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The Journal of Physiology logoLink to The Journal of Physiology
. 2013 May 20;591(Pt 14):3507–3523. doi: 10.1113/jphysiol.2013.256784

Molecular basis of activation of the arachidonate-regulated Ca2+ (ARC) channel, a store-independent Orai channel, by plasma membrane STIM1

Jill L Thompson 1, Trevor J Shuttleworth 1
PMCID: PMC3731610  PMID: 23690558

Abstract

Currently, Orai proteins are known to encode two distinct agonist-activated, highly calcium-selective channels: the store-operated Ca2+ release-activated Ca2+ (CRAC) channels, and the store-independent, arachidonic acid-activated ARC channels. Surprisingly, whilst the trigger for activation of these channels is entirely different, both depend on stromal interacting molecule 1 (STIM1). However, whilst STIM1 in the endoplasmic reticulum membrane is the critical sensor for the depletion of this calcium store that triggers CRAC channel activation, it is the pool of STIM1 constitutively resident in the plasma membrane that is essential for activation of the ARC channels. Here, using a variety of approaches, we show that the key domains within the cytosolic part of STIM1 identified as critical for the activation of CRAC channels are also key for activation of the ARC channels. However, examination of the actual steps involved in such activation reveal marked differences between these two Orai channel types. Specifically, loss of calcium from the EF-hand of STIM1 that forms the key initiation point for activation of the CRAC channels has no effect on ARC channel activity. Secondly, in marked contrast to the dynamic and labile nature of interactions between STIM1 and the CRAC channels, STIM1 in the plasma membrane appears to be constitutively associated with the ARC channels. Finally, specific mutations in STIM1 that induce an extended, constitutively active, conformation for the CRAC channels actually prevent activation of the ARC channels by arachidonic acid. Based on these findings, we propose that the likely role of arachidonic acid lies in inducing the actual gating of the channel.

Key points

  • The two known endogenous Orai channels, the calcium store-dependent CRAC channel and the store-independent ARC channel, are both regulated by the protein STIM1.

  • However, whilst CRAC channel activation is regulated by STIM1 in the endoplasmic reticulum, it is the pool of STIM1 constitutively residing in the plasma membrane that regulates the ARC channels.

  • Here we show that, although the specific regions of STIM1 critical for the regulation of these channels are largely the same, the actual mechanism of activation is markedly different.

  • Specifically, STIM1 in the plasma membrane exists in a constitutive association with the ARC channel, only requiring arachidonic acid to induce opening of the channel.

  • As these channels are known to play critical roles in the generation and modulation of key intracellular calcium signals, such distinct modes of activation are likely to have important implications for the generation and modulation of such signals in diverse cell types.

Introduction

The entry of calcium from extracellular sources plays a critical role in the initiation and regulation of the agonist-induced increases in cytosolic calcium concentrations that represent the major signalling system in a wide variety of cell types. In many cell types, particularly non-excitable cells, such entry is often a consequence of the initial depletion of intracellular calcium stores in a process originally defined by Putney (1986) as capacitative, or store-operated, calcium entry. The channels typically responsible for such entry were subsequently characterized biophysically as highly calcium-selective, low conductance channels and named CRAC channels (for calcium release-activated calcium channels; Hoth & Penner, 1992, 1993; Zweifach & Lewis, 1993). However, only in the last few years has the molecular identity of these channels and the mechanism of their store-dependent activation been revealed, first with the identification of STIM1 as the sensor of store depletion and activator of the channel (Liou et al. 2005; Roos et al. 2005; Zhang et al. 2005), followed by the discovery of the protein Orai1 as the essential pore-forming subunit of the CRAC channels (Feske et al. 2006; Prakriya et al. 2006; Vig et al. 2006a,b; Yeromin et al. 2006; Zhang et al. 2006).

