The ER/PM microdomain, PI(4,5)P₂ and the regulation of STIM1-Orai1 channel function

Abstract

All forms of cell signaling occur in discreet cellular microdomains in which the ER is the main participant and include microdomains formed by the ER with lysosomes, endosomes, the nucleus, mitochondria and the plasma membrane. In the microdomains the two opposing organelles transfer and exchange constituents including lipids and ions. As is the case for other forms of signaling pathways, many components of the receptor-evoked Ca²⁺ signal are clustered at the ER/PM microdomain, including the Orai1-STIM1 complex. This review discusses recent advances in understanding the molecular components that tether the ER and plasma membrane to form the ER/PM microdomains in which PI(4,5)P₂ is enriched, and how dynamic targeting of the Orai1-STIM1 complex to PI(4,5)P₂-poor and PI(4,5)P₂-rich microdomains controls the activity of Orai1 and its regulation by Ca²⁺ that is mediated by SARAF.

Introduction

All forms of cell signaling occur in discreet cellular microdomains, depending on the cellular function they regulate. Accordingly, Ca²⁺ signaling proteins are assembled into complexes within such microdomains. This can be seen very nicely in polarized cells, such as secretory epithelial cells, where expression of both Ca²⁺ and cAMP signaling proteins is highly polarized. These include G proteins-coupled receptors [1-3], the plasma membrane Ca²⁺ pump (PMCA) and the Endo/Sarcoplasmic reticulum Ca²⁺ pump (SERCA) [4, 5], all IP₃ receptors [6, 7], the Ca²⁺ influx channels TRPC1 [8], TRPC3 [9] and Orai1 [8], the ER Ca²⁺ sensor STIM1 [8] and several adenylyl cyclase isoforms [10]. These proteins are expressed at high levels in the apical pole, the site of many specialized activities of polarized cells. Disruption of such polarized signaling is associated with disease states (Reviewed by Petersen OH, this Special Issue of Cell Calcium).

Organization of signaling complexes in microdomains increases signaling fidelity and strength and allows cross-talk and synergism between signaling modalities. A good example is the well-established cross-talk and synergism between the cAMP and Ca²⁺ signaling pathways. Synergism in activation of exocytosis [11] and fluid secretion [12] in epithelia, exocytosis by endocrine glands [13] and other cellular functions have been known for many years. However, the molecular mechanism of synergism and cross-talk has only been understood recently with increased understanding of the organization of signaling complexes in microdomains. For example, the response of IP₃ receptors (IP₃Rs) to IP₃ is modulated by cAMP/PKA-mediated phosphorylation of the IP₃Rs on specific serine/threonine residues [14, 15]. Ca²⁺-dependent adenylyl cyclases (ACs) are regulated by specific components of the Ca²⁺ signal and both of which are localized at specific ER/PM microdomains. AC8 is associated with the N terminus of Orai1 in an endoplasmic reticulum/plasma membrane (ER/PM) microdomain [16] that may also express TRPC1 [17]. Ca²⁺ entering the cells specifically through Orai1 and TRPC1 activate AC8 [16, 17] (See also review by Cooper DM in this Special Issue of Cell Calcium). Plasma membrane ACs are also regulated by STIM1 [18]. Clustering and translocation of STIM1 to the plasma membrane in response to store depletion increases cAMP by activation of plasma membrane ACs, independent of Orai1, Ca²⁺ influx and an increase in cytoplasmic Ca²⁺ [18]. This may involve formation or expansion of ER/PM microdomains (see below discussion of STIM1 and ER/PM microdomains by Hogan PG in this Special Issue of Cell Calcium).

Synergism between the Ca²⁺ and cAMP pathways is mediated by IRBIT (IP₃Rs binding protein released with IP₃) in the apical pole microdomain of polarized cells [19]. In resting cells IRBIT binds to the IP₃ binding pocket of the IP₃Rs to inhibit Ca²⁺ signaling [20], with IP₃Rs acting to buffer and restrict availability of IRBIT for target proteins. A relatively large increase in IP₃ is required to release IRBIT from the unphosphorylated IP₃Rs. However, under physiological stimulus intensity, activation of the cAMP/PKA pathway phosphorylates the IP₃Rs at specific serine residues [14, 15]. This increases the affinity of the IP₃Rs for IP₃ and facilitates release of IRBIT from the IP₃Rs upon stimulation of Gq-coupled receptors at the apical pole [19]. IRBIT then activates transporters at the luminal membrane, such as the Cl⁻ channel CFTR [21, 22] and the Cl⁻/HCO₃⁻ exchanger slc26a6 [19], resulting in synergistic activation of fluid and HCO₃⁻ secretion by secretory ducts [23].

