Isoform-Specific Properties of Orai Homologues in Activation, Downstream Signaling, Physiology and Pathophysiology.

Ca2+ ion channels are critical in a variety of physiological events, including cell growth, differentiation, gene transcription and apoptosis. One such essential entry pathway for calcium into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel. It consists of the Ca2+ sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca2+ ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the "classical" Ca2+ ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca2+ store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca2+ permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.

Gating and regulation of the calcium release-activated calcium channel: Recent progress from experiments and molecular modeling.

Calcium release-activated calcium (CRAC) channels are highly calcium ion (Ca2+)-selective channels in the plasma membrane. The transient drop of endoplasmic reticulum Ca2+ level activates its calcium sensor stromal interaction molecule (STIM) and then triggers the gating of the CRAC channel pore unit Orai. This process involves a variety of activities of the immune system. Therefore, understanding how the activation and regulation of the CRAC channel can be accomplished is essential. Here we briefly summarize the recent progress on Orai gating and its regulation by 2-aminoethoxydiphenylborate (2-APB) obtained from structural biology studies, biochemical and electrophysiological measurements, as well as molecular modeling. Indeed, integration between experiments and computations has further deepened our understanding of the channel gating and regulation.

Inhibition of MCF-7 breast cancer cell proliferation by a synthetic peptide derived from the C-terminal sequence of Orai channel.

Intracellular Ca2+ signals play many important cellular functions such as migration, proliferation and differentiation. Store-operated Ca2+ entry (SOCE) is a major route of Ca2+ entry in nonexcitable cells. The activation of SOCE requires engagement between stromal interaction molecule 1 (STIM1) molecules on the endoplasmic reticulum and Ca2+ release-activated Ca2+ (CRAC) channel Orais (Orai1-3) on the plasma membrane. Accumulating evidence indicates that SOCE plays critical roles in cancer cell proliferation, invasion and metastasis. Here, we used the synthetic intracellular peptides derived from the C-termini of Orai channels to treat the breast cancer cells. We have found that Orai3-CT peptide exhibits stronger binding to STIM1 than Orai1-CT, and Orai3-CT peptide acts in a dominant negative fashion, blocking the STIM1-Orai1 interaction and reducing the Ca2+ entry and proliferation of breast cancer cells.

Critical parameters maintaining authentic CRAC channel hallmarks.

Ca2+ ions represent versatile second messengers that regulate a huge diversity of processes throughout the cell's life. One prominent Ca2+ entry pathway into the cell is the Ca2+ release-activated Ca2+ (CRAC) ion channel. It is fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM1) and Orai. STIM1 is a Ca2+ sensor located in the membrane of the endoplasmic reticulum, and Orai, a highly Ca2+ selective ion channel embedded in the plasma membrane. Ca2+ store-depletion leads initially to the activation of STIM1 which subsequently activates Orai channels via direct binding. Authentic CRAC channel hallmarks and biophysical characteristics include high Ca2+ selectivity with a reversal potential in the range of + 50 mV, small unitary conductance, fast Ca2+-dependent inactivation and enhancements in currents upon the switch from a Na+-containing divalent-free to a Ca2+-containing solution. This review provides an overview on the critical determinants and structures within the STIM1 and Orai proteins that establish these prominent CRAC channel characteristics.

Glu106 targeted inhibitors of ORAI1 as potential Ca2+ release-activated Ca2+ (CRAC) channel blockers - molecular modeling and docking studies.

Calcium release-activated calcium modulator 1(ORAI1) is an integral component of the calcium release-activated calcium channel (CRAC) channel complex and plays a central role in regulating Ca2 + concentrations in T-lymphocytes. It is critical for many physiological processes, including cell-proliferation, cytokine production and activation of the immune system. Loss of ORAI1 function is linked with rheumatoid arthritis (RA) and hence pharmacological blockers of ORAI1 could be potential therapeutic agents for the treatment of RA. In this study, we have used a high-throughput screening approach to inhibit the binding of Ca2+ toward ORAI1 and the interactions are verified through induced fit docking. The results hint that these compounds act by possibly binding with, and thereby blocking Ca2+-binding with ORAI1 (E106). The molecular dynamics (MD) simulations shows strong support toward the hit compounds by showing the ligand potency throughout the simulation timescale of 30 ns. We have thus identified a novel class of highly stable, potential lead compounds that directly bind with the selectivity filter region E106 and block Ca2+ binding on ORAI1. This resulting alteration in the pore geometry of ORAI1 due to the strong blocking mechanism of lead compounds will greatly diminish its function and the downstream activities that result from the same including decreased production of cytokines in autoimmune disorders. This study may lay the foundation for finding novel lead compounds for clinical trials that could positively modulate the course of autoimmune disorders with ORAI1 as its specific target.

Structural Insights into Ca2+ Permeation through Orai Channels.

Orai channels belong to the calcium release-activated calcium (CRAC) channel family. Orai channels are responsible for the influx of extracellular Ca2+ that is triggered by Ca2+ depletion from the endoplasmic reticulum (ER); this function is essential for many types of non-excitable cells. Extensive structural and functional studies have advanced the knowledge of the molecular mechanism by which Orai channels are activated. However, the gating mechanism that allows Ca2+ permeation through Orai channels is less well explained. Here, we reviewed and summarized the existing structural studies of Orai channels. We detailed the structural features of Orai channels, described structural comparisons of their closed and open states, and finally proposed a "push-pull" model of Ca2+ permeation.

Structural and Mechanistic Insights of CRAC Channel as a Drug Target in Autoimmune Disorder.

