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Biochemical and functional properties of the store-operated Ca2+ channels
Cellular Signalling 21 (2009) 457–461
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c e l l s i g
Review
Biochemical and functional properties of the store-operated Ca2+ channels Ginés M. Salido a, Stewart O. Sage b, Juan A. Rosado a,⁎ a b
Department of Physiology (Cell Physiology Research Group), University of Extremadura, Cáceres 10071, Spain Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
a r t i c l e
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Article history: Received 7 October 2008 Accepted 10 November 2008 Available online 13 November 2008 Keywords: Store-operated calcium entry STIM1 TRPC1 Orai1
a b s t r a c t Store-operated calcium entry (SOCE) is a major mechanism for Ca2+ entry in excitable and non-excitable cells. The best-characterised store-operated current is ICRAC, but other currents activated by Ca2+ store depletion have also been reported. The recent identification of the proteins stromal interaction molecule 1 (STIM1) and Orai1 has shed new light on the nature and regulation of SOC channels. STIM1 has been presented as the endoplasmic reticulum (ER) Ca2+ sensor that communicates the content of the Ca2+ stores to the storeoperated channels, a mechanism that involves redistribution of STIM1 to peripheral ER sites and coclustering with the Ca2+ channel subunit, Orai1. Interestingly, TRPC1, which has long been proposed as a SOC channel candidate, associates with Orai1 and STIM1 in a ternary complex that appears to increase the variability of SOC currents available to modulate cell function. © 2008 Elsevier Inc. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Store-operated channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Activation of store-operated channels: dynamic assembly of TRPCs, STIM1 and Orai1 for 4. Biochemical properties of the interaction between STIM1, TRPCs and Orai . . . . . . . 5. New insights into the physiological role of Orai, STIM and TRPC proteins . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The plasma membrane constitutes both a barrier that separates the intracellular and extracellular media, preventing the free movement of ions from one compartment to the other, and a means of regulating communication between these compartments. Certain integral membrane proteins provide structural channels through which ions, such as Ca2+, a universal intracellular messenger involved in essential cellular functions [1], can diffuse following an electrochemical gradient. The free Ca2+ concentration in the extracellular medium is about 1 mM, while cytosolic Ca2+ concentration ([Ca2+]c) in resting cells is approximately 100 nM and certain intracellular organelles, such as the endoplasmic reticulum (ER) have a free luminal Ca2+ concentration ([Ca2+]L) ranging from 0.2–1 mM. These Ca2+ gradients between compartments are essential to the cellular processes regulated by Ca2+. Many cellular agonists evoke increases in [Ca2+]c, which consist of an initial and transient release of Ca2+ from intracellular stores followed
⁎ Corresponding author. E-mail address: [email protected] (J.A. Rosado). 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.11.005
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by a more sustained Ca2+ entry from the extracellular medium through Ca2+-permeable channels. This Ca2+-release activated Ca2+ entry, also termed capacitative or store-operated Ca2+ entry (SOCE), is a major mechanism for Ca2+ influx [2], and the identity of the store-operated Ca2+ (SOC) channels involved has attracted much attention. In this review we consider recent work concerning the nature and biochemical characteristics of these important channels. 2. Store-operated channels SOCE involves a number of non-selective Ca2+-permeable channels with different biophysical properties. The first identified store-operated current, ICRAC, [3] is mediated through a non-voltage activated, inwardly rectifying channel that is highly selective for Ca2+ (pCa2+/pNa+ ~ 1000) [2,4]. More recently, other store-operated currents (ISOCs) of greater conductance and smaller Ca2+ selectivity than ICRAC have been identified by inhibition of the sarco-endoplasmic reticulum Ca2+ATPase (SERCA) with thapsigargin or cell dialysis with the Ca2+ chelator BAPTA. These ISOCS occur through poorly selective cation channels that have been described in vascular endothelial cells (pCa2+/pNa+ ~ 10) [5], aortic and portal vein myocytes ((pCa2+/pNa+ ~ 1 and 50, respectively)
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[6,7], pancreatic acinar cells (pCa2+/pNa+ ~ 0.07) [8], submandibular gland cells (pCa2+/pNa+ ~ 40) and parotid gland cells (pCa2+/pNa+ ~ 4) [9]. The channels conducting ISOCs are cation selective and, in some cases, such as aortic myocytes, do not discriminate between Na+, K+, Cs+, Ca2+, Ba2+ or Sr2+ [6]. The nature of the channels that conduct ICRAC and ISOCS has been a matter of intense investigation and debate. Recently, the protein Orai1 (also named CRACM1 for CRAC modulator) has been proposed to form the pore of the channel mediating ICRAC [10–14]. The involvement of Orai1 in ICRAC was identified by gene mapping in patients with an hereditary severe combined immune deficiency (SCID) syndrome attributed to loss of ICRAC. The ORAI1 gene on chromosome 12 was mutated in SCID patients, and ICRAC was restored by expression of wildtype Orai1 in T cells [10]. The role of Orai1 in ICRAC was confirmed in a whole-genome screen of Drosophila S2 cells [10] with other groups reporting similar results at almost the same time [11,12]. Orai1 is a relatively small (301 amino acid) protein with four predicted transmembrane domains and cytosolic N- and C-terminal tails (Fig. 1) [13]. Point mutations of the conserved transmembrane glutamate residues of Orai1, in positions 106 and 190, have demonstrated their involvement in ion selectivity. An E106Q mutation of Orai1 has been reported to prevent the reconstitution of large ICRAC currents. Interestingly, a charge conserving mutation of glutamate to aspartate (E106D) resulted in a change in Ca2+ selectivity and smaller currents [15]. Mutations of the glutamate residue at position 190 change the ion selectivity of ICRAC from being Ca2+-selective to being selective for monovalent cations [15,16]. The Orai1 protein has been demonstrated to form multimeric ionchannel complexes in the plasma membrane by co-overexpressing two differently tagged versions of the protein in HEK293 cells and performing reciprocal coimmunoprecipitation experiments and immunoblotting with the anti-tag antibodies [15]. The multimeric structure of the channel responsible for ICRAC has recently been demonstrated as a tetramer, where the charged residues from the four individual Orai1 subunits, essential for Ca2+ selectivity, are arranged to form a tetrameric ion pore structure [17]. The channel formed by Orai1 has been reported to be regulated by Ca2+ store depletion with the participation of the intraluminal Ca2+ sensor, STIM1, a process that is reviewed below. Although a pool of Orai1 is constitutively expressed