Many of the microdomains in which signaling complexes are assembled and communicate are formed by the ER with several organelles. This topic has been reviewed extensively in recent years (for example [24, 25]) and in other parts of this Special Issue of Cell Calcium and will be discussed briefly below.

The ER-formed microdomains

In addition to functioning as a hub for signaling pathways, cellular microdomains serve to transmit and exchange signals and molecules between organelles. Microdomains are formed in regions of close apposition of cellular membranes when the membranes of two organelles are within a distance of 10-30 nm and are tethered by structural proteins that span the distance between the two apposed membranes. The ER forms microdomains with multiple organelles, including the plasma membrane [24], the mitochondria [26, 27], lysosomes [28] and the nucleus [25]. Several of the molecular components that form the tethers have been identified in the last few years, although some of these may have additional functions like lipid metabolism and transfer [29, 30] or modulation of channel activity [31]. Another important component of the microdomains is enrichment with specific lipids, most commonly cholesterol in the plasma membrane and phosphatidylinositols in other sites beside the plasma membrane [32].

A group of proteins participating in the formation of most microdomains are the VAMP-associated proteins (VAPs) [33]. The major VAP isoforms are VAP-A and VAP-B. The VAPs are type II integral membrane proteins that are anchored in the ER and bind proteins containing the phenylalanines in an acid tract (FFAT) motifs [34]. Proteins of particular interest containing FFAT motifs that bind to the VAPs are the Oxysterol-binding protein (OSBP)-related proteins (ORPs). The ORPs have in addition to Oxysterol binding site a PH domain that binds phosphatidylinositols [35]. Accordingly, the ORPs bind and transfer lipids, including cholesterol [36] and phosphatidylinositol-4-phosphate (PI4P) [37].

Another group of proteins in the microdomains are the septins [38]. The septins are GTP-binding proteins that heteropolymerize into filaments and rings, which interact with actin filaments and with microtubules [39, 40]. The septins are present in a variety of microdomains including the ER/PM microdomain and the ER/nuclear envelope microdomain [41], the cell division site [42], base of cilia [43] and dendrites [44]. All septins have a central core domain, which contains a polybasic region that can bind phospholipids, a GTP binding domain and a septin unique element [45]. Binding to phospholipids regulates septin filament assembly and, in turn, septins appear to regulate phospholipids at the plasma membrane [46-48]. A major role of the septins is to function as a diffusion barrier to prevent mixing proteins between domains and probably regulate the size and perhaps the dimension of the microdomains [41].

Many of the proteins that function as tethers contain a conserved membrane-binding domain, called synaptotagmin-like-mitochondrial-lipid protein (SMP) domain, that targets the proteins to membrane contact sites [49]. The SMP domain is conserved and has the same function in all species, with the yeast and mammalian homologues targeted to the same site [49].

The ER-formed microdomains are tethered with proteins specific to the organelle that is in close contact with the ER. Other reviews in this Special Issue discuss the role of the various tether proteins in forming the ER/Mitochondria microdomain, the SR-Plasmalema microdomain in muscle and the ER/lysosomes/endosomes microdomain and will not be discussed further here. Below we discuss the ER/PM microdomain in relation to the function of the Orai1/STIM1 complex.

The Molecular components of the ER/PM microdomain

An ER(SR)/PM microdomain has been first noted in muscle by Porter and Palade [50], which are essential for excitation-contraction coupling. Subsequent studies revealed that in skeletal muscle the SR/PM microdomains are formed by interaction of the type 1 ryanodine receptor is the SR and the voltage-activated Ca²⁺ channels in the plasma membrane [51]. In cardiac muscle type 2 ryanodine receptor is in the SR [52]. Assembly and stabilization of the microdomain is dependent on the tethering protein Junctophilin (reviewed in this Special Issue by Takeshima et al). ER/PM microdomains have been observed in the immunological synapse [53], neurons [54] and in many cells at the site of the native and expressed STIM1 [55]. However, the proteins that tether the ER and plasma membrane and their potential functions have only been found recently and studied.

