CRAC Channel Pore Subunit Research Articles

NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca2+ entry in Tcells.

SUMMARYSustained activation of the Ca2+-release-activated Ca2+ (CRAC) channel is pivotal for effector T cell responses. The mechanisms underlying this sustainability remain poorly understood. We find that plasma membrane localization of ORAI1, the pore subunit of CRAC channels, is limited in effector T cells, with a significant fraction trapped in intracellular vesicles. From a targeted screen, we identify an essential component of ORAI1+ vesicles, naked cuticle homolog 2 (NKD2). Mechanistically, NKD2, an adaptor molecule activated by signaling pathways downstream of T cell receptors, orchestrates trafficking and insertion of ORAI1 + vesicles to the plasma membrane. Together, our findings suggest that T cell receptor (TCR)-stimulation-dependent insertion of ORAI1 into the plasma membrane is essential for sustained Ca2+ signaling and cytokine production in T cells.

NKD2 Mediates Stimulation-Dependent ORAI1 Trafficking to Augment Ca <sup>2+</sup> Entry in T Cells

Sustained activation of the Ca2+ release-activated Ca2+ (CRAC) channel is pivotal for effector T cell responses. The mechanisms underlying this sustainability remain poorly understood. We find that plasma membrane localization of ORAI1, the pore subunit of CRAC channels, is limited in effector T cells, with a significant fraction trapped in intracellular vesicles. From a targeted screen, we have identified an essential component of ORAI1+ vesicles, naked cuticle homolog 2 (NKD2). Mechanistically, NKD2, an adaptor molecule activated by signaling pathways downstream of T cell receptors, orchestrates trafficking and insertion of ORAI1+ vesicles to the plasma membrane. Together, our findings suggest that TCR stimulation-dependent insertion of ORAI1 into the plasma membrane is essential for sustained Ca2+ signaling and cytokine production in T cells.

Immunological Disorders: Regulation of Ca2+ Signaling in T Lymphocytes.

Engagement of T cell receptors (TCRs) with cognate antigens triggers cascades of signaling pathways in helper T cells. TCR signaling is essential for the effector function of helper T cells including proliferation, differentiation, and cytokine production. It also modulates effector T cell fate by inducing cell death, anergy (nonresponsiveness), exhaustion, and generation of regulatory T cells. One of the main axes of TCR signaling is the Ca2+-calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway. Stimulation of TCRs triggers depletion of intracellular Ca2+ store and, in turn, activates store-operated Ca2+ entry (SOCE) to raise the intracellular Ca2+ concentration. SOCE in T cells is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which have been very well characterized in terms of their electrophysiological properties. Identification of STIM1 as a sensor to detect depletion of the endoplasmic reticulum (ER) Ca2+ store and Orai1 as the pore subunit of CRAC channels has dramatically advanced our understanding of the regulatory mechanism of Ca2+ signaling in T cells. In this review, we discuss our current understanding of Ca2+ signaling in T cells with specific focus on the mechanism of CRAC channel activation and regulation via protein interactions. In addition, we will discuss the role of CRAC channels in effector T cells, based on the analyses of genetically modified animal models.

The STIM-Orai Pathway: Orai, the Pore-Forming Subunit of the CRAC Channel.

This chapter focuses on the Orai proteins, Orai1-Orai3, with special emphasis on Orai1, in humans and other mammals, and on the definitive evidence that Orai is the pore subunit of the CRAC channel. It begins by reviewing briefly the defining characteristics of the CRAC channel, then discusses the studies that implicated Orai as part of the store-operated Ca2+ entry pathway and as the CRAC channel pore subunit, and finally examines ongoing work that is providing insights into CRAC channel structure and gating.

Junctate is a Ca 2 + -sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1)

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.

Permeation and gating mechanisms in store-operated CRAC channels

Ca2+ is a ubiquitous signaling messenger mediating many essential cellular functions such as excitability, exocytosis and transcription. Among the different pathways by which cellular Ca2+ signals are generated, the entry of Ca2+ through store-operated Ca2+ release-activated Ca2+ (CRAC) channels has emerged as a widespread mechanism for regulating Ca2+ signaling in many eukaryotic cells. CRAC channels are implicated in the physiology and pathophysiology of numerous cell types, underlie several disease processes including a severe combined immunodeficiency syndrome, and have emerged as major targets for drug development. Although little was known of the molecular mechanisms of CRAC channels for several decades, the discovery of Orai1 as a prototypic CRAC channel pore-subunit, and the identification of STIM1 as the ER Ca2+ sensor, have led to rapid progress in our understanding of many aspects of CRAC channel behavior. This review examines the molecular features of the STIM and Orai proteins that regulate the activation and conduction mechanisms of CRAC channels.

Molecular Determinants of CRAC Channel Gating

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels regulate numerous cellular functions including transcription, motility, and proliferation in many cell types. The discoveries of Orai1, the CRAC channel pore subunit, and STIM1, the ER Ca2+ sensor, have produced rapid progress in our understanding of the molecular features of the CRAC channel pore and the cellular events involved in channel activation. It is now known that following depletion of intracellular Ca2+ stores, STIM1 activates CRAC channels by interacting with the cytoplasmic N- and C-terminal domains of Orai1. Orai1 and Orai3 CRAC channels can additionally be activated in a store-independent fashion by the compound 2-APB. However, the molecular and structural mechanisms of STIM1- and 2-APB-mediated gating remain poorly understood. Using a combination of site-directed mutagenesis and cysteine accessibility analysis, we are probing the structural alterations in the pore that occur during STIM1- or 2-APB-mediated activation of Orai1 and Orai3 channels. Our data indicate that the pore of the CRAC channel changes significantly as it transitions from the closed to the open state. Specifically, STIM1 binding causes large modification of the pore architecture, giving rise to features classically associated with CRAC channels such as its narrow pore and high Ca2+ selectivity. Thus, in addition to serving as the ER Ca2+ sensor and activator of the CRAC channel, STIM1 appears to function as a channel subunit, modifying the structural features of the pore to confer its unique permeation profile in the active state.