in the plasma membrane, translocation of further Orai1 subunits to the membrane has been reported upon store depletion [18]. Two homologues of Orai1, Orai2 and Orai3 have been described. These can form heteromultimeric complexes with Orai1 [19]. Orai1, Orai2 and Orai3 are able to form store-operated channels that share a similar high Ca2+ selectivity. In contrast, channels formed by the three homologues exhibit slightly different selectivities for Na+ ions [19]. The Na+ currents through Orai1, Orai2 and Orai3 have been shown to be inhibited by extracellular Ca2+ with a half-maximal concentration of ~ 20 μM [20]. In addition to the three Orai homologues, two spliced variants of Orai2, Orai2 short (Orai2S) and Orai2 long (Orai2L), have been identified in mice. Orai2S is strongly sensitive to inactivation by internal Ca2+ and plays a negative dominant role in the formation of Ca2+ channels when co-expressed with Orai1 [21]. The transient receptor potential (TRP) proteins have also been long suggested as components of the SOC channels. Mutation of the TRP and TRPL genes selectively abolish the delayed, light-sensitive and sustained depolarization due to Na+ and Ca2+ influx in Drosophila photoreceptors [22]. In addition, expression of Drosophila TRP in Sf9 insect cells and Xenopus oocytes resulted in greater Ca2+ influx following store depletion with thapsigargin [23,24]. A number of mammalian homologues of TRP have been found and are classified into three major subfamilies closely related to TRP (TRPC, TRPV and TRPM), two subfamilies that are more distantly related to TRP (TRPP and TRPML), and a less related TRPN group that is expressed in flies and worms [25]. TRP channels are mostly nonselective for monovalent and divalent cations (pCa2+/pNa+ ≤ 10). Exceptions are TRPM4 and TRPM5, which shows a great selectivity for monovalent cations, and the Ca2+-selective TRPV5 and TRPV6, which shows a Ca2+ to Na+ permeability ratio N100. As with Orai proteins, TRP channels lack voltage sensitivity [26]. There is now considerable evidence supporting a role for TRP proteins in the conduction of Ca2+ entry during SOCE. Particular attention has been paid to members of the canonical TRP (TRPC) subfamily. Using different approaches, from overexpression of specific TRP proteins to knockdown of endogenous TRPs and pharmacological studies, it has been suggested that several TRPC proteins can be activated by Ca2+ store depletion. There are seven related members of the TRPC family, designated TRPC1-7. TRPC1, 4 and 5, as well as TRPC3,
Fig. 1. Schematic diagram of the positions of key domains in STIM1, STIM2 and Orai1. The following abbreviations are used: EF: EF hand motif; SAM: sterile alpha motif (SAM) domain; TM: transmembrane region; ERM: ezrin/radixin/moesin domain; S/P: serine/proline-rich region; K: lysine-rich region; E-rich: glutamate-rich motif. The cytosolic region of the proteins is depicted in the figures.
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6 and 7 can be grouped into two major categories based on biochemical and functional similarities [2]. The remaining TRPC2 is a pseudogene in humans [27]. TRPC1 is suggested to be involved in SOCE by antisense experiments in human salivary glands [28] and vascular endothelial cells [29]. In support of this, antibodies directed to the pore-forming region of TRPC1 have been shown to reduce SOCE in vascular smooth muscle cells and human platelets [30,31]. In addition to TRPC1, TRPC3 overexpression enhances SOCE in HEK293 cells, and, although this effect has been attributed to some constitutive activity in TRPC3-expressing cells, receptor stimulation increased the size of the current [32,33]. Suppression of TRPC3 has recently been reported to lead to the disappearance of SOC channels in A431 cells [34]. TRPC4 is suggested to be an important component of the channel supporting ICRAC-like currents, small currents activated by extracellular Ca2+, in human gingival keratinocytes [35]. A role for TRPC6 in SOCE has also been proposed in human platelets. Electrotransjection of a TRPC6 antibody directed towards the C-terminus of the protein reduces both Ca2+ and Mn2+ entry induced by store depletion or stimulation with agonists [36]. In addition to TRPC channels, other members of the TRP family, such as TRPV6, have been suggested as potential mediators of SOCE [37]. The expression of TRPV6 increases a current with similar properties to ICRAC in CHO cells. TRPV6 has also been suggested as a potential component of the protein complex forming SOC channels in lymph node prostate cancer cells [38]. 