Water in peripheral TM-interfaces of Orai1-channels triggers pore opening.

The activation of the Ca-channel Orai1 via the physiological activator stromal interaction molecule 1 (STIM1) requires structural rearrangements within the entire channel complex involving a series of gating checkpoints. Focusing on the gating mechanism operating along the peripheral transmembrane domain (TM) 3/TM4-interface, we report here that some charged substitutions close to the center of TM3 or TM4 lead to constitutively active Orai1 variants triggering nuclear factor of activated T-cell (NFAT) translocation into the nucleus. Molecular dynamics simulations unveil that this gain-of-function correlates with enhanced hydration at peripheral TM-interfaces, leading to increased local structural flexibility of the channel periphery and global conformational changes permitting pore opening.

Essential role of N-terminal SAM regions in STIM1 multimerization and function.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca concentration and directly binds to the store-operated Ca channel Orai1 for its activation when Ca recedes. At high resting ER-Ca concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels.

Photocrosslinking-induced CRAC channel-like Orai1 activation independent of STIM1.

Ca release-activated Ca (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca-sensor STIM1 in the ER membrane and the Ca ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation.

A single amino acid deletion in the ER Ca sensor STIM1 reverses the in vitro and in vivo effects of the Stormorken syndrome-causing R304W mutation.

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca sensor protein STIM1, which forms the Ca release-activated Ca (CRAC) channel together with the plasma membrane channel Orai1. ER Ca store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca store depletion, causing constitutive binding to Orai1 and CRAC channel activation.

Swing-out opening of stromal interaction molecule 1.

Stromal interaction molecule 1 (STIM1) resides in the endoplasmic reticulum (ER) membrane and senses luminal calcium (Ca ) concentration. STIM1 activation involves a large-scale conformational transition that exposes a STIM1 domain termed "CAD/SOAR", - which is required for activation of the calcium channel Orai. Under resting cell conditions, STIM1 assumes a quiescent state where CAD/SOAR is suspended in an intramolecular clamp formed by the coiled-coil 1 domain (CC1) and CAD/SOAR.

Science CommuniCation Developing Scientific Literacy on Calcium: The Involvement of CRAC Currents in Human Health and Disease.

All human life starts with a calcium (Ca) 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.

Calcium Signals during SARS-CoV-2 Infection: Assessing the Potential of Emerging Therapies.

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.

Orai1 Boosts SK3 Channel Activation.

The interplay of SK3, a Ca sensitive K ion channel, with Orai1, a Ca ion channel, has been reported to increase cytosolic Ca 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 Ca levels that decrease SK3 K permeation, co-expressed Orai1 potentiates SK3 currents.

The many states of STIM1.

Harnessing single-molecule FRET illuminates the structural changes necessary for a protein to fine-tune the influx of calcium when reserves inside a cell run low.

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 Ca sensor in the endoplasmic reticulum (ER), and Orai1, the Ca ion channel in the plasma membrane. Ca 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.

Resonance assignment of coiled-coil 3 (CC3) domain of human STIM1.

The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties.

Transmembrane Domain 3 (TM3) Governs Orai1 and Orai3 Pore Opening in an Isoform-Specific Manner.

STIM1-mediated activation of calcium selective Orai channels is fundamental for life. The three Orai channel isoforms, Orai1-3, together with their multiple ways of interplay, ensure their highly versatile role in a variety of cellular functions and tissues in both, health and disease. While all three isoforms are activated in a store-operated manner by STIM1, they differ in diverse biophysical and structural properties.

STIM Proteins: An Ever-Expanding Family.

Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel.

CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions.

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca) ion channels represents the activation of the Ca-sensor protein STIM1 upon Ca store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex.

Mechanism of STIM activation.

Store-operated calcium entry (SOCE) through Orai ion channels is an intracellular signaling pathway that is initiated by ligand-induced depletion of calcium from the endoplasmic reticulum (ER) store. The molecular link between SOCE and ER store depletion is thereby provided by a distinct class of single pass ER transmembrane proteins known as stromal interaction molecules (STIM). STIM proteins are equipped with a precise N-terminal calcium sensing domain that enables them to react to changes of the ER luminal calcium concentration.

Interhelical interactions within the STIM1 CC1 domain modulate CRAC channel activation.

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity.

Oxidative Stress-Induced STIM2 Cysteine Modifications Suppress Store-Operated Calcium Entry.

Store-operated calcium entry (SOCE) through STIM-gated ORAI channels governs vital cellular functions. In this context, SOCE controls cellular redox signaling and is itself regulated by redox modifications. However, the molecular mechanisms underlying this calcium-redox interplay and the functional outcomes are not fully understood.

Sequential activation of STIM1 links Ca with luminal domain unfolding.

The stromal interaction molecule 1 (STIM1) has two important functions, Ca sensing within the endoplasmic reticulum and activation of the store-operated Ca channel Orai1, enabling plasma-membrane Ca influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca ions.

A dual mechanism promotes switching of the Stormorken STIM1 R304W mutant into the activated state.

STIM1 and Orai1 are key components of the Ca-release activated Ca (CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca sensor STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant.

Rapid NMR-scale purification of N,C isotope-labeled recombinant human STIM1 coiled coil fragments.

We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels.

Authentic CRAC channel activity requires STIM1 and the conserved portion of the Orai N terminus.

Calcium (Ca) is an essential second messenger required for diverse signaling processes in immune cells. Ca release-activated Ca (CRAC) channels represent one main Ca entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca sensor in the endoplasmic reticulum, and the Ca ion channel Orai in the plasma membrane.

Communication between N terminus and loop2 tunes Orai activation.

Ca release-activated Ca (CRAC) channels constitute the major Ca entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca-selective Orai channel and the Ca-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating.

Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins.

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3.