The Orai family of proteins, of which there are two additional members (Orai2 and Orai3), are unique in that they bear no obvious relationship to any other known channel protein family (Feske et al. 2006; Zhang et al. 2006; Gwack et al. 2007). To date, these proteins have been shown to be the essential pore-forming subunits of at least two distinct endogenously expressed channels: the calcium release-activated calcium (CRAC) channels (Prakriya et al. 2006; Vig et al. 2006a), and the arachidonate-regulated calcium (ARC) channels (Mignen et al. 2008, 2009; Shuttleworth, 2009). Both of these channels are widely expressed in a variety of different cell types, often co-existing in the same cell, and both have been shown to play key roles in the modulation of agonist-induced calcium signals, although often acting via entirely separate mechanisms (see, for example, Thompson & Shuttleworth, 2011). More critically, their modes of activation are entirely distinct. Whereas the CRAC channels are activated as a result of the depletion of endoplasmic reticulum (ER) calcium stores, ARC channel activation is entirely independent of such store depletion and is induced as a result of the generation of low levels of intracellular arachidonic acid (Mignen & Shuttleworth, 2000; Mignen et al. 2003a). Surprisingly, despite this clear difference, the activation of both channels is dependent on STIM1 which, as noted above, was initially identified as the activator of CRAC channels as a result of its location in the ER membrane where it acts to sense the depletion of calcium from ER stores. However, STIM1 was originally identified as a plasma membrane protein (Manji et al. 2000; Williams et al. 2002), and it is this pool of STIM1, typically representing some 10–25% of the total cellular STIM1, that is specifically responsible for the activation of the store-independent ARC channels (Mignen et al. 2007).

Recently, considerable progress has been made in discerning the molecular and structural processes underlying the activation of the CRAC channels by the ER resident STIM1 (see recent reviews from Lewis, 2011; Muik et al. 2012; Soboloff et al. 2012). However, corresponding studies on the activation of the ARC channels are complicated by the relatively small proportion of total cellular STIM1 involved in the regulation of these channels. As a means of addressing this issue, we have recently developed a unique construct of STIM1 that allows the exclusive activation of the ARC channels, whether by exogenous arachidonic acid or by an appropriate physiological agonist, without any ability to activate the co-existing CRAC channels (Thompson & Shuttleworth, 2012). This construct consists of just the cytosolic portion of the STIM1 molecule, anchored to the inner face of the plasma membrane via an N-terminally attached Lck domain sequence, and was designated as Lck-STIM1-C. The decision to exclude all N-terminal regions of STIM1 in this construct was based on the realization that such regions, specifically the calcium-binding EF-hand that plays such a key role in the activation of the CRAC channels by sensing the depletion of intracellular calcium stores, would be located in the extracellular environment. Here, it would never normally lose its bound calcium, and was therefore considered unlikely to play any significant role in the activation of the ARC channels, as confirmed in our earlier study (Thompson & Shuttleworth, 2012). Critically, we have shown that this construct fulfils three criteria that we considered to be essential for its use in studies of the regulation of ARC channel activity by plasma membrane STIM1. First, it should enable the exclusive activation of ARC channels, thereby avoiding any possible contributions from the biophysically similar CRAC channels. Second, it should be capable of supporting measurable activation of the endogenous channels, so that any complications arising from the overexpression of proteins that clearly have, at least, some degree of affinity for each other can be avoided. Finally, it should include the means to minimize any involvement of the endogenous STIM1 so that the actions of various experimental modifications (mutations, deletions etc.) can be assessed independently of any complications arising from the endogenous wild-type STIM1. This last criterion is fulfilled by the incorporation of a silent mutation in the Lck-STIM1-C construct that renders it resistant to knockdown when expressed in cells treated with small interfering RNA (siRNA) against the endogenous STIM1. In addition, we have shown that all the basic properties of the ARC channel currents that result from their activation in the Lck-STIM1-C-expressing cells essentially duplicate those seen in cells expressing the wild-type STIM1. These include the magnitude of the resulting whole-cell currents, their rate of activation, their relative sensitivity to both arachidonic acid and carbachol, and their characteristic current–voltage relationship displaying marked inward rectification and a very positive reversal potential indicative of a highly calcium-selective conductance (Thompson & Shuttleworth, 2012).

In the study reported here, we have now made extensive use of this construct to examine the key components and actions of STIM1 that underlie the activation of the ARC channels, and compare them with those previously reported to be involved in the activation of CRAC channels. The results obtained indicate many features that are common to the activation of both channels. However, they also serve to highlight certain specific marked differences in such activation, differences that can undoubtedly be used as a basis for further studies to define the unique processes underlying the activation of these two distinct, physiologically important, Orai channels.