3. Activation of store-operated channels: dynamic assembly of TRPCs, STIM1 and Orai1 for store-operated calcium influx SOCE was first proposed two decades ago by Putney as a mechanism for receptor-regulated Ca2+ influx that allows refilling of the intracellular Ca2+ pool once agonist stimulation has finished [39,40]. Hence, the regulation of SOC channel gating by the filling state of the intracellular stores is one of the fundamental characteristics of the mechanisms underlying the activation of SOCE and the Ca2+ sensor of the intracellular Ca2+ stores has long been searched for. Using an RNA interference-based screen to identify genes that alter SOCE, STIM1, a transmembrane protein located in the Ca2+ stores, has been identified as the intraluminal Ca2+ sensor that communicates the amount of stored Ca2+ to plasma membrane SOC channels [41,42]. STIM1 has a single transmembrane domain with an EF hand motif near the N-terminus, which is located in the lumen of the endoplasmic reticulum (Fig. 1). STIM1 enhances SOCE when co-expressed with Orai1 [13,43,44], as well as with Orai2 [43], which suggests that these combinations of proteins are sufficient to mediate the process of SOCE. Consistent with this, mutation of the Ca2+ binding EF hand domain of STIM1 resulted in constitutive SOC channel activation without changing the content of the Ca2+ stores [45]. Following Ca2+ store depletion, STIM1 has been shown to move from locations throughout the membrane of the Ca2+ stores to accumulate in regions close to the plasma membrane [46]. Aggregation of STIM1 underneath the plasma membrane induces Orai1 clustering at discrete sites in the plasma membrane directly opposite the STIM1 clusters, resulting in the activation of SOCE [47,48]. STIM1 translocation and formation of STIM1-Orai1 clusters have been reported to occur in an ATP-independent manner [49]. A pool of STIM1 proteins has also been reported to migrate from the Ca2+ stores to the plasma membrane with the EF hand domain facing the extracellular medium in Drosophila S2 cells, human T lymphocytes [45], smooth muscle cells [50] and human platelets [51], where STIM1 might sense extracellular Ca2+ and regulate the opening of SOC channels [52]. In contrast, expression of STIM1 in the plasma membrane has not been detected in HEK293, Jurkat and HeLa cells [42,43]. Like STIM1, STIM2 has been reported to be involved in the activation of SOCE. STIM2 is suggested to be a feedback regulator that keeps basal [Ca2+]c and [Ca2+]L within tight limits [53]. STIM2 activates
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two distinct Ca2+ currents: a store-operated current activated by receptor-induced, IP3-dependent, Ca2+ store depletion and a storeindependent current mediated by cell dialysis during whole-cell perfusion [54]. STIM1 and STIM2 are type I transmembrane proteins containing 685 and 833 amino acids, respectively. The N-terminus is in the lumen of the Ca2+ stores or in the extracellular space when STIM1 localizes in the plasma membrane, and contains a single EFhand Ca2+-binding motif and a sterile alpha motif (SAM) domain, a protein–protein interaction module. The C-terminus of both proteins is in the cytoplasm and consists of two coiled-coil domains, a Ser/Prorich region, and a Lys-rich region [55]. In addition, the C-terminus of STIM1 contains a conserved ERM (ezrin/radixin/moesin) domain and a glutamate-rich region (Fig. 1) [56]. Recent studies have revealed a structural stability difference in the EF-SAM region (residues 58–201 in STIM1 and 149–292 in STIM2) between STIM1 and STIM2, which may account for their different biological functions. For both proteins the EF-SAM region shows a low Ca2+-binding affinity (Kd 0.5 mM). STIM2 EF-SAM is appreciably more stable than STIM1 EF-SAM [53]. The involvement of TRP proteins as components of SOC channels has received support from studies reporting that STIM1 directly or indirectly regulates all TRPC proteins, with the exception of TRPC7, in cells with depleted Ca2+ stores. The interaction between STIM1 and TRPC1 upon Ca2+ store depletion was first presented in human platelets endogenously expressing both proteins [51] and HEK293 cells expressing STIM1 [56]. Expression of the EF hand mutant (D76A) STIM1, which constitutively induces STIM1 clustering into punctae near the plasma membrane and Ca2+ entry, activates TRPC1 and increases its spontaneous activity [56]. In addition to TRPC1, STIM1 has been reported to interact directly with TRPC2, TRPC4 and TRPC5. Indirect regulation of TRPC proteins by STIM1 has been recently proposed. STIM1 induces heteromultimerization of TRPC proteins, conferring on them SOC channel properties. Intracellular STIM1, rather than plasma membrane STIM1, regulates TRPC3 and TRPC6, as well as their role in SOCE, by mediating the interaction of TRPC1 with TRPC3 and of TRPC4 with TRPC6. TRPC hetermultimerization has been shown to be enhanced by Ca2+-mobilizing agonists suggesting that store depletion-induced clustering of STIM1 is required for the formation of heteromeric SOC channels that provide the cell with a variety of ISOCS [57]. The number of putative SOC channels formed by heteromultimerization of channel subunits has been considerably enhanced by the demonstration that Orai proteins interact with TRPCs. The interaction of Orai proteins with TRPCs has been reported to confer STIM1mediated store depletion sensitivity to SOC channels [58]. Especially relevant is the dynamic formation of an Orai1-TRPC1-STIM1 ternary complex stimulated de novo by store depletion [59]. These findings have recently received support from experiments in human platelets where Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1 subunits [60]. The mode of activation of hTRPC forming channels has been suggested to depend on an adequate stoichiometric relationship between the amounts of Orai, STIM and TRPC proteins, so that overexpression of STIM1 and Orai1 would recruit all of the TRPCs in the SOCE pathway; in contrast, overexpression of either STIM or Orai is expected to have no effect since Orai or STIM, respectively, would be limiting [61]. This phenomenon might explain the differences reported concerning the role of TRPCs in store- or receptor-operated Ca2+ entry. In addition, TRPC1-containing heteromultimeric channels have been proposed to be activated by extracellular activators such as thioredoxin [62]. Together these findings suggest that TRPC1 is a point of convergence between intra and extracellular signals. 4. Biochemical properties of the interaction between STIM1, TRPCs and Orai A number of recent studies have addressed the identity of protein regions that are important for SOC channel activation. The EF-SAM
