Signalling to transcription: Store-operated Ca2+ entry and NFAT activation in lymphocytes (original) (raw)
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A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function
Nature, 2006
Antigen stimulation of immune cells triggers Ca 2þ entry through Ca 2þ release-activated Ca 2þ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca 2þ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca 2þ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca 2þ influx and the CRAC current (I CRAC ). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.
Journal of Biological Chemistry, 2009
- Nature 441, 179 -185). Although heterozygous carriers of the mutation show no clinical symptoms of immunodeficiency, store-operated Ca 2؉ entry in their T cells is impaired, suggesting a gene-dosage effect of the mutation. We have recently demonstrated that the functional CRAC channel pore is composed of a tetrameric assembly of Orai1 subunits (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2008) J. Physiol. 586, 419 -425).
Changing calcium: CRAC channel (STIM and Orai) expression, splicing, and posttranslational modifiers
American journal of physiology. Cell physiology, 2016
A wide variety of cellular function depends on the dynamics of intracellular Ca(2+) signals. Especially for relatively slow and lasting processes such as gene expression, cell proliferation, and often migration, cells rely on the store-operated Ca(2+) entry (SOCE) pathway, which is particularly prominent in immune cells. SOCE is initiated by the sensor proteins (STIM1, STIM2) located within the endoplasmic reticulum (ER) registering the Ca(2+) concentration within the ER, and upon its depletion, cluster and trap Orai (Orai1-3) proteins located in the plasma membrane (PM) into ER-PM junctions. These regions become sites of highly selective Ca(2+) entry predominantly through Orai1-assembled channels, which, among other effector functions, is necessary for triggering NFAT translocation into the nucleus. What is less clear is how the spatial and temporal spread of intracellular Ca(2+) is shaped and regulated by differential expression of the individual SOCE genes and their splice varian...
The Orai1 SCID mutation and CRAC channel function in the heterozygous condition
- Nature 441, 179 -185). Although heterozygous carriers of the mutation show no clinical symptoms of immunodeficiency, store-operated Ca 2؉ entry in their T cells is impaired, suggesting a gene-dosage effect of the mutation. We have recently demonstrated that the functional CRAC channel pore is composed of a tetrameric assembly of Orai1 subunits (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2008) J. Physiol. 586, 419 -425).
Nature Cell Biology, 2010
Orai1 and STIM1 are critical components of Ca 2+ release-activated Ca 2+ (CRAC) channels that mediate store-operated Ca 2+ entry (SOCE) in immune cells. While Orai1 and STIM1 co-cluster and physically interact to mediate SOCE, the cytoplasmic machinery modulating these functions remains poorly understood. We sought to find modulators of Orai1 and STIM1 using affinity protein purification and identified a novel EF-hand protein, CRACR2A (CRAC regulator 2A, EFCAB4B, FLJ33805). We show that CRACR2A directly interacts with Orai1 and STIM1, forming a ternary complex that dissociates at elevated Ca 2+ concentrations. Studies using siRNAmediated knockdown and mutagenesis show that CRACR2A is important for clustering of Orai1 and STIM1 upon store depletion. Expression of an EF-hand mutant of CRACR2A enhanced STIM1 clustering, elevated cytoplasmic Ca 2+ and induced cell death, suggesting its active interaction with CRAC channels. These observations implicate CRACR2A, a novel Ca 2+ binding protein, highly expressed in T cells and conserved in vertebrates, as a key regulator of CRAC channel-mediated SOCE.
Nature, 2005
As the sole Ca 2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx plays an important role in Ca 2+ signaling and many other cellular processes 1-3 . A calcium release-activated calcium (CRAC) channel in T lymphocytes is the best characterized SOC influx channel 4-6 and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation 7-10 . The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously, we identified Stim and the mammalian homolog STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes 11 . Here, we show that expression of EF hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca 2+ store content. By immunofluorescence, EM localization, and surface biotinylation we demonstrate that STIM1 migrates from ER-like sites to the plasma membrane upon depletion of the Ca 2+ store. We propose that STIM1 functions as the missing link between Ca 2+ store depletion and SOC influx, serving as a Ca 2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.
Structural and Functional Mechanisms of CRAC Channel Regulation
Journal of Molecular Biology, 2015
In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca 2+ influx through store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels. The ensuing Ca 2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. The physiological importance of CRAC channels for human health is underscored by studies indicating that mutations in CRAC channel genes produce a spectrum of devastating diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Moreover, from a basic science perspective, CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca 2+-selectivity, store-operated gating, and distinct pore properties and therefore serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Studies in the last two decades have revealed the cellular and molecular choreography of the CRAC channel activation process, and it is now established that opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca 2+ sensors, STIM1 and STIM2. In this review, we summarize the functional and structural mechanisms of CRAC channel regulation, focusing on recent advances in our understanding of the conformational and structural dynamics of CRAC channel gating.
The molecular physiology of CRAC channels
Immunological Reviews, 2009
The Ca 2+ release-activated Ca 2+ (CRAC) channel is a highly Ca 2+ -selective store-operated channel expressed in T cells, mast cells, and various other tissues. CRAC channels regulate critical cellular processes such as gene expression, motility, and the secretion of inflammatory mediators. The identification of Orai1, a key subunit of the CRAC channel pore, and STIM1, the endoplasmic reticulum (ER) Ca 2+ sensor, have provided the tools to illuminate the mechanisms of regulation and the pore properties of CRAC channels. Recent evidence indicates that the activation of CRAC channels by store depletion involves a coordinated series of steps, which include the redistributions of STIM1 and Orai1, direct physical interactions between these proteins, and conformational changes in Orai1, culminating in channel activation. Additional studies have revealed that the high Ca 2+ selectivity of CRAC channels arises from the presence of an intrapore Ca 2+ binding site, the properties of which are finely honed to occlude the permeation of the much more prevalent Na + . Structure-function studies have led to the identification of the potential porebinding sites for Ca 2+ , providing a firm framework for understanding the mechanisms of selectivity and gating of the CRAC channel. This review summarizes recent progress in understanding the mechanisms of CRAC channel activation, pore properties, and modulation.
The Journal of General Physiology, 2007
Ca 2+ entry through store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identifi cation of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca 2+ selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I-II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca 2+ selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca 2+ block, and channel regulation by Ca 2+ . We fi nd that alteration of the acidic residues lowers Ca 2+ selectivity and results in striking increases in Cs + permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs + permeation. Ca 2+ binding to the selectivity fi lter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca 2+ binding. Unexpectedly, the mutations diminish Ca 2+ -mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca 2+ selectivity, increase in Cs + permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels. Correspondence to Murali Prakriya: m-prakriya@northwestern.edu The online version of this article contains supplemental material.