Structural Determinants of Ion Permeation in Crac Channels

CRAC channels generate Ca2+ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca2+ selectivity, low Cs+ permeability, and small unitary conductance. To identify the conduction pathway of the transported ions and gain insight into the structural bases of these characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca2+ selectivity is conferred mainly by E106 in the TM1 segment. The pore is sufficiently narrow along much of its length to permit stable coordination of Cd2+ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. Together, these results reveal new insights into the long-sought structural basis for the unique permeation properties of CRAC channels.

Structural determinants of ion permeation in CRAC channels

CRAC channels generate Ca(2+) signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca(2+) selectivity, low Cs(+) permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca(2+) selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd(2+) by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.

Orai1 Mutations Alter Ion Permeation and Ca2+-dependent Fast Inactivation of CRAC Channels: Evidence for Coupling of Permeation and Gating

Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca2+ 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 Ca2+ 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 Ca2+ block, and channel regulation by Ca2+. We find that alteration of the acidic residues lowers Ca2+ 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. Ca2+ binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca2+ binding. Unexpectedly, the mutations diminish Ca2+-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca2+ 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.

Murine ORAI2 Splice Variants Form Functional Ca2+ Release-activated Ca2+ (CRAC) Channels

The stimulation of membrane receptors coupled to the phopholipase C pathway leads to activation of the Ca(2+) release-activated Ca(2+) (CRAC) channels. Recent evidence indicates that ORAI1 is an essential pore subunit of CRAC channels. STIM1 is additionally required for CRAC channel activation. The present study focuses on the genomic organization, tissue expression pattern, and functional properties of the murine ORAI2. Additionally, we report the cloning of the murine ORAI1, ORAI3, and STIM1. Two chromosomal loci were identified for the murine orai2 gene, one containing an intronless gene and a second locus that gives rise to the splice variants ORAI2 long (ORAI2L) and ORAI2 short (ORAI2S). Northern blots revealed a prominent expression of the ORAI2 variants in the brain, lung, spleen, and intestine, while ORAI1, ORAI3, and STIM1 appeared to be near ubiquitously expressed in mice tissues. Specific antibodies detected ORAI2 in RBL 2H3 but not in HEK 293 cells, whereas both cell lines appeared to express ORAI1 and STIM1 proteins. Co-expression experiments with STIM1 and either ORAI1 or ORAI2 variants showed that ORAI2L and ORAI2S enhanced substantially CRAC current densities in HEK 293 but were ineffective in RBL 2H3 cells, whereas ORAI1 strongly amplified CRAC currents in both cell lines. Thus, the capability of ORAI2 variants to form CRAC channels depends strongly on the cell background. Additionally, CRAC channels formed by ORAI2S were strongly sensitive to inactivation by internal Ca(2+). When co-expressed with STIM1 and ORAI1, ORAI2S apparently plays a negative dominant role in the formation of CRAC channels.

Identifying the CRAC Channel

Release of Ca 2+ from intracellular stores elicits Ca 2+ entry through plasma membrane Ca 2+ release-activated Ca 2+ (CRAC) channels. Stromal interaction molecule (Stim in Drosophila , STIM1 in mammals) and Orai (in Drosophila , Orai1 in humans, also known as CRACM1) have been implicated in mediating the CRAC current; however, the precise relation of Orai to the CRAC channel has been unclear. Two groups now use mutational analysis to identify Orai as forming the CRAC channel pore subunit. Yeromin et al . overexpressed wild-type or mutant Orai together with Stim in S1 cells. Store depletion promoted association of the wild-type proteins and elicited a large CRAC current; coexpression of Stim with Orai mutants altered CRAC current properties. A glutamate to aspartate mutation at position 180 (E180D) altered ion selectivity and rectification; a glutamate to alanine mutation at the same location acted as a dominant negative to block native CRAC current; and mutations of aspartate to alanine at 184 and 186 or asparagine to alanine at 188 reduced sensitivity to Gd 3+ block. Prakirya et al . used both biotinylation with a cell-impermeant reagent and immunocytochemical staining of tagged Orai1 expressed in human embryonic kidney (HEK) 293 cells to show that it was a plasma membrane protein. They found that a glutamate to alanine mutation at position 106 (E106D, corresponding to Yeromin et al .’s E180D mutation) altered ion selectivity of Orai1 expressed in T cells deficient in CRAC current and Ca 2+ -influx, as did a glutamate to glutamine mutation at position 190. Thus, both groups conclude that Orai forms the CRAC channel pore subunit. M. Prakriya, S. Feske, Y. Gwack, S. Srikanth, A. Rao, P. G. Hogan, Orai1 is an essential pore subunit of the CRAC channel. Nature 443 , 230-233 (2006). [PubMed] A. V. Yeromin, S. L. Zhang, W. Jiang, Y. Yu, O. Safrina, M. D. Cahalan, Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443 , 226-229 (2006). [PubMed]