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region of STIM1 has been reported to induce a monomer-to-oligomer transition upon Ca2+ dissociation from the EF-hand motif, where hydrophobic areas in the EF-SAM domain participate in STIM1 oligomerisation [63]. Recent studies have reported that STIM1 oligomerisation is essential for the communication between the Ca2+ stores and the plasma membrane [64]. It has been reported that the Cterminus of STIM1 is able to activate endogenous SOC channels when endogenously expressed STIM1 was effectively knocked down. An interesting finding that deletion analyses have revealed is that the STIM1 ERM domain mediates the selective binding of STIM1 to TRPC1, 2, 4 and 5 but not to the other TRPC isoforms. Another interesting region of STIM1 is the cationic lysine-rich tail. Deletion of this region or substitution of all of the lysine residues inhibits the ability of STIM1 to gate TRPC1 [56]. Finally, the ability of STIM1 to aggregate in clusters close to the plasma membrane has also been reported to reside in the ERM region. Deletion of part of the first coiled-coil domain, located in the ERM region, that includes a glutamate-rich region, and deletion of the S/P-rich region in the C-terminus of STIM1, impairs the translocation of STIM1 towards the plasma membrane in response to depletion of the Ca2+ stores [65]. The cytoplasmic coiled-coil domains of STIM1 have also been suggested to interact with those of Orai1. Coiled-coil regions are known to mediate homotypic as well as heterotypic protein interactions, facilitating the Orai1-STIM1 interactions required for the activation of ICRAC [63]. The N-terminus of Orai1 has been demonstrated to be important for the interaction with STIM1. Orai1 contains a proline- and arginine-rich N-terminal cytoplasmic sequence. Coexpression of STIM1 with full length Orai1 or with a chimeric Orai2 containing the Orai1 N-terminus resulted in enhanced SOCE, while a truncated version of Orai1 containing the N-terminus without the pore-forming transmembrane domain had a dominant negative effect on SOCE [66]. In addition, the C-terminal coiled-coil motif of Orai1 has been presented as a key domain for dynamic coupling to STIM1 [67]. Recent studies have reported that Orai1 is able to form relatively stable complexes with TRPC proteins [58,59], sometimes involving other cytoplasmic Ca2+ handling proteins, such as SERCA [68]. Orai1 has been reported to interact with both TRPC1 N- and C-termini in vitro, although there seems to be a preference for interaction with the C-termini [58]. Altogether, these findings indicate that the EF hand domain of STIM1, which resides within the endoplasmic reticulum, senses [Ca2+]L and inhibits STIM1 activity when stores are filled. When [Ca2+]L decreases Ca2+ dissociates from the EF hand motif and STIM1 activates SOC channels by a mechanism that involves ERM-dependent interaction with the channel subunits and lysine-rich region-dependent activation of the channel. Orai1 and TRPC proteins can independently mediate ICRAC and ISOCs or might interact to form SOC channels with different biophysical properties, providing the cell with valuable tools to regulate particular Ca2+ signals. Orai proteins, by interaction with TRPCs, have been shown to act as regulatory subunits that confer STIM1-mediated store depletion sensitivity to SOC channels composed of pore-forming TRPC subunits [58].
in HEK293 cells supports the involvement of SOCE in Ca2+ oscillations [72]. In particular, a major finding is the essential role for STIM1 and Orai1 in the mechanism of Ca2+ signalling with physiological stimulus strength, when [Ca2+]c levels undergo oscillations [72]. A number of studies have demonstrated the involvement of proteins of the SOCE machinery in cellular functions. The original study identifying Orai as a pore-forming SOC subunit indicated that it is involved in T cell function [10]. In addition, Orai1 has been shown to be crucial in mouse mast cell effector function [73]. On the other hand, STIM1 has also been presented as an essential positive regulator of mast cell activation. This protein is required for Ca2+ influx and subsequent activation of NF-B and NFAT induced by activation of the high-affinity IgE receptor FcRI [65]. STIM1 and Orai1 have also been reported to be essential for the activation of SOCE in skeletal muscle [74] and platelets, where mutations affecting the EF hand motif of human STIM1 have been suggested to be involved in inherited thrombocytopenias [75] and impaired platelet function has been demonstrated in Orai1-deficient mice [76]. There is an overwhelming amount of evidence supporting a role for TRP-containing SOC channels in cellular physiology, particularly in various modalities of sensory transduction. Drosophila TRP is involved in phototransduction, conducting the main component of the lightactivated current in the photoreceptors. A number of TRP channels, such as Drosophila TRPN, some TRPV members, TRPA or the ubiquitous TRPC1 channel are involved in mechanotransduction, where they are likely to be directly activated by membrane lipid tension or by mechanical force delivered through structural proteins. In addition, TRP proteins are involved in thermoreception. TRPM8 is activated by low temperature and TRPV1, TRPV2, TRPV3 and TRPV4 are activated by heat (see [2,77]. TRP-forming channels are also important for a number of developmental processes that require Ca2+ entry, such as the acrosome reaction, spermatogenesis or the determination of left– right asymmetry during embryogenesis [77]. Hence SOC channels are involved in a number of cellular functions, and abnormalities in SOC components might lead to pathological consequences that need to be further investigated. Acknowledgements Supported by M.E.C.-FEDER (BFU2007-60104/BFI). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
5. New insights into the physiological role of Orai, STIM and TRPC proteins
[12] [13]
Physiological concentrations of agonists induce complex and repetitive cycling of [Ca2+]c, known as [Ca2+]c oscillations, spatiotemporal patterns of Ca2+ signals that are recognized as physiological processes [69–72]. Pharmacological evidence indicates that in HEK293 cells, maintenance of Ca2+ oscillations requires SOCE [69]. This observation has been questioned because of the pharmacological approach employed [70] and by studies reporting that SOCE is not required for Ca2+ signalling under physiological conditions in intestinal cells of C. elegans [71]. Nevertheless, a recent study performing functional knockdown of several components of the SOCE machinery