The proteins that tether the ER and plasma membrane (PM) to form the ER/PM microdomain have been examined more extensively in yeast than in mammalian cells, and even now only some of them are known [24, 25, 29]. The first protein to be identified is Ist2 (increased sodium tolerance 2), which was identified as a protein that is asymmetrically distributed to the bud and its polarized localization required border formation by septin [56]. A subsequent important finding was that Ist2 is targeted to the peripheral ER in yeast, with its C terminus polybasic domain tethering the ER to the plasma membrane [57-59]. Moreover, the Ist2 C terminus is sufficient to target several ER and plasma membrane proteins to the ER/PM microdomain [57, 60]. Ist2 is predicted to have eight transmembrane domains and is homologous to the mammalian TMEM16 family proteins [24] that are also known as Anoctamines [61, 62]. However, recent crystallization of TMEM16F indicates that the family has ten transmembrane domains [63], raising the possibility that Ist2 also has ten transmembrane domains. TMEM16A (ANO1) [61, 64, 65] and TMEM16B (ANO2) [66] function as a Ca²⁺-activated Cl⁻ channel and TMEM16F (ANO6) functions as phospholipids scramblase [67] and anion and cation channel [68, 69]. The role and isoform of the TMEM16 (ANO) proteins in tethering the ER/PM is unknown at this time and so is the function of Ist2 besides tethering the ER to the plasma membrane.

A second important group of proteins in the ER/PM microdomain are the Tricalbins (Tcbs) in yeast [49, 70, 71] and their mammalian homologues the Extended-Synaptotagmins (E-Syts) [72, 73]. The Tcbs/E-Syts are integral ER membrane proteins with a membrane anchor hydrophobic patch [73], a conserved SMP domain [49] and multiple Ca²⁺ and PI(4,5)P₂ binding C2 domains [49, 73]. The Tcbs are localized in the peripheral ER at the ER/PM junction [49]. Similarly, all three E-Syts localize to the ER/PM junction and expression of all E-Syts markedly increases the number of the ER/PM microdomains, with E-Syt2 and E-Syt3 being particularly efficient in generating ER/PM microdomains [73]. Interestingly, targeting the E-Syts to the ER/PM microdomain requires PI(4,5)P₂,. It is dependent and is mediated by Ca²⁺ binding to the fifth C2 domain [73].

The last proteins known to be present in and required for formation of the ER/PM microdomains are the VAP proteins discussed above [33]. In yeasts the VAPs bind the Oxysterol binding protein related proteins (ORPs) through an FFAT motif and recruit it to the ER/PM microdomains [74]. The ORPs have a PH domain that anchors them by binding PI4P in the plasma membrane [75]. The ORPs can also interact with the PI4P phosphatase Sac1 and recruit it to the ER/PM microdomain to control PI metabolism at this site [76, 77].

Considering the many functions of the ER/PM microdomains and the functional specificity of three E-Syts with respect to regulation of the STIM1-Orai1 gating (see below), it is likely that additional proteins are present and function in the microdomain and are still to be discovered. A common feature of these proteins might be binding to polar lipids in the plasma membrane. Indeed the E-Syts, Ist2, the ORPs and Sac1 all bind PI(4,5)P₂ and/or PI4P, which serve to anchor them to the plasma membrane. This also allows regulation of proteins in the ER/PM microdomains or their function by PI4P and PI(4,5)P₂.

The Orai1-STIM1 complex in the PI(4,5)P₂ microdomain

To illustrate the functional importance of localization to the ER/PM microdomain below we discuss how localization of the Orai1-STIM1 complex in the ER/PM microdomain affects regulation of channel function. The Orai1-STIM1 complex mediates an essential component of the receptor-evoked Ca²⁺ signal. The receptor-evoked Ca²⁺ signal is initiated by PLC-mediated generation of IP₃ [78] and generation of other second messengers, like cADPR and NAADP [79], to release Ca²⁺ from the ER and the endolysosomal system, respectively. Ca²⁺ release from the ER causes co-clustering of STIM1 and the pore forming Ca²⁺-selective Orai1 channel at the ER/PM microdomain, resulting in activation of Ca²⁺ influx across the plasma membrane. Ca²⁺ influx sustain the Ca²⁺ signal during cell stimulation and replenishes the Ca²⁺ lost from the ER during both physiological Ca²⁺ oscillations and maximal activation of Ca²⁺ release and efflux [80].

