**2. General overview of STIM and ORAI proteins**

In humans, there are two different genes coding for STIM proteins: STIM1 and STIM2. STIM1 gene shows three known transcriptional variants that generate the proteins STIM1 (canonical),


composition is different across the plasma membrane. This strategy is expensive in terms of the consumption of energy, since the ionic composition of the intracellular medium is modified by pumping out some ions from the cytosol. However, this is cost-efficient because it provided the possibility to proliferate and to gain cellular specialization. In this regard, free calcium (Ca2+) concentration in the cytosol of cells is much lower than that observed in the external medium, so there are mechanisms to remove the excess of free Ca2+ from the cytosol, such as extruding Ca2+ to the extracellular medium or to intracellular Ca2+ stores. This pumping is carried out by plasma membrane Ca2+ pumps and by endoplasmic reticulum Ca2+ pumps, respectively. Also, buffering of Ca2+ with Ca2+-binding proteins is another strategy to keep cytosolic free Ca2+ concentration ([Ca2+]i) within the low nanomolar range (~100 nM). The reason why the [Ca2+]i is tightly controlled is because this level is a second messenger in cell signaling, that is, transient variations of [Ca2+]i communicate a signal to be transmitted. For instance, during fertilization of mammalian oocytes, a series of short-term cytosolic increases of [Ca2+]i occurs in the oocyte for ~20 h after the fusion with sperm. These transient and short spikes are required to release the arrest of the cell cycle and to stimulate the transition from the fertilized oocyte to 1-cell embryo (zygote). The level of [Ca2+]i is also involved in many other cellular events, like the control of gene expression, vesicular trafficking, neurotransmit-

Cytosolic Ca2+ spikes and Ca2+ waves are generated by the opening of Ca2+-specific ion channels located at the plasma membrane and subcellular organelles. When they become activated, plasma membrane Ca2+ channels let the influx of extracellular Ca2+ so the [Ca2+]i rapidly increases, triggering the activation of Ca2+-sensitive effectors. As the main intracellular Ca2+ store, the endoplasmic reticulum (ER) also contains Ca2+ channels that become activated upon certain stimuli to let the transient release of Ca2+ to the cytosol. Then, elevated [Ca2+] i activates Ca2+ pumps to reduce the level of free Ca2+ in the cytosol, making possible the temporal increase of [Ca2+]i which is essential for its role as a messenger. The speed of the Ca2+ rise, as well as the Ca2+ removal, together with the time that this elevation lasts, define the temporal Ca2+ signaling, or Ca2+ signature, a critical point in the activation of subsequent events. Similarly, the specific distribution of Ca2+ channels and pumps define the spatial Ca2+ signature. The spatiotemporal control of the Ca2+ signaling is relevant for determining the regulation of different signaling pathways that finally lead to diverse actions. In summary, it is not only important to know how Ca2+ levels are altered upon specific stimuli, but also their

In this chapter, we summarize the current knowledge regarding the role of the STIM and ORAI proteins family. Because of their role as ER intraluminal Ca2+ sensors, STIM proteins have been recently involved in the modulation of several Ca2+-dependent signaling pathways. ORAI proteins are Ca2+ channels located at the plasma membrane that regulate the influx of Ca2+, in some cases under the control of STIM proteins. Thus, cooperation of both proteins is

In humans, there are two different genes coding for STIM proteins: STIM1 and STIM2. STIM1 gene shows three known transcriptional variants that generate the proteins STIM1 (canonical),

ter release, cytoskeletal dynamics, and so on.

4 Calcium and Signal Transduction

specific duration, shape, and subcellular localization.

critical for Ca2+ influx, Ca2+ signaling, and cell physiology.

**2. General overview of STIM and ORAI proteins**

**Table 1.** Accession number for genes and reference sequences (RefSeq) of transcriptional variants and proteins.

STIM1L (the longest isoform), and STIM1S (the shortest isoform). For STIM2 gene, also three transcriptional variants have been described coding for proteins STIM2, STIM2.1 (or STIM2 beta), and STIM2.2 (or STIM2 alpha) (see **Table 1**).

Also in humans, three different genes code for ORAI proteins: ORAI1, ORAI2, and ORAI3. ORAI1 gene yields a single product (ORAI1 protein, also known as calcium release-activated calcium channel protein 1), whereas ORAI2 gene produces two variants (isoforms 1 and 2), and ORAI3 gene generates a single transcriptional variant and a single protein isoform (**Table 1**).

STIM1 protein is a positive regulator of store-operated Ca2+ entry (SOCE) [1, 2], a Ca2+ influx pathway regulated by the filling status of intracellular Ca2+ stores, mainly the ER. Although there is a significant pool of STIM1 at the plasma membrane, most STIM1 is ER-resident. When located at the ER, STIM1 shows a single transmembrane domain (TM) with the N-terminus toward the intraluminal space of this organelle. The Ca2+-sensitive EF-hand domain, together with a sterile-α-motif (SAM), constitute an intraluminal Ca2+ sensor, with an apparent dissociation constant for Ca2+ of 250 μM [3]. When the intraluminal Ca2+ concentration drops below this Kd, the dissociation of Ca2+ from the EF-hand domain is transmitted to the SAM domain, and to the cytosolic domain of the protein leading to its activation [4]. The cytosolic domain shows a well-studied calcium release-activated calcium (CRAC) activation domain (CAD), with a series of short coiled-coil (CC) domains that bind to ORAI1 plasma membrane channels to activate Ca2+ influx [5]. STIM1 protein also shows a Ser/Pro rich domain, close to a short sequence of four amino acids that binds to the microtubule plus-end binding protein EB1 [6], and finally a terminal Lys-rich domain which is critical for the activation of non-ORAI1 Ca2+ channels, such as TRPCs [7].

STIM2 and STIM1 share >60% sequence identity, and STIM2 also senses intraluminal Ca2+ concentration although with different sensitivity, since the dissociation constant for Ca2+ (~500 μM) is twofold higher than that of STIM1 [8], suggesting that STIM2 becomes activated with smaller changes in intraluminal Ca2+ levels, whereas STIM1 activates Ca2+ entry upon more severe conditions [9].

ORAI1 is a plasma membrane protein with four transmembrane domains with the N- and C-termini oriented to the cytosol. The Ca2+ channel is formed by a hexamer of ORAI1 monomers, with the Ca2+ pore in the center of the hexamer [10, 11]. Both the N- and C-terminal domains are involved in the binding to STIM1 [12]. The paralogues ORAI2 and ORAI3 share 63% and 58% sequence identity with ORAI1, being the extracellular loop 3 that connects TM domain 3 and 4, significantly larger in ORAI3.
