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	Figure 1. The molecular choreography of SOCE mediated by ORAI1 and STIM1. At resting condition, the STIM1 luminal EF‐SAM domain is loaded with Ca2+ and remains largely as a monomer. The STIM1 cytoplasmic domain (STIM1ct), consisting of a long coiled‐coil (CC1), a minimal ORAI1 activating region (SOAR or CAD) and a C‐terminal poly‐basic C‐tail (K), likely stays as a dimer and adopts a folded back configuration that keeps itself inactive. Upon ER Ca2+ store depletion, dissociation of Ca2+ from the EF‐SAM domain initiates a destabilization‐coupled oligomerization process in the ER lumen. Conformational changes in the canonical EF‐hand Ca2+‐binding motif disrupt the intramolecular interaction between the EF‐hands and SAM domains, thereby causing aggregation of the luminal EF‐SAM domains. The luminal domain oligomerization further triggers conformational changes that propagate throughout STIM1ct. STIM1ct redeploys itself and adopts a more extended conformation by exposing the SOAR/CAD domain, as well as the poly‐basic C‐tail. Next, activated STIM1 multimerizes and moves toward the ER‐PM junctional sites, where it recruits and directly gates ORAI1 channels possibly through direct physical contacts with both termini of ORAI1. This process is also facilitated by the interaction between its poly‐basic C‐tail and the negatively charged phosphoinositides in the inner leaflet of the plasma membrane. Sustained Ca2+ influx through ORAI1 channels activates downstream effectors such as calcineurin, a Ca2+‐dependent phosphatase that dephosphorylates the nuclear factor of activated T cells (NFAT) and triggers the nuclear translocation of NFAT to regulate gene expression during lymphocyte activation. Ultimately, activated T cells differentiate into various effector cells, including Th1, Th2, Th17, and iTregs.
	Figure 2. Domain organization and structural features of STIM1. (A) Domain architecture of human STIM1. The STIM1 luminal domain consists of a signal peptide (SP), a canonical EF‐hand Ca2+‐binding motif (cEF), a hidden non‐Ca2+‐binding EF‐hand (49), and a sterile alpha motif (SAM). The cytoplasmic domain (STIM1ct) includes a long coiled‐coil (CC1), the SOAR/ CAD domain composed of four alpha helices, an inhibitory region (ID), a proline/ serine rich region (PS), and a polybasic C‐tail (K). Disease‐associated mutations in STIM1 are indicated below the cartoon. Red, gain‐of‐function mutations; blue, loss‐of‐function or hypomorphic mutations. (B) The NMR solution structure of Ca2+‐bound EF‐SAM domain (PDB entry: 2K60). Residues involved in the hydrophobic interactions between the EF‐hand pair (green) and the SAM domain (magenta) are shown as spheres. (C) The X‐ray crystal structure of CC1. Boxed area represents an autoinhibitory region (IH) within CC1. Spheres stand for key residues contributing to coiled coil interactions. (D) The ORAI1 activating domain SOAR forms V‐shaped dimers, each of which consists of four helices to adopt a configuration resembling the letter “R” (PDB entry: 3TEQ). (E) A modeled structure of hexameric human ORAI1 docked by three pairs of STIM1 CC1‐CC2 homodimer (residues 312‐387; PDB entry: 2MAK). The lower panel is a close‐up view of the coiled‐coil interplays between the STIM1 CC1‐CC2 homodimer (green) and the ORAI1 C‐terminal coiled‐coil (yellow). Residues participating in the hydrophobic or potential electrostatic interactions are indicated.
	Figure 3. ORAI1 as the pore‐forming subunit of CRAC channels. (A) The primary sequence and membrane topology of the four‐pass transmembrane protein ORAI1. Disease‐associated mutations (red circle: gain‐of‐function mutation; blue circles: loss‐of‐function mutations) and the Ca2+‐coordinating residue (E106) are indicated. The boundary of transmembrane segments (TM) is deduced on the basis of sequence alignment results between the human ORAI1 and Drosophila Orai. (B) The X‐ray crystal structure of Drosophila Orai that likely represents an inactive conformation (PDB entry: 4HKR). Drosophila Orai exists as a hexamer with the Ca2+ ion (red sphere) coordinated by a glutamate ring formed by E106. The Orai C‐termini (TM4 extension helices, boxed area) in each of the three symmetric units form antiparallel coiled‐coils. (C) The ion conduction pathway of Drosophila Orai. Only two subunits of Orai are shown to aid visualization. Residues lining one side of the TM1 helix from each subunit constitute the ion permeation pathway. The pore contains a glutamate ring, a relatively rigid hydrophobic region and a more flexible basic region that likely binds anions. The corresponding positions in human ORAI1 are indicated in parentheses. (D) The TM4 extension forms an antiparallel coiled coil that is stabilized through intermolecular hydrophobic interactions (equivalent positions in human ORAI1, L273, and L276 are indicated in parentheses).
	Figure 4. Physiological and pathophysiological roles of CRAC channel in human.