Methods

Cells lines, constructs and transfections

Culture conditions for the Flp-In-293 cells (Invitrogen, Grand Island, NY), procedures for the depletion of endogenous STIM1 using siRNA duplexes, and details of the Lck-STIM1-C construct were as previously described (Thompson & Shuttleworth, 2012). Cells from the human embryonic kidney cell line HEK293 stably expressing the muscarinic m3 receptor were as described previously (Thompson & Shuttleworth, 2011). Details of the specific constructs used in this report can be found online in the Supplemental material. Constructs were prepared using a variety of molecular approaches including sub-cloning, QuikChange II (Stratagene, La Jolla, CA) site-directed mutagenesis, and PCR. All PCR-modified constructs were confirmed by sequencing. Cells were transfected using an Amaxa Nucleofector II (Gaithersburg, MD) following the manufacturer's guidelines.

Electrophysiology

Procedures for patch clamp experiments were essentially as previously described (Thompson & Shuttleworth, 2011). Briefly, 18–24 h after plating on polylysine-coated coverslips, whole-cell current recordings were performed on cells at room temperature (20–22°C) using 250 ms voltage pulses to −80 mV delivered every 2 s from a holding potential of 0 mV. To obtain current–voltage relationships, 10 ms pulses to potentials between −100 mV and +60 mV were applied at 20 mV intervals. The standard internal (pipette) solution contained 140 mm CsC2H3O2, 3.5 mm CaCl2, 3.72 mm MgCl2, 10 mm EGTA and 10 mm Hepes (pH 7.2). The calculated free Ca2+ concentration of this solution was 100 nm. The standard extracellular solution contained 140 mm NaCl, 5 mm CsCl, 1.2 mm MgCl2, 10 mm CaCl2, 30 mm glucose, and 10 mm Hepes (pH 7.4). Where currents were measured under divalent-free extracellular conditions this was changed to a solution containing 140 mm NaCl, 5 mm CsCl, 0.5 mm EDTA, 0.5 mm EGTA, 25 mm glucose, and 10 mm Hepes (pH 7.4). In situations where the presence of constitutive currents induced by the various constructs was being examined, currents obtained at the end of each experiment measured in an external solution containing La3+ (100 μm) were used for leak subtraction. In the cases where either arachidonic acid-activated currents or agonist-activated currents were determined, the initial currents obtained before activation of the channel were used for leak subtraction.

Cell imaging

All images were obtained on an Olympus Fluoview 1000 multiphoton/confocal laser scanning microscope system (FV1000 MPE) consisting of an Olympus BX61WI upright microscope coupled to a suite of diode and gas lasers (405, 440, 515, 565 and 633 nm) operated and controlled by the Olympus FV10-ASW software. Imaging was performed using a ×60 water-immersion objective (UPLSAPO; numeric aperture, 1.2) for the bimolecular fluorescence complementation (BiFC) and sensitized emission fluorescence resonance energy transfer (FRET) assays, whilst a ×40 water-immersion objective (LUMPLFLN; numeric aperture, 0.8) was used for the time-dependent ‘Live FRET’ assays. Relevant constructs of Lck-STIM1-C and Orai3 for use in BiFC experiments were produced as described in the online Supplemental material.