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Contents lists available at ScienceDirect
Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c e l l s i g
Review
Biochemical and functional properties of the store-operated Ca2+ channels Ginés M. Salido a, Stewart O. Sage b, Juan A. Rosado a,⁎ a b
Department of Physiology (Cell Physiology Research Group), University of Extremadura, Cáceres 10071, Spain Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
a r t i c l e
i n f o
Article history: Received 7 October 2008 Accepted 10 November 2008 Available online 13 November 2008 Keywords: Store-operated calcium entry STIM1 TRPC1 Orai1
a b s t r a c t Store-operated calcium entry (SOCE) is a major mechanism for Ca2+ entry in excitable and non-excitable cells. The best-characterised store-operated current is ICRAC, but other currents activated by Ca2+ store depletion have also been reported. The recent identification of the proteins stromal interaction molecule 1 (STIM1) and Orai1 has shed new light on the nature and regulation of SOC channels. STIM1 has been presented as the endoplasmic reticulum (ER) Ca2+ sensor that communicates the content of the Ca2+ stores to the storeoperated channels, a mechanism that involves redistribution of STIM1 to peripheral ER sites and coclustering with the Ca2+ channel subunit, Orai1. Interestingly, TRPC1, which has long been proposed as a SOC channel candidate, associates with Orai1 and STIM1 in a ternary complex that appears to increase the variability of SOC currents available to modulate cell function. © 2008 Elsevier Inc. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Store-operated channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Activation of store-operated channels: dynamic assembly of TRPCs, STIM1 and Orai1 for 4. Biochemical properties of the interaction between STIM1, TRPCs and Orai . . . . . . . 5. New insights into the physiological role of Orai, STIM and TRPC proteins . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The plasma membrane constitutes both a barrier that separates the intracellular and extracellular media, preventing the free movement of ions from one compartment to the other, and a means of regulating communication between these compartments. Certain integral membrane proteins provide structural channels through which ions, such as Ca2+, a universal intracellular messenger involved in essential cellular functions [1], can diffuse following an electrochemical gradient. The free Ca2+ concentration in the extracellular medium is about 1 mM, while cytosolic Ca2+ concentration ([Ca2+]c) in resting cells is approximately 100 nM and certain intracellular organelles, such as the endoplasmic reticulum (ER) have a free luminal Ca2+ concentration ([Ca2+]L) ranging from 0.2–1 mM. These Ca2+ gradients between compartments are essential to the cellular processes regulated by Ca2+. Many cellular agonists evoke increases in [Ca2+]c, which consist of an initial and transient release of Ca2+ from intracellular stores followed
⁎ Corresponding author. E-mail address: [email protected] (J.A. Rosado). 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.11.005
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by a more sustained Ca2+ entry from the extracellular medium through Ca2+-permeable channels. This Ca2+-release activated Ca2+ entry, also termed capacitative or store-operated Ca2+ entry (SOCE), is a major mechanism for Ca2+ influx [2], and the identity of the store-operated Ca2+ (SOC) channels involved has attracted much attention. In this review we consider recent work concerning the nature and biochemical characteristics of these important channels. 2. Store-operated channels SOCE involves a number of non-selective Ca2+-permeable channels with different biophysical properties. The first identified store-operated current, ICRAC, [3] is mediated through a non-voltage activated, inwardly rectifying channel that is highly selective for Ca2+ (pCa2+/pNa+ ~ 1000) [2,4]. More recently, other store-operated currents (ISOCs) of greater conductance and smaller Ca2+ selectivity than ICRAC have been identified by inhibition of the sarco-endoplasmic reticulum Ca2+ATPase (SERCA) with thapsigargin or cell dialysis with the Ca2+ chelator BAPTA. These ISOCS occur through poorly selective cation channels that have been described in vascular endothelial cells (pCa2+/pNa+ ~ 10) [5], aortic and portal vein myocytes ((pCa2+/pNa+ ~ 1 and 50, respectively)
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[6,7], pancreatic acinar cells (pCa2+/pNa+ ~ 0.07) [8], submandibular gland cells (pCa2+/pNa+ ~ 40) and parotid gland cells (pCa2+/pNa+ ~ 4) [9]. The channels conducting ISOCs are cation selective and, in some cases, such as aortic myocytes, do not discriminate between Na+, K+, Cs+, Ca2+, Ba2+ or Sr2+ [6]. The nature of the channels that conduct ICRAC and ISOCS has been a matter of intense investigation and debate. Recently, the protein Orai1 (also named CRACM1 for CRAC modulator) has been proposed to form the pore of the channel mediating ICRAC [10–14]. The involvement of Orai1 in ICRAC was identified by gene mapping in patients with an hereditary severe combined immune deficiency (SCID) syndrome attributed to loss of ICRAC. The ORAI1 gene on chromosome 12 was mutated in SCID patients, and ICRAC was restored by expression of wildtype Orai1 in T cells [10]. The role of Orai1 in ICRAC was confirmed in a whole-genome screen of Drosophila S2 cells [10] with other groups reporting similar results at almost the same time [11,12]. Orai1 is a relatively small (301 amino acid) protein with four predicted transmembrane domains and cytosolic N- and C-terminal tails (Fig. 1) [13]. Point mutations of the conserved transmembrane glutamate residues of Orai1, in positions 106 and 190, have demonstrated their involvement in ion selectivity. An E106Q mutation of Orai1 has been reported to prevent the reconstitution of large ICRAC currents. Interestingly, a charge conserving mutation of glutamate to aspartate (E106D) resulted in a change in Ca2+ selectivity and smaller currents [15]. Mutations of the glutamate residue at position 190 change the ion selectivity of ICRAC from being Ca2+-selective to being selective for monovalent cations [15,16]. The Orai1 protein has been demonstrated to form multimeric ionchannel complexes in the plasma membrane by co-overexpressing two differently tagged versions of the protein in HEK293 cells and performing reciprocal coimmunoprecipitation experiments and immunoblotting with the anti-tag antibodies [15]. The multimeric structure of the channel responsible for ICRAC has recently been demonstrated as a tetramer, where the charged residues from the four individual Orai1 subunits, essential for Ca2+ selectivity, are arranged to form a tetrameric ion pore structure [17]. The channel formed by Orai1 has been reported to be regulated by Ca2+ store depletion with the participation of the intraluminal Ca2+ sensor, STIM1, a process that is reviewed below. Although a pool of Orai1 is constitutively expressed