Teaching Material

N. T. Nguyen, W. Han, W.-M. Cao, Y. Wang, S. Wen, Y. Huang, M. Li, L. Du, Y. Zhou. Store-Operated Calcium Entry Mediated by ORAI and STIM. Compr Physiol 8: 2018, 981-1002.

Didactic Synopsis

Major Teaching Points:

1. The structure-function relation of calcium release-activated calcium (CRAC) channel composed of ORAI and stromal interaction molecules (STIM) that mediates store-operated calcium entry (SOCE).
2. Molecular steps underlying the functional STIM-ORAI coupling during SOCE activation
   1. The STIM1 EF-SAM domain senses the Ca²⁺ store depletion within the endoplasmic reticulum (ER) lumen and subsequent undergoes structural changes to induce EF-sterile alpha motif aggregation.
   2. The STIM1 luminal domain aggregation prompts a conformational switch in the cytoplasmic domain of STIM1 to overcome autoinhibition and expose the ORAI-activating domain SOAR/CAD.
   3. Activated STIM1 molecules further oligomerize and migrate toward the ER-plasma membrane junctions to directly engage and gate ORAI1 channels.
3. The physiological roles of STIM-ORAI signaling in various tissues and organs, as well as the major pathological conditions arising from loss- or gain-of-function mutations in human ORAI1 and STIM1.

Didactic Legends

The figures—in a freely downloadable PowerPoint format—can be found on the Images tab along with the formal legends published in the article. The following legends to the same figures are written to be useful for teaching.

Figure 1 Teaching points: Understanding the sequential steps of SOCE activation mediated by STIM1 and ORAI1. At resting condition, the STIM1 luminal EF-SAM domain is bound with Ca²⁺ and remains largely as a monomer. The dissociation of Ca²⁺ from the EF-SAM domain due to Ca²⁺ depletion within the ER lumen initiates the oligomerization of luminal EF-SAM domains. The structural rearrangement within ER lumen further triggers the reorganization of the STIM1 transmembrane domain to transduce the signal toward the cytosolic region of STIM1 (STIM1ct). STIM1ct overcomes its autoinhibition-mediated by the CC1 and SOAR coiled coil interactions. As a consequence, the ORAI-activating domain, SOAR/CAD, is exposed, along with the poly-basic C-tail that facilitates STIM1 translocation toward the plasma membrane. Next, STIM1 molecules multimerize to form oligomers larger than dimers and migrate to the ER-PM junctional sites, where they directly gate ORAI channels to induce Ca²⁺ influx.

Figure 2 Teaching points: Understanding the structure of human STIM1, which correlates to its function. STIM1 contains the following essential domains: a signal peptide (SP) that retains STIM1 in ER, a canonical EF-hand Ca²⁺-binding motif (cEF) that senses ER Ca²⁺ fluctuations, a hidden non-Ca²⁺-binding EF-hand, a sterile alpha motif (SAM) involved mediating EF-SAM oligomerization, transmembrane domain (TM) for signal transduction across the ER membrane, a long coiled-coil (CC1) that cages SOAR at rest, the SOAR/ CAD domain composed of four alpha helices that directly activates ORAI, an inhibitory region (ID), a proline/serine-rich region (PS), and a polybasic C-tail (K) that interacts with negatively charged phosphoinositides in the plasma membrane. The disease-associated STIM1 mutations are highlighted. The NMR solution structure and the X-ray crystal structure of STIM1 domains are described in the formal figure legend.

Figure 3 Teaching points: Understanding the 3D structure of Drosophila Orai, which forms the pore-forming subunit of CRAC channel to conduct Ca²⁺ permeation. Drosophila Orai exists as a hexamer with each subunit contains four-pass transmembrane segments. A distinctive feature of the Orai channel is represented by its selective filter, which is formed by a ring of glutamate side chains (E106), a relatively rigid hydrophobic region and a more flexible basic region that likely binds anions.

Figure 4 Teaching points: Physiological and pathophysiological roles of STIM-ORAI signaling in human.