FRET assays

Image analysis and determination of FRET signals were carried out using the Fluoview software (v. 2.0a). For FRET analysis using the sensitized emission approach, three images are taken for each field of view (cyan fluorescent protein (CFP) excitation/CFP emission, yellow fluorescent protein (YFP) excitation/YFP emission, and CFP excitation/YFP emission) using the following automatically selected optical parameters: 440 nm excitation/405–440 nm dichroic/465–495 nm emission, 515 nm excitation/515 nm dichroic/535–565 nm emission, and 440 nm excitation/405–440 nm dichroic/ 535–565 nm emission, respectively. All acquired images were background subtracted, and the analysis software then used an algorithm-based process to correct for both spectral bleed-through and uncontrolled variations in fluorophore expression on a pixel-by-pixel basis, to obtain a value of FRET efficiency (E%; see Elangovan et al. 2003; Sekar & Periasamy, 2003 for details). Further analysis of the FRET efficiency data for the plasma membrane-associated proteins in the various experiments was performed in ImageJ using an approach based on that described by Covington et al. (2010). In this, for each cell analysed, an 8 pixel-wide (1.6 μm) region of interest (ROI) incorporating the cell periphery including the plasma membrane was drawn on either the CFP or YFP image using the ‘Make Band’ function in ImageJ. This ROI was then copied to the FRET image, and values for the total area and average intensity from this ROI used to obtain the average FRET efficiency value for this region of the cell. The mean data presented were recorded for the number of cells indicated (n), obtained from a minimum of three separate transfections per experimental treatment. To obtain an estimate of an approximate ‘maximal’ membrane-associated FRET signal for the specific system used, we expressed a Lck-tagged construct consisting of Venus and Cerulean fluorophores connected by a flexible linker formed by five glycines (Lck-Ven5GCer; Koushik et al. 2006). Examination of HEK cells expressing this construct gave a FRET signal (E%), calculated as described above, of 52.2 ± 5.6 (n= 13). It should be noted that the FRET efficiency values determined by the above protocol potentially incorporate multiple variables, including the orientation, stoichiometry and intermolecular distance of the STIM1–Orai complex formed, in addition to strict single protein-to-protein association. This renders detailed quantitative comparison between the different treatments difficult. Consequently, in this study, we evaluated the mean FRET values obtained only as ‘significant’versus‘weak or negligible’ signals based on values that were greater than 25%versus less than 5%, respectively, of the mean maximal value obtained with the Lck-Ven5GCer positive control construct. In addition, where we required examination of dynamic effects of addition of arachidonic acid on the recorded FRET signals, we performed a time-dependent ‘Live FRET’ analysis. This simply involved recording the changes in ratio of the background-subtracted acceptor fluorescence signal under donor excitation, divided by the similarly corrected donor fluorescence signal under donor excitation. The extracellular solution used in these studies contained 132.5 mm NaCl, 4.8 mm KCl, 1.2 KH2PO4, 1.2 mm MgCl2, 1.3 mm CaCl2, 6 mm glucose, 5.95 mm Hepes free acid and 9.05 mm Na-Hepes (pH 7.4).

Western blotting and co-immunoprecipitation

Endogenous Orai3 was immunoprecipitated from cell lysates obtained from the relevant cells under the specific conditions described below. To minimize non-specific associations, all lysates were prepared in an immunoprecipitation buffer containing 1% Triton X-100, and unsolubilized membrane fragments were removed by centrifugation (100,000 g for 40 min). For examining constitutive co-immunoprecipitation of STIM1 with Orai3 (Fig. 5A, top), lysates were prepared from approximately 107 cells, and 450 μl (585 μg protein) were used per sample. For each experiment, 20 μl (20 μg) of an Orai3 C-terminal antibody (Ab) (ProSci, Poway, CA, cat. no. 4215), was added and incubated overnight at 4°C. As a control, the C-terminal Ab was pre-incubated (30 min at room temp) with 4 μg of the Orai3 C-terminal peptide antigen. A 50 μl volume of prewashed A/G agarose beads was added to each of the samples and incubated at 4°C for 2.5 h. The beads were then washed six times with lysis buffer and the protein eluted with 50 μl of 2X sample loading buffer plus β-mercaptoethanol (βME) and heated to 70°C for 10 min. Each sample (40 μl) was run on a 7% SDS-PAGE gel, transferred to nitrocellulose membrane and blotted with 1:300 STIM1 mAb (BD Biosciences, San Jose, CA, cat. no. 610954) followed by 1:10,000 of an anti-rabbit IgG, horseradish peroxidase-linked secondary Ab and detected using Pierce enhanced chemiluminescence. For examining the effects of arachidonic acid exposure on the co-immunoprecipitation (Fig. 5A bottom), HEK cells stably expressing STIM1 were incubated with or without 8 μm arachidonic acid for 5 min at 37°C prior to lysate preparation (from approximately 2 × 106 STIM1-stable cells). The volume of lysate used was adjusted as appropriate to give 80 μg protein per sample, and was incubated overnight at 4°C with 20 μl (20 μg) of the same Orai3 C-terminal Ab used above. The Western blot procedure was as described above, except a sample volume of 50 μl was used for loading onto the gel.