Over activation of Ca²⁺ influx causes Ca²⁺ toxicity, which is mediated by excesive activation of TRPC [81] and Orai channels [82]. Orai channels have a dominant role in Ca²⁺ toxicity since Orai1 is essential for Ca²⁺ influx and regulates the activity of the TRPC channels [83, 84]. Hence, cells strictly regulate the activity of the Orai1 channel. The most potent and immediate form of regulator of Orai1 is by Ca²⁺ itself. Two modes of regulation of Orai1 by an increase in cytoplasmic Ca²⁺ have been described and extensively studied; fast Ca²⁺-dependent inactivation (FCDI), which occurs within milliseconds after channel activation, and slow Ca²⁺-dependent inactivation (SCDI) that requires several minutes to be completed (reviewed in [85, 86]). A stretch of negatively charged residues in STIM1 appears to participate in FCDI of Orai1 [87-89], and a protein interacting with STIM1 named SARAF was shown to be essential for SCDI of Orai1 [90]. SARAF interacts with the STIM1 SOAR domain [91], which is regulated by a STIM1 domain downstream of SOAR that we named C terminal inhibitory domain (CTID) [92]. More recently, we discovered that interaction of SARAF with STIM1 requires the presence of the STIM1-Orai1 complex at the ER/PM microdomain, which was used to reveal important features of the ER/PM microdomains and its role in regulating Orai1 function [31]. Interestingly, SARAF regulated both modes of Ca²⁺-mediated inhibition of Orai1, the FCDI and SCDI [31]. How SARAF regulates FCDI remains to be studied.

Since the discovery of STIM1 [93, 94] and Orai1 [95-97] it was clear that the Orai1-STIM1 complex is clustered in ER/PM microdomain and that STIM1 mediates the clustering and targets the complex to the ER/PM microdomain [55, 98]. The proteins that form the microdomain and whether targeting into the microdomain has a regulatory function besides determining the Ca²⁺ influx sites has just begun to be revealed. A significant recent finding was that septin 4 (and likely septin 5) forms a PI(4,5)P₂ microdomain around the Orai1-STIM1 complex [46]. The role of PI(4,5)P₂ in regulation of Orai1 was unclear since PI(4,5)P₂ does not regulate the activity of store-operated Ca²⁺ influx or of Orai1 current [31, 99], although a role for plasma membrane PI4P in targeting of proteins that interact with acidic lipids, like STIM1, has been suggested [99, 100]. Another recent finding showed that altering the ER/PM microdomain by knockout of E-Syt1 prolonged the receptor-evoked Ca²⁺ signal upon repeated stimulation [101], although another study concluded that knockout of E-Syt1 and E-Syt2 alone or together did not affect polarized localization of IP₃Rs and the receptor-stimulated Ca²⁺ signal in hepatocytes [102].

Recently, we reexamined the role of the ER/PM microdomain in the regulation of the Orai1-STIM1 complex and in the role of SARAF [31]. We used inhibition by SARAF as a readout of the STIM1 conformation and localization of the Orai1-STIM1 complex in plasma membrane microdomains. In the resting state SARAF weakly interacts with STIM1 in the ER and only to a limited extent. In response to Ca²⁺ store depletion and formation of the Orai1-STIM1 complex SARAF is recruited to the complex and specifically interacts with STIM1 [31]. Requirement of SARAF and interaction with STIM1 is delayed and its characterization revealed that it requires the presence of the Orai1-STIM1 complex in a PI(4,5)P₂-rich microdomain. This is illustrated in Figure 1. Measurement of FRET between STIM1 and SARAF in the presence of Orai1 to assay the interaction between them shows that the interaction required PI(4,5)P₂ and the PI(4,5)P₂ binding polybasic domain of STIM1 (Fig. 1a). Similarly, the SARAF-mediated SCDI of Orai1 current was eliminated by depletion of PI(4,5)P₂ and by deletion of the STIM1 polybasic domain (Fig. 1b). Importantly, when the Orai1-STIM1 complex was in a PI(4,5)P₂-poor microdomain it was still fully active. It just lost the regulation by SARAF.

Fig. 1. Interaction of SARAF with STIM1 and the SARAF-mediated SCDI requires the STIM1 polybasic domain and plasma membrane PI(4,5)P₂.

