Orai1, STIM1, and their associating partners

Abstract

Store-operated Ca(2+) (SOC) entry is one of the major mechanisms to raise intracellular Ca(2+) concentration in non-excitable cells. Ca(2+)-release-activated Ca(2+) (CRAC) channels are a subtype of SOC channels that are extensively characterized in immune cells. Identification of STIM1 as an endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore subunit has dramatically advanced the molecular understanding of CRAC channels. Recent efforts have focused on understanding the physiological aspects of CRAC channels at an organism level using transgenic animal models and at a molecular level using electrophysiological and biochemical tools. In this review, we summarize our current understanding of the interacting partners of Orai and STIM proteins in the regulation of CRAC channel activity and other non-CRAC channel-related functions.

Figure 1. Molecular components of CRAC channels in T cells

Antigen engagement of T cell receptor stimulates phospholipase C (PLC) that hydrolyses PIP₂ (phosphatidylinositol 4,5-bisphosphate) into IP₃ (inositol 1,4,5 trisphosphate) and diacyl glycerol. Generated IP₃ binds to the IP₃ receptor (IP₃R) on the ER (endoplasmic reticulum) membrane to empty the ER Ca²⁺ store (Phase I, store depletion). ER Ca²⁺ depletion is sensed by STIM1, an EF-hand protein localized in the ER membrane, which translocates into the junctions of plasma membrane and ER membrane. STIM1 couples with Orai1 at the junctions by protein interaction and induces opening of CRAC channels (Phase II, store-operated Ca²⁺ entry). Opening of Orai1 raises the intracellular Ca²⁺ concentration and enhances mitochondrial Ca²⁺ uptake. The increased Ca²⁺ ions trigger a broad range of downstream signalling pathways including the Ca²⁺–calmodulin/calcineurin-NFAT (nuclear factor of activated T cells) pathway (Phase III, transcriptional events). Ca²⁺-bound calmodulin (CaM) forms a complex with a protein phosphatase, calcineurin (Cn), and dephosphorylates the heavily phosphorylated, cytoplasmic NFAT leading to its nuclear translocation. Nuclear NFAT forms a multimeric protein complex of itself or with other transcription factors (e.g. AP-1) to induce gene transcription involved in cytokine production, cell proliferation and cell death depending on the amplitude or duration of [Ca²⁺]_i and the associating partners.

Figure 2. Summary of associating partners of Orai1 and STIM1

A, schematic diagram of Orai1 structure and its interacting partners. Orai1 has four transmembrane segments (TM1–TM4). It has two extracellular domains and the second extracellular domain between TM3 and TM4 contains the asparagine (N²²³) residue involved in glycosylation. The TM1 lines the pore. Orai1 contains three intracellular domains including the N terminus, intracellular loop and C-terminal coiled-coil domain. The interacting partners for each domain such as STIM1, SPCA2, CaM, CRACR2A, and caveolin are indicated. Arginine at position 91 was mutated in patients (R91W) with non-functional CRAC channels suffering from severe combined immunodeficiency (indicated in red). B, associating partners of STIM1. STIM1 contains an ER-luminal N terminus, a single transmembrane segment, and a cytoplasmic region. The cytoplasmic region has three coiled-coil domains: a serine/threonine-rich domain, a proline/glutamate/serine/threonine-rich (PEST) domain, and a poly-basic region at the C terminus. The protein interaction domains of each partner determined by functional analyses or pulldown studies are indicated. Golli protein was shown to interact with the cytoplasmic domain of STIM1, but the exact binding site information is not known (dashed line).

Figure 3. Molecular mechanism of CRAC channel regulation by multiple Ca²⁺-sensing molecules

Schematic diagram showing binding partners of CRAC channels during different stages of activation. Under resting conditions, Orai1 and STIM1 are distributed at the PM and the ER membrane. The subunit stoichiometry of Orai1 under resting conditions is currently contentious (indicated with a question mark). Upon store depletion, STIM1 oligomerizes and translocates to form clusters at the ER–PM junctions. Some junctional proteins well studied in excitable cells (e.g. junctophilins and junctate) may play a role in determining the ER–PM junctions (sites of Orai and STIM clustering) in non-excitable cells. The interaction between the polybasic residues of STIM1 and PM phosphoinositides as well as protein interaction between Orai1 and STIM1 plays an important role in their translocation. Several protein interactors including ERp57, P100, golli and CRACR2A have been identified to modulate STIM1 translocation. By physical interactions with Orai1 through the CAD/SOAR domain (coloured in red), clustered STIM1 recruits and activates Orai1 in the PM–ER junctions. After [Ca²⁺]_i increases, Ca²⁺-bound calmodulin interacts with the N terminus of Orai1 and inactivates CRAC channels (bottom). When ER Ca²⁺ is re-filled by sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), the protein complex dissociates, and Orai1 and STIM1 redistribute to their respective membrane.