Figure 5. Evidence for a constitutive association between Lck-STIM1-C and Orai3.

Figure 5

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A, top panel, representative immunoblot obtained from untransfected cells showing the co-immunoprecipitation of endogenous STIM1 with Orai3. Endogenous Orai3 was immunoprecipitated from cell lysates using a C-terminal antibody, run on a gel and probed with an antibody to STIM1. As a control, the same lysate was treated with Orai3 antibody that had been preincubated with the relevant C-terminal peptide antigen prior to immunoprecipitation (‘preab’). Bottom panel, representative immunoblot as above, except that it was from cells stably expressing STIM1, and which had been incubated either with (‘+AA’) or without (‘cont’) exogenous arachidonic acid (8 μm for 5 min) as indicated. Also shown are lanes for the different lysates (5 μg) probed directly with the STIM1Ab. B, representative images of a cell co-expressing a Lck-STIM1-C construct bearing a C-terminal eGFP tag (top image), and an Orai3 construct bearing an N-terminal mCherry tag (middle image). The bottom image shows an overlay of the above eGFP and mCherry images. Scale bar is 5 μm. C, representative BiFC image of cells co-expressing a Lck-STIM1-C construct bearing a C-terminal tag composed of residues 1–158 of eYFP and an Orai3 construct bearing the complementary 159–238 eYFP sequence as an N-terminal tag. Scale bar is 5 μm. D, representative images of cells co-expressing various Lck-STIM1-C constructs bearing a C-terminal Cerulean tag (top row) together with an Orai3 construct N-terminally tagged with Venus (middle row), and the resulting FRET signal (bottom row). The relevant Lck-STIM1 constructs are indicated above each column of images. Scale bars are 5 μm.

Results

Loss of calcium from the EF-hand of plasma membrane STIM1 does not activate the ARC channels

As already discussed, the normal processes by which STIM1 molecules in the ER membrane act to induce the activation of the CRAC channels is fairly well defined, with the loss of calcium from the luminal EF-hand of STIM1 inducing the oligomerization of the protein, followed by the translocation of the STIM1 oligomers within the ER membrane to sites close to the plasma membrane where they interact with, and activate, the channels. In contrast, the activation of the ARC channels is exclusively dependent on the pool of STIM1 in the plasma membrane and requires the presence of low levels of intracellular arachidonic acid. It would seem that the simplest way in which these two very different processes might be reconciled would be if arachidonic acid, in some way, duplicated or mimicked the effect of loss of calcium from the EF-hand of STIM1. If this were so, then the prediction would be that preventing calcium from binding to the EF-hand of the STIM1 in the plasma membrane should result in the activation of the ARC channels in the absence of arachidonic acid. One way in which this can be achieved would be to express a STIM1 construct bearing mutations at two sites within the EF-hand of STIM1 (D76A and E87A) which render the EF-hand of STIM1 unable to bind calcium. As would be predicted, studies from several groups have shown that such mutations induce the constitutive activation of CRAC channels in the absence of any depletion of the ER stores (Liou et al. 2005; Zhang et al. 2005; Mignen et al. 2007). Unfortunately, we have previously demonstrated that such sustained constitutive activity of the CRAC channels in cells results in a reciprocal inhibition of the ARC channels (Luo et al. 2001; Mignen et al. 2001), clearly eliminating the use of such mutations in any attempt to examine their effect on ARC channel activation. An alternative approach would be to simply reduce extracellular calcium concentrations to levels where the bound calcium is released from the EF-hand of the STIM1 located in the plasma membrane. However, given the estimated Kd of the EF-hand for calcium (∼200–600 μm, Stathopulos et al. 2006; 210 μm, Brandman et al. 2007; 187 μm, Luik et al. 2008), reducing extracellular calcium to levels where the EF-hand of STIM1 in the plasma membrane would lose its bound calcium would result in extremely small calcium currents through the ARC channels. However, studies on both CRAC and ARC channels have shown that removal of all extracellular divalent cations results in the development of significantly larger monovalent cation currents through these channels when activated. Based on this, we argued that exposure of cells to such divalent-free external solutions should, in theory, result in the activation of the endogenous STIM1 in the plasma membrane, much as depletion of intracellular calcium stores results in the activation of STIM1 in the ER membrane. Given the strict dependence of ARC channel activity on plasma membrane STIM1, any such activation induced by the loss of calcium from the extracellular EF-hand should result in the corresponding activation of the ARC channels which, in the divalent-free extracellular medium, would now be detectible as a large monovalent cation current.

