scholarly journals STIM1, an essential and conserved component of store-operated Ca2+ channel function

2005 ◽  
Vol 169 (3) ◽  
pp. 435-445 ◽  
Author(s):  
Jack Roos ◽  
Paul J. DiGregorio ◽  
Andriy V. Yeromin ◽  
Kari Ohlsen ◽  
Maria Lioudyno ◽  
...  
Keyword(s):  
T Cells ◽  
Mammalian Cells ◽  
Hek293 Cells ◽  
Complete Suppression ◽  
Patch Clamp Recording ◽  
Common Component ◽  
Cellular Processes ◽  
Crac Channels ◽  
Crac Channel ◽  
Molecular Components

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

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 Single Channel Properties and Regulated Expression of Ca2+ Release-Activated Ca2+ (Crac) Channels in Human T Cells

2000 ◽  
Vol 150 (6) ◽  
pp. 1435-1444 ◽  
Author(s):  
Alla F. Fomina ◽  
Christopher M. Fanger ◽  
J. Ashot Kozak ◽  
Michael D. Cahalan
Keyword(s):  
T Cells ◽  
Single Channel ◽  
Immunosuppressive Drug ◽  
Kinetic Properties ◽  
Activated T Cells ◽  
Expression Levels ◽  
Crac Channels ◽  
Human T Cells ◽  
Channel Expression ◽  
Crac Channel

Although the crucial role of Ca2+ influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca2+ channels in normal human T lymphocytes. The use of Na+ as the permeant ion in divalent-free solution permitted Ca2+ release-activated Ca2+ (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca2+ store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca2+ in the micromolar range, selective Ca2+ permeation in the millimolar range, and inactivation that depended upon intracellular Mg2+ ions. The number of CRAC channels per cell increased greatly from ∼15 in resting T cells to ∼140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to ∼60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 μM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca2+ influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca2+ influx in human resting T cells, and that the expression of CRAC channels increases ∼10-fold during activation, resulting in enhanced Ca2+ signaling.

scholarly journals Identification of Phosphotyrosine Binding Domain-Containing Proteins as Novel Downstream Targets of the EphA8 Signaling Function

2007 ◽  
Vol 27 (23) ◽  
pp. 8113-8126 ◽  
Author(s):  
Jongdae Shin ◽  
Changkyu Gu ◽  
Eunjeong Park ◽  
Soochul Park
Keyword(s):  
Cell Migration ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Dominant Negative Mutant ◽  
Eph Receptors ◽  
Protein Protein Interaction ◽  
Cellular Processes ◽  
Phosphotyrosine Binding Domain ◽  
Ptb Domain

ABSTRACT Eph receptors and ephrins have been implicated in a variety of cellular processes, including morphology and motility, because of their ability to modulate intricate signaling networks. Here we show that the phosphotyrosine binding (PTB) domain-containing proteins AIDA-1b and Odin are tightly associated with the EphA8 receptor in response to ligand stimulation. Both AIDA-1b and Odin belong to the ankyrin repeat and sterile alpha motif domain-containing (Anks) protein family. The PTB domain of Anks family proteins is crucial for their association with the juxtamembrane domain of EphA8, whereas EphA8 tyrosine kinase activity is not required for this protein-protein interaction. In addition, we found that Odin is a more physiologically relevant partner of EphA8 in mammalian cells. Interestingly, overexpression of the Odin PTB domain alone attenuated EphA8-mediated inhibition of cell migration in HEK293 cells, suggesting that it acts as a dominant-negative mutant of the endogenous Odin protein. More importantly, small interfering RNA-mediated Odin silencing significantly diminished ephrinA5-induced EphA8 signaling effects, which inhibit cell migration in HEK293 cells and retract growing neurites of Neuro2a cells. Taken together, our findings support a possible function for Anks family proteins as scaffolding proteins of the EphA8 signaling pathway.

scholarly journals Molecular mechanisms of STIM/Orai communication

2016 ◽  
Vol 310 (8) ◽  
pp. C643-C662 ◽  
Author(s):  
Isabella Derler ◽  
Isaac Jardin ◽  
Christoph Romanin
Keyword(s):  
Molecular Mechanisms ◽  
Signaling Cascades ◽  
Signaling Cascade ◽  
Cellular Processes ◽  
Crac Channels ◽  
Key Players ◽  
Intracellular Ca ◽  
Crac Channel ◽  
Structural Insights ◽  
Molecular Picture

