Auxora Vs. Placebo for the Treatment of Patients with Severe COVID-19 Pneumonia: A Randomized Clinical Trial

Background: Calcium release-activated calcium (CRAC) channel inhibitors block proinflammatory cytokine release, preserve endothelial integrity and may effectively treat patients with severe COVID-19 pneumonia. Methods: CARDEA was a phase 2, randomized, double-blind, placebo-controlled trial evaluating the addition of Auxora, a CRAC channel inhibitor, to corticosteroids and standard of care in adults with severe COVID-19 pneumonia. The primary endpoint was time to recovery through Day 60, with secondary endpoints of all-cause mortality at Day 60 and Day 30. Due to declining rates of COVID-19 hospitalizations and encroachment of prohibited medications as standard of care, the trial was stopped early. Results: The pre-specified efficacy set consisted of the 261 patients with a baseline imputed PaO2/FiO2 £200 with 130 and 131 in the Auxora and placebo groups, respectively. Time to recovery was 7 vs. 10 days (P=0.0979) for patients who received Auxora vs. placebo, respectively. The all-cause mortality rate at Day 60 was 13.8% with Auxora vs. 20.6% with placebo (P=0.1449); Day 30 all-cause mortality was 7.7% and 17.6%, respectively (P=0.0165). Similar trends were noted in patients on high flow nasal cannula at baseline or those with a baseline imputed PaO2/FiO2 ≤100. Serious adverse events occurred less frequently in patients treated with Auxora vs. placebo. Conclusions: Auxora was safe and well tolerated with strong signals in both time to recovery and all-cause mortality through Day 60 in patients with severe COVID-19 pneumonia. Further studies of Auxora in combination with corticosteroids and other immunomodulatory agents are warranted.Trial registration: NCT04345614

Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

Unveiling the Gating Mechanism of CRAC Channel: A Computational Study

CRAC channel is ubiquitous and its importance in the regulation of the immune system is testified by the severe immunodeficiencies caused by its mutations. In this work we took advantage of the availability of open and closed structures of this channel to run for the first time simulations of the whole gating process reaching the relevant time-scale with an enhanced sampling technique, Targeted Molecular Dynamics. Our simulations highlighted a complex allosteric propagation of the conformational change from peripheral helices, where the activator STIM1 binds, to the central pore helices. In agreement with mutagenesis data, our simulations revealed the key role of residue H206 whose displacement creates an empty space behind the hydrophobic region of the pore, thus releasing a steric brake and allowing the opening of the channel. Conversely, the process of pore closing culminates with the formation of a bubble that occludes the pore even in the absence of steric block. This mechanism, known as “hydrophobic gating”, has been observed in an increasing number of biological ion channels and also in artificial nanopores. Our study therefore shows promise not only to better understand the molecular origin of diseases caused by disrupted calcium signaling, but also to clarify the mode of action of hydrophobically gated ion channels, possibly even suggesting strategies for the biomimetic design of synthetic nanopores.

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.

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

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.

CRAC Channel Controls the Differentiation of Pathogenic B Cells in Lupus Nephritis

Store-operated Ca2+ release-activated Ca2+ (CRAC) channel is the main Ca2+ influx pathway in lymphocytes and is essential for immune response. Lupus nephritis (LN) is an autoimmune disease characterized by the production of autoantibodies due to widespread loss of immune tolerance. In this study, RNA-seq analysis revealed that calcium transmembrane transport and calcium channel activity were enhanced in naive B cells from patients with LN. The increased expression of ORAI1, ORAI2, and STIM2 in naive B cells from patients with LN was confirmed by flow cytometry and Western blot, implying a role of CRAC channel in B-cell dysregulation in LN. For in vitro study, CRAC channel inhibition by YM-58483 or downregulation by ORAI1-specific small-interfering RNA (siRNA) decreased the phosphorylation of Ca2+/calmodulin-dependent protein kinase2 (CaMK2) and suppressed Blimp-1 expression in primary human B cells, resulting in decreased B-cell differentiation and immunoglobulin G (IgG) production. B cells treated with CaMK2-specific siRNA showed defects in plasma cell differentiation and IgG production. For in vivo study, YM-58483 not only ameliorated the progression of LN but also prevented the development of LN. MRL/lpr lupus mice treated with YM-58483 showed lower percentage of plasma cells in the spleen and reduced concentration of anti-double-stranded DNA antibodies in the sera significantly. Importantly, mice treated with YM-58483 showed decreased immune deposition in the glomeruli and alleviated kidney damage, which was further confirmed in NZM2328 lupus mice. Collectively, CRAC channel controlled the differentiation of pathogenic B cells and promoted the progression of LN. This study provides insights into the pathogenic mechanisms of LN and that CRAC channel could serve as a potential therapeutic target for LN.

Physiological Functions of CRAC Channels

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol-1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Defects in the STIM1 SOARα2 domain affect multiple steps in the CRAC channel activation cascade

The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners. Graphic abstract Legend: Upon intracellular Ca2+ store depletion of the endoplasmic reticulum (ER), Ca2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca2+-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca2+ store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).

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.