Matthias Sallinger

All human life starts with a calcium (Ca²⁺) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca²⁺ network are store-operated calcium channels in the cells’ membrane. The best-characterised store-operated channel is the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca²⁺ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca²⁺ signalling through CRAC channels is involved in the hallmarks of cancer development: uncontrolled cell growth, resistance to cell death, migration, invasion, and metastasis. In this work we highlight the importance of Ca²⁺ and its role in human health and disease with focus on CRAC channels.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). This respiratory illness was declared a pandemic by the world health organization (WHO) in March 2020, just a few weeks after being described for the first time. Since then, global research effort has considerably increased humanity’s knowledge about both viruses and disease. It has also spawned several vaccines that have proven to be key tools in attenuating the spread of the pandemic and severity of COVID-19. However, with vaccine-related skepticism being on the rise, as well as breakthrough infections in the vaccinated population and the threat of a complete immune escape variant, alternative strategies in the fight against SARS-CoV-2 are urgently required. Calcium signals have long been known to play an essential role in infection with diverse viruses and thus constitute a promising avenue for further research on therapeutic strategies. In this review, we introduce the pivotal role of calcium signaling in viral infection cascades. Based on this, we discuss prospective calcium-related treatment targets and strategies for the cure of COVID-19 that exploit viral dependence on calcium signals.

The interplay of SK3, a Ca²⁺ sensitive K⁺ ion channel, with Orai1, a Ca²⁺ ion channel, has been reported to increase cytosolic Ca²⁺ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K⁺ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca²⁺ levels that decrease SK3 K⁺ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3–CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role in SK3–Orai1 regulation. Under physiological ionic conditions, STIM1 is able to impede SK3–Orai1 interplay by significantly decreasing their co-localization. Forced STIM1–Orai1 activity and associated Ca²⁺ influx promote SK3 K⁺ currents. The dynamic regulation of Orai1 to boost endogenous SK3 channels was also determined in the human prostate cancer cell line LNCaP.

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca²⁺ from the endoplasmic reticulum (ER), is critical for Ca²⁺ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca²⁺ concentration, and in turn, are responsible for relaying the signal of Ca²⁺ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca²⁺ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca²⁺ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics.

The Ca²⁺ sensor STIM1 and the Ca²⁺ channel Orai1 that form the store-operated Ca²⁺ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca²⁺ selectivity and permeation of Na⁺ in the absence of Ca²⁺. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca²⁺ channel by a high-affinity pore blocker.

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca²⁺ sensor protein that induces permeation of Orai Ca²⁺ channels upon endoplasmic reticulum Ca²⁺-store depletion. A drop in luminal Ca²⁺ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca²⁺ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca²⁺-release-activated Ca²⁺ currents (I_CRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca²⁺ down-stream activation of autophagic processes.