Panel (a): FRET was measured between STIM1 (green) or STIM1(ΔK) (red) and SARAF and in the absence of PI(4,5)P₂ (blue). Panel (b): Orai1 current was measured in the presence of STIM1 (black) alone, STIM1+SARAF (red), STIM1(ΔK) (green) and in the presence of STIM1+SARAF and absence of PI(4,5)P₂ (blue). The results are reproduced from [31].

The PI(4,5)P₂-rich microdomain in which SARAF interacts with STIM1 is demarcated and tethered by septin 4 and E-Syt1. Septin 4 was shown to form a PI(4,5)P₂-rich domain around a Orai1-STIM1 complex [46], whereas E-Syt1 in addition to tethering the ER and plasma membrane may also transport PI(4,5)P₂ to the domain, as suggested by the resolved structure of the lipid accommodating barrel shape of E-Syt2 lipid interacting domain [103]. Hence, depletion of septin 4 and E-Syt1 eliminated the STIM1-SARAF FRET and SARAF-mediated SCDI [31]. The role of all E-Syts was examined further by their knockdown and by their overexpression. Remarkably, both the knockdown [31] and the over-expression (Figure 2) showed that the PI(4,5)P₂ rich microdomain at which the Orai1-STIM1 complex is regulated by SARAF is specifically tethered by E-Syt1 with no role for E-Syt2 and E-Syt3. This was quite unexpected considering that all E-Syts tether the ER to the plasma membrane and that E-Syt2 and E-Syt3 appear to do be more efficient than E-Syt1 [73].

Fig. 2. Only E-syt1 formed tethers regulate the Orai1-STIM1 complex.

Panel (a): Orai1 current was measured in the presence of STIM1 (black), STIM1+E-Syt1 (red) and in cells treated with siE-Syt1 (green). Panels (b, c): Orai1 current was measured in the presence of STIM1 (black), STIM1+E-Syt2 (red in b) or E-Syt3 (red in c). Note that only E-Syt1 increased SCDI and knockdown of E-Syt1 reduced SCDI. The results in panel (a) are reproduced from [31].

Together, the findings suggest diversity and specificity in the properties of the ER/PM microdomains. Thus, all E-Syts interact with PI(4,5)P₂ [73] and many processes and proteins have been shown to be regulated by phosphatidylinositols [32]. The ER/PM microdomains also mediate exchange of lipids and other constituents between organelles [25, 29], are the site for initiation of endocytosis [104] and the localization of the various Ras proteins [105]. It should be of interest to determine which of the E-Syts participate in each of these activities that take place at the ER/PM microdomains. However, it is important to note that a single E-Syt is not sufficient to form a defined an ER/PM microdomain. In fact, disruption of the ER/PM microdomain in yeast required deletion of six proteins: the three tricalbins, Ist2, VAP-A and VAP-B [77]. This can also explain the minor phenotype of the E-Syt2/E-Syt3 double knockout mice [106] and the luck of effect of knockdown of E-Syt1/E-Syt2 on localization of IP₃Rs in hepatocytes [102].

An interesting aspect of the ER/PM microdomains is their dynamic nature. This is clearly demonstrated in the formation of the Orai1-STIM1 complex in response to store depletion. Another aspect of the dynamic nature of the microdomains is that they can be poor or rich in particular lipids such as PI(4,5)P₂. It turned that the PI(4,5)P₂-poor and PI(4,5)P₂-rich domains can be selectively accessed by the targeting motifs of the Ras and Lyn proteins, where the Kras motif targets proteins to the PI(4,5)P₂-rich domain and the Hras and Lys motifs target proteins to a PI(4,5)P₂-poor domain. Using these tools we were able to provide evidence to suggest that after its formation the Orai1-STIM1 complex is targeted first to a PI(4,5)P₂-poor domain and is then translocated to a PI(4,5)P₂-rich domain. It is also possible that the PI(4,5)P₂-rich domain is formed around the Orai1-STIM1 complex after its formation in the PI(4,5)P₂-poor domain. Once the Orai1-STIM1 complex is in a PI(4,5)P₂-rich domain SARAF accesses STIM1 to mediated gating of Orai1 by SCDI. This is illustrated in Fig. 3, which shows that upon store depletion clustered STIM1 is transiently present in a PI(4,5)P₂-poor domain and only after a 30-60 seconds delay the complex is seen in a PI(4,5)P₂-rich domain. The overall findings lead to the model in Figure 4 illustrating assembly and targeting of the Orai1-STIM1 complex to a PI(4,5)P₂-poor domain that is free of septin 4 and E-Syt1. In this location the channel is fully active and is not inhibited by Ca²⁺ to allow maximal Ca²⁺ influx. Once cytoplasmic Ca²⁺ raises to a desired concentration, the Orai1-STIM1 complex is translocated to a PI(4,5)P₂-rich domain (or a PI(4,5)P₂-rich domain is formed around it) and now SARAF interacts with STIM1 to initiate SCDI that markedly reduces Ca²⁺ influx to match the Ca²⁺ loss by PMCA and guard against Ca²⁺ toxicity.

