Studies of Structure-Function and Subunit Composition of Orai/STIM Channel

Among all known second messengers in eukaryotic cells, Ca is one of the most versatile and is involved in a multitude of physiological and cellular processes including cell proliferation, growth, gene expression, muscle contraction, and exocytosis/secretion [1,2]. To act as an intracellular signal molecule, Ca has to enter the cell at specific physiological/cellular situations and time points. One major pathway that allows Ca entry into the cells involves the Ca release-activated Ca (CRAC) channels, which belong to the group of store-operated channels (SOC) [3–14]. In the beginning of the CRAC/SOC channel analysis, these channels were studied and characterized using mainly cells of the immune system, that is, T-lymphocytes and mast cells [9,10,14,15]. Finally, in 2005–2006, the major key players forming the functional CRAC channel complex were identified [16–27]: first, the stromal interaction molecule (STIM), which represents the Ca sensor in the endoplasmic reticulum (ER), and second, Orai, which is located in the plasma membrane (PM) and builds the ion-conducting transmembrane (TM) protein complex. Feske and colleagues [16] had studied a defect in CRAC channel function linked to one form of hereditary severe combined immune deficiency (SCID) syndrome, which allowed the identification of the Orai1 (also initially termed CRACM1) channel protein and its mutated form (Orai1 R91W) in SCID patients. By successfully employing and combining a modified linkage analysis with single-nucleotide polymorphism arrays and a RNA interference screen, light was shed on the gene and protein that forms the Ca conducting CRAC channel [16]. Furthermore, the search for homologous proteins using a sequence database research revealed Orai1, Orai2, and Orai3 in higher vertebrates. The three members of the Orai protein family have been analyzed with bioinformatics methods showing that they represent TM proteins with 4 PM spanning domains connected by one intracellular and two extracellular loops and cytosolic N-and C-termini [16,20,28,29]. Several research groups have concentrated on the electrophysiological examination and characterization of Orai proteins revealing the typical high Ca selectivity and low single-channel conductance, concluding that these proteins unequivocally represent the pore-forming entity of the CRAC channel. The CRAC channel-activating protein—stromal interaction molecule (STIM)—has been presented and published by Liou et al. as well as Roos et al. in 2005 [18,19]. Screening about 2300 signaling proteins in S2 cells and HeLa cells using an RNA interference-based gene knockdown approach, 2 homologous proteins highly involved in ER store depletion-mediated Ca influx were elucidated—STIM1 and STIM2. These proteins serve as ER-resident Ca sensors, which closely communicate with the CRAC channels upon Ca depletion of the ER [18,19]. Both STIM1 and STIM2 are single-pass TM proteins with the N-terminus in the ER lumen and the larger C-terminal part facing the cytosol. The ER luminal part, which functions as a Ca sensor of [Ca], contains, among other parts, a Ca-sensing EF hand followed by the α-helical TM domain. The larger cytosolic part of STIM is responsible for coupling to and activation of Orai channels [6,30–33]. Confocal microscopy images reveal an intracellular tubular distribution of STIM1 under resting conditions with full ER Ca stores; however, a small percentage of STIM1 has also been detected in the PM [34]. Lowering the ER-intraluminal Ca concentration represents the initial trigger for STIM1 activation. In the course of store depletion, Ca is released from the STIM1 EF hand domain followed by STIM1 homomerization and translocation to the cell periphery into the so-called ER-PM junctions—regions where the ER membrane is in tight proximity to the plasma membrane. Low [Ca] finally leads to the formation of oligomeric STIM1 clusters/punctae in these microdomains where Orai channels localize as well. This physical coupling of STIM1 to Orai channels therefore induces Ca influx linked to specific downstream signaling and ER store refilling [30,35–39]. Besides the activation of CRAC channels, STIM1 has been shown to play a role in arachidonate as well as leukotriene C4-stimulated Ca channels (see Chapter 11) as well as TRP channel regulation [40]. After the initial characterization of STIM and Orai with limited structural knowledge based on bioinformatics predictions, in 2012, the crystal structures of cytosolic fragments of STIM1 and full-length Orai were reported, allowing new and more focused studies of STIM1 and Orai related to their intra- and intermolecular interactions [41].