in the plasma membrane, translocation of further Orai1 subunits to the membrane has been reported upon store depletion [18]. Two homologues of Orai1, Orai2 and Orai3 have been described. These can form heteromultimeric complexes with Orai1 [19]. Orai1, Orai2 and Orai3 are able to form store-operated channels that share a similar high Ca2+ selectivity. In contrast, channels formed by the three homologues exhibit slightly different selectivities for Na+ ions [19]. The Na+ currents through Orai1, Orai2 and Orai3 have been shown to be inhibited by extracellular Ca2+ with a half-maximal concentration of ~ 20 μM [20]. In addition to the three Orai homologues, two spliced variants of Orai2, Orai2 short (Orai2S) and Orai2 long (Orai2L), have been identified in mice. Orai2S is strongly sensitive to inactivation by internal Ca2+ and plays a negative dominant role in the formation of Ca2+ channels when co-expressed with Orai1 [21]. The transient receptor potential (TRP) proteins have also been long suggested as components of the SOC channels. Mutation of the TRP and TRPL genes selectively abolish the delayed, light-sensitive and sustained depolarization due to Na+ and Ca2+ influx in Drosophila photoreceptors [22]. In addition, expression of Drosophila TRP in Sf9 insect cells and Xenopus oocytes resulted in greater Ca2+ influx following store depletion with thapsigargin [23,24]. A number of mammalian homologues of TRP have been found and are classified into three major subfamilies closely related to TRP (TRPC, TRPV and TRPM), two subfamilies that are more distantly related to TRP (TRPP and TRPML), and a less related TRPN group that is expressed in flies and worms [25]. TRP channels are mostly nonselective for monovalent and divalent cations (pCa2+/pNa+ ≤ 10). Exceptions are TRPM4 and TRPM5, which shows a great selectivity for monovalent cations, and the Ca2+-selective TRPV5 and TRPV6, which shows a Ca2+ to Na+ permeability ratio N100. As with Orai proteins, TRP channels lack voltage sensitivity [26]. There is now considerable evidence supporting a role for TRP proteins in the conduction of Ca2+ entry during SOCE. Particular attention has been paid to members of the canonical TRP (TRPC) subfamily. Using different approaches, from overexpression of specific TRP proteins to knockdown of endogenous TRPs and pharmacological studies, it has been suggested that several TRPC proteins can be activated by Ca2+ store depletion. There are seven related members of the TRPC family, designated TRPC1-7. TRPC1, 4 and 5, as well as TRPC3,
Fig. 1. Schematic diagram of the positions of key domains in STIM1, STIM2 and Orai1. The following abbreviations are used: EF: EF hand motif; SAM: sterile alpha motif (SAM) domain; TM: transmembrane region; ERM: ezrin/radixin/moesin domain; S/P: serine/proline-rich region; K: lysine-rich region; E-rich: glutamate-rich motif. The cytosolic region of the proteins is depicted in the figures.
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6 and 7 can be grouped into two major categories based on biochemical and functional similarities [2]. The remaining TRPC2 is a pseudogene in humans [27]. TRPC1 is suggested to be involved in SOCE by antisense experiments in human salivary glands [28] and vascular endothelial cells [29]. In support of this, antibodies directed to the pore-forming region of TRPC1 have been shown to reduce SOCE in vascular smooth muscle cells and human platelets [30,31]. In addition to TRPC1, TRPC3 overexpression enhances SOCE in HEK293 cells, and, although this effect has been attributed to some constitutive activity in TRPC3-expressing cells, receptor stimulation increased the size of the current [32,33]. Suppression of TRPC3 has recently been reported to lead to the disappearance of SOC channels in A431 cells [34]. TRPC4 is suggested to be an important component of the channel supporting ICRAC-like currents, small currents activated by extracellular Ca2+, in human gingival keratinocytes [35]. A role for TRPC6 in SOCE has also been proposed in human platelets. Electrotransjection of a TRPC6 antibody directed towards the C-terminus of the protein reduces both Ca2+ and Mn2+ entry induced by store depletion or stimulation with agonists [36]. In addition to TRPC channels, other members of the TRP family, such as TRPV6, have been suggested as potential mediators of SOCE [37]. The expression of TRPV6 increases a current with similar properties to ICRAC in CHO cells. TRPV6 has also been suggested as a potential component of the protein complex forming SOC channels in lymph node prostate cancer cells [38]. 