BACKGROUND: Calcium (Ca2+) ion is a major intracellular signaling messenger, controlling a diverse array of cellular functions like gene expression, secretion, cell growth, proliferation, and apoptosis. The major mechanism controlling this Ca2+ homeostasis is store-operated Ca2+ release-activated Ca2+ (CRAC) channels. CRAC channels are integral membrane protein majorly constituted via two proteins, the stromal interaction molecule (STIM) and ORAI. Following Ca2+ depletion in the Endoplasmic reticulum (ER) store, STIM1 interacts with ORAI1 and leads to the opening of the CRAC channel gate and consequently allows the influx of Ca2+ ions. A plethora of studies report that aberrant CRAC channel activity due to Loss- or gain-of-function mutations in ORAI1 and STIM1 disturbs this Ca2+ homeostasis and causes several autoimmune disorders. Hence, it clearly indicates that the therapeutic target of CRAC channels provides the space for a new approach to treat autoimmune disorders. OBJECTIVE: This review aims to provide the key structural and mechanical insights of STIM1, ORAI1 and other molecular modulators involved in CRAC channel regulation. RESULTS AND CONCLUSION: Understanding the structure and function of the protein is the foremost step towards improving the effective target specificity by limiting their potential side effects. Herein, the review mainly focusses on the structural underpinnings of the CRAC channel gating mechanism along with its biophysical properties that would provide the solid foundation to aid the development of novel targeted drugs for an autoimmune disorder. Finally, the immune deficiencies caused due to mutations in CRAC channel and currently used pharmacological blockers with their limitation are briefly summarized.

CRAC channel-based optogenetics.

Store-operated Ca²+ entry (SOCE) constitutes a major Ca2+ influx pathway in mammals to regulate a myriad of physiological processes, including muscle contraction, synaptic transmission, gene expression, and metabolism. In non-excitable cells, the Ca²+ release-activated Ca²+ (CRAC) channel, composed of ORAI and stromal interaction molecules (STIM), constitutes a prototypical example of SOCE to mediate Ca2+ entry at specialized membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and the plasma membrane (PM). The key steps of SOCE activation include the oligomerization of the luminal domain of the ER-resident Ca2+ sensor STIM1 upon Ca²+ store depletion, subsequent signal propagation toward the cytoplasmic domain to trigger a conformational switch and overcome the intramolecular autoinhibition, and ultimate exposure of the minimal ORAI-activating domain to directly engage and gate ORAI channels in the plasma membrane. This exquisitely coordinated cellular event is also facilitated by the C-terminal polybasic domain of STIM1, which physically associates with negatively charged phosphoinositides embedded in the inner leaflet of the PM to enable efficient translocation of STIM1 into ER-PM MCSs. Here, we present recent progress in recapitulating STIM1-mediated SOCE activation by engineering CRAC channels with optogenetic approaches. These STIM1-based optogenetic tools make it possible to not only mechanistically recapture the key molecular steps of SOCE activation, but also remotely and reversibly control Ca²+-dependent cellular processes, inter-organellar tethering at MCSs, and transcriptional reprogramming when combined with CRISPR/Cas9-based genome-editing tools.

Engineered Cross-Linking to Study the Pore Architecture of the CRAC Channel.

ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a prototypical store-operated channel that is essential for the activation of cells of the immune system. Here we describe a convenient yet powerful cross-linking approach to examine the pore architecture of CRAC channels using ORAI1 proteins engineered to contain one or two cysteine residues. The generalizable cross-linking in situ approach can also be readily extended to study other integral membrane proteins expressed in various types of cells.

Molecular Dynamics Simulations of Orai Reveal How the Third Transmembrane Segment Contributes to Hydration and Ca2+ Selectivity in Calcium Release-Activated Calcium Channels.

Calcium release-activated calcium (CRAC) channels open upon depletion of Ca2+ from the endoplasmic reticulum, and when open, they are permeable to a selective flux of calcium ions. The atomic structure of Orai, the pore domain of CRAC channels, from Drosophila melanogaster has revealed many details about conduction and selectivity in this family of ion channels. However, it is still unclear how residues on the third transmembrane helix can affect the conduction properties of the channel. Here, molecular dynamics and Brownian dynamics simulations were employed to analyze how a conserved glutamate residue on the third transmembrane helix (E262) contributes to selectivity. The comparison between the wild-type and mutated channels revealed a severe impact of the mutation on the hydration pattern of the pore domain and on the dynamics of residues K270, and Brownian dynamics simulations proved that the altered configuration of residues K270 in the mutated channel impairs selectivity to Ca2+ over Na+. The crevices of water molecules, revealed by molecular dynamics simulations, are perfectly located to contribute to the dynamics of the hydrophobic gate and the basic gate, suggesting a possible role in channel opening and in selectivity function.

Three-dimensional spatio-temporal modelling of store operated Ca2+ entry: Insights into ER refilling and the spatial signature of Ca2+ signals.

The spatial organisation of Orai channels and SERCA pumps within ER-PM junctions is important for enhancing the versatility and specificity of sub-cellular Ca2+ signals generated during store operated Ca2+ entry (SOCE). In this paper, we present a novel three dimensional spatio-temporal model describing Ca2+ dynamics in the ER-PM junction and sub-PM ER during SOCE. We investigate the role of Orai channel and SERCA pump location to provide insights into how these components shape the Ca2+ signals generated and affect ER refilling. We find that the organisation of Orai channels within the ER-PM junction controls the amplitude and shape of the Ca2+ profile but does not enhance ER refilling. The model shows that ER refilling is only weakly affected by the location of SERCA2b pumps within the ER-PM junction and that the placement of SERCA2a pumps within the ER-PM junction has much greater control over ER refilling.