcium-activated potassium current modulates excitability in isolated canine intracardiac neurons

AJP Cell Physiology ◽

10.1152/ajpcell.00148.2012 ◽

2013 ◽

Vol 304 (3) ◽

pp. C280-C286 ◽

Cited By ~ 8

Author(s):

Guillermo J. Pérez ◽

Mayurika Desai ◽

Seth Anderson ◽

Fabiana S. Scornik

Keyword(s):

Membrane Potential ◽

Calcium Release ◽

Single Channel ◽

Calcium Influx ◽

Ryanodine Receptors ◽

Bk Channels ◽

Release Mechanism ◽

Dependent Manner ◽

Whole Cell ◽

Intracardiac Neurons

We studied principal neurons from canine intracardiac (IC) ganglia to determine whether large-conductance calcium-activated potassium (BK) channels play a role in their excitability. We performed whole cell recordings in voltage- and current-clamp modes to measure ion currents and changes in membrane potential from isolated canine IC neurons. Whole cell currents from these neurons showed fast- and slow-activated outward components. Both current components decreased in the absence of calcium and following 1–2 mM tetraethylammonium (TEA) or paxilline. These results suggest that BK channels underlie these current components. Single-channel analysis showed that BK channels from IC neurons do not inactivate in a time-dependent manner, suggesting that the dynamic of the decay of the fast current component is akin to that of intracellular calcium. Immunohistochemical studies showed that BK channels and type 2 ryanodine receptors are coexpressed in IC principal neurons. We tested whether BK current activation in these neurons occurred via a calcium-induced calcium release mechanism. We found that the outward currents of these neurons were not affected by the calcium depletion of intracellular stores with 10 mM caffeine and 10 μM cyclopiazonic acid. Thus, in canine intracardiac neurons, BK currents are directly activated by calcium influx. Membrane potential changes elicited by long (400 ms) current injections showed a tonic firing response that was decreased by TEA or paxilline. These data strongly suggest that the BK current present in canine intracardiac neurons regulates action potential activity and could increase these neurons excitability.

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P2 receptor-mediated afferent arteriolar vasoconstriction during calcium blockade

AJP Renal Physiology ◽

10.1152/ajprenal.0038.2001 ◽

2002 ◽

Vol 282 (2) ◽

pp. F245-F255 ◽

Cited By ~ 21

Author(s):

Edward W. Inscho ◽

Anthony K. Cook

Keyword(s):

Calcium Channels ◽

Calcium Channel ◽

Calcium Release ◽

Calcium Influx ◽

Receptor Activation ◽

P2 Receptor ◽

Channel Blockade ◽

Calcium Channel Blockade ◽

Vasoconstrictor Response ◽

Arteriolar Diameter

Experiments were performed to determine the role of L-type calcium channels on the afferent arteriolar vasoconstrictor response to ATP and UTP. With the use of the blood-perfused juxtamedullary nephron technique, kidneys were perfused at 110 mmHg and the responses of arterioles to α,β-methylene ATP, ATP, and UTP were determined before and during calcium channel blockade with diltiazem. α,β-Methylene ATP (1.0 μM) decreased arteriolar diameter by 8 ± 1% under control conditions. This response was abolished during calcium channel blockade. In contrast, 10 μM UTP reduced afferent arteriolar diameter to a similar degree before (20 ± 4%) and during (14 ± 4%) diltiazem treatment. Additionally, diltiazem completely prevented the vasoconstriction normally observed with ATP concentrations below 10 μM and attenuated the response obtained with 10 μM ATP. These data demonstrate that L-type calcium channels play a significant role in the vasoconstrictor influences of α,β-methylene ATP and ATP but not UTP. The data also suggest that other calcium influx pathways may participate in the vasoconstrictor response evoked by P2 receptor activation. These observations support previous findings that UTP-mediated elevation of intracellular calcium concentration in preglomerular vascular smooth muscle cells relies primarily on calcium release from intracellular pools, whereas ATP-mediated responses involve both voltage-dependent calcium influx, through L-type calcium channels, and the release of calcium from intracellular stores. These results support the argument that P2X and P2Y receptors influence the diameter of afferent arterioles through activation of disparate signal transduction mechanisms.

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Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1523410113 ◽

2015 ◽

Vol 113 (2) ◽

pp. 440-445 ◽

Cited By ~ 35

Author(s):

Joseph L. Dynes ◽

Anna Amcheslavsky ◽

Michael D. Cahalan

Keyword(s):