Ca2+entry into the cell via store-operated Ca2+release-activated Ca2+(CRAC) channels triggers diverse signaling cascades that affect cellular processes like cell growth, gene regulation, secretion, and cell death. These store-operated Ca2+channels open after depletion of intracellular Ca2+stores, and their main features are fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM) and Orai. STIM represents an endoplasmic reticulum-located Ca2+sensor, while Orai forms a highly Ca2+-selective ion channel in the plasma membrane. Functional as well as mutagenesis studies together with structural insights about STIM and Orai proteins provide a molecular picture of the interplay of these two key players in the CRAC signaling cascade. This review focuses on the main experimental advances in the understanding of the STIM1-Orai choreography, thereby establishing a portrait of key mechanistic steps in the CRAC channel signaling cascade. The focus is on the activation of the STIM proteins, the subsequent coupling of STIM1 to Orai1, and the consequent structural rearrangements that gate the Orai channels into the open state to allow Ca2+permeation into the cell.

scholarly journals A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients

2005 ◽  
Vol 202 (5) ◽  
pp. 651-662 ◽  
Author(s):  
Stefan Feske ◽  
Murali Prakriya ◽  
Anjana Rao ◽  
Richard S. Lewis
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
T Cell Response ◽  
Activation Mechanism ◽  
K Channel ◽  
Voltage Sensitivity ◽  
Functional Link ◽  
Crac Channels ◽  
Crac Channel

Engagement of the TCR triggers sustained Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, which helps drive gene expression underlying the T cell response to pathogens. The identity and activation mechanism of CRAC channels at a molecular level are unknown. We have analyzed ion channel expression and function in T cells from SCID patients which display 1–2% of the normal level of Ca2+ influx and severely impaired T cell activation. The lack of Ca2+ influx is not due to deficient regulation of Ca2+ stores or expression of several genes implicated in controlling Ca2+ entry in lymphocytes (kcna3/Kv1.3, kcnn4/IKCa1, trpc1, trpc3, trpv6, stim1). Instead, electrophysiologic measurements show that the influx defect is due to a nearly complete absence of functional CRAC channels. The lack of CRAC channel activity is correlated with diminished voltage sensitivity and slowed activation kinetics of the voltage-dependent Kv1.3 channel. These results demonstrate that CRAC channels provide the major, if not sole, pathway for Ca2+ entry activated by the TCR in human T cells. They also offer evidence for a functional link between CRAC and Kv1.3 channels, and establish a model system for molecular genetic studies of the CRAC channel.

scholarly journals Knockdown of the Ribosomal Protein eL29 in Mammalian Cells Leads to Significant Changes in Gene Expression at the Transcription Level

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1228 ◽  
Author(s):  
Alexander V. Gopanenko ◽  
Alena V. Kolobova ◽  
Maria I. Meschaninova ◽  
Alya G. Venyaminova ◽  
Alexey E. Tupikin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Protein A ◽  
Hek293 Cells ◽  
Transcription Level ◽  
Noticeable Effect ◽  
Cellular Processes ◽  
The One

An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.

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 Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 275 ◽  
Author(s):  
Lili Yue ◽  
Liuqing Wang ◽  
Yangchun Du ◽  
Wei Zhang ◽  
Kozo Hamada ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Channel Activity ◽  
Double Knockout ◽  
Over Expression ◽  
Inositol 1 ◽  
Crac Channels ◽  
Crac Channel ◽  
Trisphosphate Receptor ◽  
Type 3

Being the largest the Ca2+ store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca2+ signalling often involves both Ca2+ release via inositol 1, 4, 5-trisphosphate receptors (IP3R) and store operated Ca2+ entries (SOCE) through Ca2+ release activated Ca2+ (CRAC) channels on plasma membrane (PM). IP3Rs are functionally coupled with CRAC channels and other Ca2+ handling proteins. However, it still remains less well defined as to whether IP3Rs could regulate ER-mediated Ca2+ signals independent of their Ca2+ releasing ability. To address this, we generated IP3Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP3Rs-TKO, IP3Rs-DKO), and systemically examined ER Ca2+ dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca2+ leakage and refilling, as well as SOCE were all significantly reduced in IP3Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP3R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP3R3 is one crucial player in coordinating ER-mediated Ca2+ signalling.

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.

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