Fig. 3. Dynamic translocation of STIM1 between PI(4,5)P₂-poor and PI(4,5)P₂-rich microdomains in response to Ca²⁺ store depletion.

The time course of the translocation of STIM1 to the PI(4,5)P₂-poor and PI(4,5)P₂-rich domains was evaluated by following FRET between CFP-STIM1 and Lys-GFP and Kras-YFP, respectively, and in the presence or absence of PI(4,5)P₂ depletion. Note that store depletion results in a rapid by transient translocation to the Lyn domain (orange) that became more sustained upon PI(4,5)P₂ depletion (green), while the translocation to the Kras domain is delayed (red) and is inhibited by deletion of PI(4,5)P₂ (blue). The results are reproduced from [31].

Fig. 4. Gating of Orai1 by translocation between PI(4,5)P₂-poor and PI(4,5)P₂-rich microdomains.

The ER/PM microdomain is formed by E-Syt1-mediated tethering of the ER and plasma membrane and by other yet unknown proteins that most likely include the VAPs and TMAM16 isoform. This domain is rich in PI(4,5)P₂ that may be calculated by E-Syt1 and controlled/stabilized by septin 4. Adjust to the PI(4,5)P₂-rich microdomain exist a domain that is poor with PI(4,5)P₂ . The two domains can be included within the same Orai1-STIM1 puncta. When the stores are depleted of Ca²⁺ STIM1 opens and form clusters within the ER and then translocates first to the PI(4,5)P₂-poor microdomain to cluster and activate Orai1. At this domain the STIM1 polybasic domain is shielded and does not interact with plasma membrane PI(4,5)P₂. After initiation of Ca²⁺ influx by Orai1 the Orai1-STIM1 complex gradually translocates to the PI(4,5)P₂-rich microdomain and the complex is stabilize in this domain by interaction of STIM1 polybasic domain with PI(4,5)P₂. In this domain STIM1 resumes a conformation that binds SARAF. Binding of SARAF to STIM1 SOAR domain initiates both FCDI and SCDI to reduce Orai1 activity, limiting Ca²⁺ influx to guard against Ca²⁺-dependent toxicity.

Regulation by PI(4,5)P₂ has been demonstrated for many channels and transporters (reviewed in [32, 107]). For most cases it was assumed and in few cases it was shown that the transporters are regulated by direct interaction with the phosphoinositides that bind to a positively charged stretch of amino acids. The findings with the Orai1-STIM1 complex of the role of E-Syt1-formed PI(4,5)P₂-rich domain in channel regulation would suggest that another form of regulation is not by synthesis and breakdown of PI(4,5)P₂, but rather by translocation of the regulated channels and transporters between PI(4,5)P₂ poor and rich microdomains. Now that we know some of the players like E-Syts, septins and other ER/PM microdomain proteins to be found, it should be informative to determine the role of translocation between PI(4,5)P₂ domains in the regulation of the various channels and transporters by PI(4,5)P₂ and other phosphoinositides.

Highlights.

• 1)ER/PM microdomains are formed by tethering proteins such as the E-syts VAPs and the yeast homologues of Ist2
• 2)The ER/PM microdomains are divided to PI(4,5)P₂-poor and PI(4,5)P₂-rich domains
• 3)Dynamic translocation between the PI(4,5)P₂-poor and PI(4,5)P₂-rich domains is a new form of regulation of proteins in the ER/PM microdomains.
• 4)Translocation of the Orai1-STIM1 complex from the PI(4,5)P₂-poor to the PI(4,5)P₂-rich domains determines STIM1 conformation and Orai1 gating by Ca²⁺.