Single Channel ◽

Calcium Influx ◽

Optical Recording ◽

Second Phase ◽

Channel Output ◽

Crac Channels ◽

Cell Recording ◽

Crac Channel ◽

Single Channel Currents ◽

Fluorescence Transients

Orai1 comprises the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel. When bound and activated by stromal interacting molecule 1 (STIM1), an endoplasmic reticulum (ER)-resident calcium sensor, Orai1 channels possess high selectivity for calcium but extremely small conductance that has precluded direct recording of single-channel currents. We have developed an approach to visualize Orai1 activity by fusing Orai1 to fluorescent, genetically encoded calcium indicators (GECIs). The GECI–Orai1 probes reveal local Ca2+ influx at STIM1–Orai1 puncta. By whole cell recording, these fusions are fully functional as CRAC channels. When GECI–Orai1 and the CRAC-activating domain (CAD) of STIM1 were coexpressed at low levels and imaged using a total internal reflectance fluorescence microscope, cells exhibited sporadic fluorescence transients the size of diffraction-limited spots and the brightness of a few activated GECI proteins. Transients typically rose rapidly and fell into two classes according to duration: briefer “flickers” lasting only a few hundred milliseconds, and longer “pulses” lasting one to several seconds. The size, intensity, trace shape, frequency, distribution, physiological characteristics, and association with CAD binding together demonstrate that GECI–Orai1 fluorescence transients correspond to single-channel Orai1 responses. Single Orai1 channels gated by CAD, and small Orai1 puncta gated by STIM1, exhibit repetitive fluctuations in single-channel output. CAD binding supports a role in open state maintenance and reveals a second phase of CAD/STIM1 binding after channel opening. These first recordings of single-channel Orai1 currents reveal unexpected dynamics, and when paired with CAD association, support multiple single-channel states.

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

eLife ◽

10.7554/elife.00802 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 31

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.

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

Stroke ◽

10.1161/str.48.suppl_1.tp272 ◽

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.202104007 ◽

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.

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Thimerosal reveals calcium-induced calcium release in unfertilised sea urchin eggs

Zygote ◽

10.1017/s0967199400001271 ◽

1993 ◽

Vol 1 (1) ◽

pp. 35-42 ◽

Cited By ~ 27

Author(s):

Alex McDougall ◽

Isabelle Gillot ◽

Michael Whitaker

Keyword(s):

Sea Urchin ◽

Calcium Release ◽

Calcium Influx ◽

Calcium Transient ◽

Cell Types ◽

Egg Activation ◽

Free Calcium ◽

Slow Increase ◽

Sea Urchin Eggs ◽

Calcium Induced Calcium Release

SummaryThe fertilisation calcium wave in sea urchin eggs triggers the onset of development. The wave is an explosive increase in intracellular free calcium concentration that begins at the point of sperm entry and crosses the egg in about 20 s. Thimerosal is a sulphydryl reagent that sensitises calcium release from intracellular stores in a variety of cell types. Treatment of unfertilised eggs with thimerosal causes a slow increase that results eventually in a large, spontaneous calcium transient and egg activation. At shorter times after thimerosal treatment, egg activation and the calcium transient can be triggered by calcium influx through voltage-gated calcium channels, a form of calcium-induced/calcium release (CICR). Thimerosal treatment also reduces the latency of the fertilisation calcium response and increases the velocity of the fertilisation wave. These results indicate that thimerosal can unmask CICR in sea urchin eggs and suggest that the ryanodine receptor channel based CICR may contribute to explosive calcium release during the fertilisation wave.

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Distinct Structural Domains of Caveolin-1 Independently Regulate Ca2+Release-Activated Ca2+Channels and Ca2+Microdomain-Dependent Gene Expression

Molecular and Cellular Biology ◽

10.1128/mcb.01068-14 ◽

2015 ◽

Vol 35 (8) ◽

pp. 1341-1349 ◽

Cited By ~ 19

Author(s):

Yi-Chun Yeh ◽

Anant B. Parekh

Keyword(s):

Gene Expression ◽

Immune Cell ◽

Nuclear Gene ◽

Calcium Entry ◽

Scaffolding Protein ◽

Activated T Cells ◽

Caveolin 1 ◽

Cell Functions ◽

Crac Channels ◽

Crac Channel

In eukaryotic cells, calcium entry across the cell surface activates nuclear gene expression, a process critically important for cell growth and differentiation, learning, and memory and immune cell functions. In immune cells, calcium entry occurs through store-operated Ca2+release-activated Ca2+(CRAC) channels, comprised of STIM1 and Orai1 proteins. Local calcium entry through CRAC channels activates expression of c-fos- and nuclear factor of activated T cells (NFAT)-dependent genes. Although c-fos and NFAT often interact to activate gene expression synergistically, they can be activated independently of one another to regulate distinct genes. This raises the question of how one transcription factor can be activated and not the other when both are stimulated by the same trigger. Here, we show that the lipid raft scaffolding protein caveolin-1 interacts with the STIM1-Orai1 complex to increase channel activity. Phosphorylation of tyrosine 14 on caveolin-1 regulates CRAC channel-evoked c-fos activation without impacting the NFAT pathway or Orai1 activity. Our results reveal that structurally distinct domains of caveolin-1 selectively regulate the ability of local calcium to activate distinct transcription factors. More generally, our findings reveal that modular regulation by a scaffolding protein provides a simple, yet effective, mechanism to tunnel a local signal down a specific pathway.

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More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms22010471 ◽

2021 ◽

Vol 22 (1) ◽

pp. 471

Author(s):

Sascha Berlansky ◽

Christina Humer ◽

Matthias Sallinger ◽

Irene Frischauf

Keyword(s):

Calcium Release ◽

Expression Patterns ◽

Direct Interaction ◽

Cell Types ◽

Cell Type ◽

Channel Function ◽

Crac Channel ◽

Cell Type Specific ◽

Interacting Networks ◽

Signalling Cascade

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca2+ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics.

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