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.

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.

The Role of Lipids in CRAC Channel Function.

The composition and dynamics of the lipid membrane define the physical properties of the bilayer and consequently affect the function of the incorporated membrane transporters, which also applies for the prominent Ca release-activated Ca ion channel (CRAC). This channel is activated by receptor-induced Ca store depletion of the endoplasmic reticulum (ER) and consists of two transmembrane proteins, STIM1 and Orai1. STIM1 is anchored in the ER membrane and senses changes in the ER luminal Ca concentration.

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.

Deciphering Molecular Mechanisms and Intervening in Physiological and Pathophysiological Processes of Ca Signaling Mechanisms Using Optogenetic Tools.

Calcium ion channels are involved in numerous biological functions such as lymphocyte activation, muscle contraction, neurotransmission, excitation, hormone secretion, gene expression, cell migration, memory, and aging. Therefore, their dysfunction can lead to a wide range of cellular abnormalities and, subsequently, to diseases. To date various conventional techniques have provided valuable insights into the roles of Ca signaling.

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.

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.

The Orai Pore Opening Mechanism.

Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca concentrations. The primary source of Ca entry into the cell is mediated by the Ca release-activated Ca (CRAC) channel. Its action is stimulated in response to internal Ca store depletion.

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.

Critical parameters maintaining authentic CRAC channel hallmarks.

Ca ions represent versatile second messengers that regulate a huge diversity of processes throughout the cell's life. One prominent Ca entry pathway into the cell is the Ca release-activated Ca (CRAC) ion channel. It is fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM1) and Orai.