3. Activation of store-operated channels: dynamic assembly of TRPCs, STIM1 and Orai1 for store-operated calcium influx SOCE was first proposed two decades ago by Putney as a mechanism for receptor-regulated Ca2+ influx that allows refilling of the intracellular Ca2+ pool once agonist stimulation has finished [39,40]. Hence, the regulation of SOC channel gating by the filling state of the intracellular stores is one of the fundamental characteristics of the mechanisms underlying the activation of SOCE and the Ca2+ sensor of the intracellular Ca2+ stores has long been searched for. Using an RNA interference-based screen to identify genes that alter SOCE, STIM1, a transmembrane protein located in the Ca2+ stores, has been identified as the intraluminal Ca2+ sensor that communicates the amount of stored Ca2+ to plasma membrane SOC channels [41,42]. STIM1 has a single transmembrane domain with an EF hand motif near the N-terminus, which is located in the lumen of the endoplasmic reticulum (Fig. 1). STIM1 enhances SOCE when co-expressed with Orai1 [13,43,44], as well as with Orai2 [43], which suggests that these combinations of proteins are sufficient to mediate the process of SOCE. Consistent with this, mutation of the Ca2+ binding EF hand domain of STIM1 resulted in constitutive SOC channel activation without changing the content of the Ca2+ stores [45]. Following Ca2+ store depletion, STIM1 has been shown to move from locations throughout the membrane of the Ca2+ stores to accumulate in regions close to the plasma membrane [46]. Aggregation of STIM1 underneath the plasma membrane induces Orai1 clustering at discrete sites in the plasma membrane directly opposite the STIM1 clusters, resulting in the activation of SOCE [47,48]. STIM1 translocation and formation of STIM1-Orai1 clusters have been reported to occur in an ATP-independent manner [49]. A pool of STIM1 proteins has also been reported to migrate from the Ca2+ stores to the plasma membrane with the EF hand domain facing the extracellular medium in Drosophila S2 cells, human T lymphocytes [45], smooth muscle cells [50] and human platelets [51], where STIM1 might sense extracellular Ca2+ and regulate the opening of SOC channels [52]. In contrast, expression of STIM1 in the plasma membrane has not been detected in HEK293, Jurkat and HeLa cells [42,43]. Like STIM1, STIM2 has been reported to be involved in the activation of SOCE. STIM2 is suggested to be a feedback regulator that keeps basal [Ca2+]c and [Ca2+]L within tight limits [53]. STIM2 activates
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two distinct Ca2+ currents: a store-operated current activated by receptor-induced, IP3-dependent, Ca2+ store depletion and a storeindependent current mediated by cell dialysis during whole-cell perfusion [54]. STIM1 and STIM2 are type I transmembrane proteins containing 685 and 833 amino acids, respectively. The N-terminus is in the lumen of the Ca2+ stores or in the extracellular space when STIM1 localizes in the plasma membrane, and contains a single EFhand Ca2+-binding motif and a sterile alpha motif (SAM) domain, a protein–protein interaction module. The C-terminus of both proteins is in the cytoplasm and consists of two coiled-coil domains, a Ser/Prorich region, and a Lys-rich region [55]. In addition, the C-terminus of STIM1 contains a conserved ERM (ezrin/radixin/moesin) domain and a glutamate-rich region (Fig. 1) [56]. Recent studies have revealed a structural stability difference in the EF-SAM region (residues 58–201 in STIM1 and 149–292 in STIM2) between STIM1 and STIM2, which may account for their different biological functions. For both proteins the EF-SAM region shows a low Ca2+-binding affinity (Kd 0.5 mM). STIM2 EF-SAM is appreciably more stable than STIM1 EF-SAM [53]. The involvement of TRP proteins as components of SOC channels has received support from studies reporting that STIM1 directly or indirectly regulates all TRPC proteins, with the exception of TRPC7, in cells with depleted Ca2+ stores. The interaction between STIM1 and TRPC1 upon Ca2+ store depletion was first presented in human platelets endogenously expressing both proteins [51] and HEK293 cells expressing STIM1 [56]. Expression of the EF hand mutant (D76A) STIM1, which constitutively induces STIM1 clustering into punctae near the plasma membrane and Ca2+ entry, activates TRPC1 and increases its spontaneous activity [56]. In addition to TRPC1, STIM1 has been reported to interact directly with TRPC2, TRPC4 and TRPC5. Indirect regulation of TRPC proteins by STIM1 has been recently proposed. STIM1 induces heteromultimerization of TRPC proteins, conferring on them SOC channel properties. Intracellular STIM1, rather than plasma membrane STIM1, regulates TRPC3 and TRPC6, as well as their role in SOCE, by mediating the interaction of TRPC1 with TRPC3 and of TRPC4 with TRPC6. TRPC hetermultimerization has been shown to be enhanced by Ca2+-mobilizing agonists suggesting that store depletion-induced clustering of STIM1 is required for the formation of heteromeric SOC channels that provide the cell with a variety of ISOCS [57]. The number of putative SOC channels formed by heteromultimerization of channel subunits has been considerably enhanced by the demonstration that Orai proteins interact with TRPCs. The interaction of Orai proteins with TRPCs has been reported to confer STIM1mediated store depletion sensitivity to SOC channels [58]. Especially relevant is the dynamic formation of an Orai1-TRPC1-STIM1 ternary complex stimulated de novo by store depletion [59]. These findings have recently received support from experiments in human platelets where Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1 subunits [60]. The mode of activation of hTRPC forming channels has been suggested to depend on an adequate stoichiometric relationship between the amounts of Orai, STIM and TRPC proteins, so that overexpression of STIM1 and Orai1 would recruit all of the TRPCs in the SOCE pathway; in contrast, overexpression of either STIM or Orai is expected to have no effect since Orai or STIM, respectively, would be limiting [61]. This phenomenon might explain the differences reported concerning the role of TRPCs in store- or receptor-operated Ca2+ entry. In addition, TRPC1-containing heteromultimeric channels have been proposed to be activated by extracellular activators such as thioredoxin [62]. Together these findings suggest that TRPC1 is a point of convergence between intra and extracellular signals. 4. Biochemical properties of the interaction between STIM1, TRPCs and Orai A number of recent studies have addressed the identity of protein regions that are important for SOC channel activation. The EF-SAM
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region of STIM1 has been reported to induce a monomer-to-oligomer transition upon Ca2+ dissociation from the EF-hand motif, where hydrophobic areas in the EF-SAM domain participate in STIM1 oligomerisation [63]. Recent studies have reported that STIM1 oligomerisation is essential for the communication between the Ca2+ stores and the plasma membrane [64]. It has been reported that the Cterminus of STIM1 is able to activate endogenous SOC channels when endogenously expressed STIM1 was effectively knocked down. An interesting finding that deletion analyses have revealed is that the STIM1 ERM domain mediates the selective binding of STIM1 to TRPC1, 2, 4 and 5 but not to the other TRPC isoforms. Another interesting region of STIM1 is the cationic lysine-rich tail. Deletion of this region or substitution of all of the lysine residues inhibits the ability of STIM1 to gate TRPC1 [56]. Finally, the ability of STIM1 to aggregate in clusters close to the plasma membrane has also been reported to reside in the ERM region. Deletion of part of the first coiled-coil domain, located in the ERM region, that includes a glutamate-rich region, and deletion of the S/P-rich region in the C-terminus of STIM1, impairs the translocation of STIM1 towards the plasma membrane in response to depletion of the Ca2+ stores [65]. The cytoplasmic coiled-coil domains of STIM1 have also been suggested to interact with those of Orai1. Coiled-coil regions are known to mediate homotypic as well as heterotypic protein interactions, facilitating the Orai1-STIM1 interactions required for the activation of ICRAC [63]. The N-terminus of Orai1 has been demonstrated to be important for the interaction with STIM1. Orai1 contains a proline- and arginine-rich N-terminal cytoplasmic sequence. Coexpression of STIM1 with full length Orai1 or with a chimeric Orai2 containing the Orai1 N-terminus resulted in enhanced SOCE, while a truncated version of Orai1 containing the N-terminus without the pore-forming transmembrane domain had a dominant negative effect on SOCE [66]. In addition, the C-terminal coiled-coil motif of Orai1 has been presented as a key domain for dynamic coupling to STIM1 [67]. Recent studies have reported that Orai1 is able to form relatively stable complexes with TRPC proteins [58,59], sometimes involving other cytoplasmic Ca2+ handling proteins, such as SERCA [68]. Orai1 has been reported to interact with both TRPC1 N- and C-termini in vitro, although there seems to be a preference for interaction with the C-termini [58]. Altogether, these findings indicate that the EF hand domain of STIM1, which resides within the endoplasmic reticulum, senses [Ca2+]L and inhibits STIM1 activity when stores are filled. When [Ca2+]L decreases Ca2+ dissociates from the EF hand motif and STIM1 activates SOC channels by a mechanism that involves ERM-dependent interaction with the channel subunits and lysine-rich region-dependent activation of the channel. Orai1 and TRPC proteins can independently mediate ICRAC and ISOCs or might interact to form SOC channels with different biophysical properties, providing the cell with valuable tools to regulate particular Ca2+ signals. Orai proteins, by interaction with TRPCs, have been shown to act as regulatory subunits that confer STIM1-mediated store depletion sensitivity to SOC channels composed of pore-forming TRPC subunits [58].
in HEK293 cells supports the involvement of SOCE in Ca2+ oscillations [72]. In particular, a major finding is the essential role for STIM1 and Orai1 in the mechanism of Ca2+ signalling with physiological stimulus strength, when [Ca2+]c levels undergo oscillations [72]. A number of studies have demonstrated the involvement of proteins of the SOCE machinery in cellular functions. The original study identifying Orai as a pore-forming SOC subunit indicated that it is involved in T cell function [10]. In addition, Orai1 has been shown to be crucial in mouse mast cell effector function [73]. On the other hand, STIM1 has also been presented as an essential positive regulator of mast cell activation. This protein is required for Ca2+ influx and subsequent activation of NF-B and NFAT induced by activation of the high-affinity IgE receptor FcRI [65]. STIM1 and Orai1 have also been reported to be essential for the activation of SOCE in skeletal muscle [74] and platelets, where mutations affecting the EF hand motif of human STIM1 have been suggested to be involved in inherited thrombocytopenias [75] and impaired platelet function has been demonstrated in Orai1-deficient mice [76]. There is an overwhelming amount of evidence supporting a role for TRP-containing SOC channels in cellular physiology, particularly in various modalities of sensory transduction. Drosophila TRP is involved in phototransduction, conducting the main component of the lightactivated current in the photoreceptors. A number of TRP channels, such as Drosophila TRPN, some TRPV members, TRPA or the ubiquitous TRPC1 channel are involved in mechanotransduction, where they are likely to be directly activated by membrane lipid tension or by mechanical force delivered through structural proteins. In addition, TRP proteins are involved in thermoreception. TRPM8 is activated by low temperature and TRPV1, TRPV2, TRPV3 and TRPV4 are activated by heat (see [2,77]. TRP-forming channels are also important for a number of developmental processes that require Ca2+ entry, such as the acrosome reaction, spermatogenesis or the determination of left– right asymmetry during embryogenesis [77]. Hence SOC channels are involved in a number of cellular functions, and abnormalities in SOC components might lead to pathological consequences that need to be further investigated. Acknowledgements Supported by M.E.C.-FEDER (BFU2007-60104/BFI). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
5. New insights into the physiological role of Orai, STIM and TRPC proteins
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Physiological concentrations of agonists induce complex and repetitive cycling of [Ca2+]c, known as [Ca2+]c oscillations, spatiotemporal patterns of Ca2+ signals that are recognized as physiological processes [69–72]. Pharmacological evidence indicates that in HEK293 cells, maintenance of Ca2+ oscillations requires SOCE [69]. This observation has been questioned because of the pharmacological approach employed [70] and by studies reporting that SOCE is not required for Ca2+ signalling under physiological conditions in intestinal cells of C. elegans [71]. Nevertheless, a recent study performing functional knockdown of several components of the SOCE machinery
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