To examine this, we began by determining the magnitude of endogenous ARC channel currents in untransfected cells in either normal or divalent-free external media. Addition of arachidonic acid (8 μm) to cells in the normal, calcium-containing, external medium resulted in inward ARC channel currents measured at −80 mV of 0.51 ± 0.10 pA pF−1 (n= 6). These currents increased to a value of 17.41 ± 2.04 pA pF−1 (n= 6) on exposure to a divalent-free external medium (Fig. 1A), and displayed the unique current–voltage relationship of monovalent currents through activated ARC channels reported previously (Fig. 1B; Mignen et al. 2003a). We next examined the effect of exposure to the divalent-free external medium in the absence of addition of exogenous arachidonic acid. Based on the argument presented above, the prediction would be that this should result in the loss of calcium from the externally exposed EF-hand of endogenous plasma membrane STIM1, presumably rendering it in an ‘activated’ state, equivalent to STIM1 in the ER following store depletion. However, exposure to the divalent-free external medium resulted in inward currents measured at −80 mV of only 0.88 ± 0.15 pA pF−1 (n= 4; Fig. 1A). In marked contrast, subsequent addition of exogenous arachidonic acid (8 μm) resulted in the appearance of large inward currents (18.87 ± 1.80 pA pF−1 at −80 mV, n= 5), bearing the characteristic current–voltage relationship of monovalent currents through the ARC channels (Fig. 1A and B). This value was not significantly different from that obtained in the cells described above, in which the channels were activated first by addition of arachidonic acid, and only then exposed to divalent-free conditions (P= 0.61). More importantly, the value of the monovalent ARC channel currents induced by exposure to divalent-free conditions, when calcium should be lost from the EF-hand of the endogenous plasma membrane STIM1, amounted to less than 5% of that seen on activation by arachidonic acid. Together, these data would seem to indicate that the loss of calcium from the EF-hand of endogenous STIM1 in the plasma membrane is not sufficient to induce significant activation of the ARC channels.

Figure 1. Effects of removing Ca2+ from the extracellular EF-hand of plasma membrane STIM1.

Figure 1

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A, comparison of the effects of exposure to a divalent-free extracellular medium (DF) on arachidonic acid-induced currents. Shown are values of inward currents at −80 mV in cells stimulated with arachidonic acid (AA, 8 μm) followed by exposure to the divalent-free medium (AA > DF; grey columns), and in cells first exposed to the divalent-free medium (DF) followed by the addition of arachidonic acid (DF>AA; black columns). All values are mean ± SEM with values of n (number of cells) shown in parentheses. B, mean (±SEM) current–voltage (I–V) curves of the arachidonic acid-stimulated, divalent-free currents from cells exposed first to arachidonic acid (grey symbols), and cells exposed to divalent-free conditions and subsequently stimulated with arachidonic acid (black symbols).

Cytosolic domains of STIM1 critical for ARC channel activation

The data obtained above indicate that the actual process by which STIM1 induces the activation of Orai channels is likely to be markedly different for the ARC channels compared to those for the CRAC channels. To explore the possible underlying nature of such differences, we began by examining the essential domains and specific residues within the cytosolic part of the STIM1 molecule that are required for the activation of these two distinct channels using the novel Lck-STIM1-C construct. Clearly, the ability of this construct to effectively, and selectively, activate endogenous ARC channels (Thompson & Shuttleworth, 2012) demonstrates that the N-terminal regions of the STIM1 molecule are not required for activation of these channels. Thus, although the sensing of store depletion and the initiation of the resulting response of STIM1 depends on regions within the N-terminal part of the molecule, the actual activation of CRAC channels by ER-resident STIM1 following store depletion depends on certain specific regions within the cytosolic C-terminus. Primary amongst these are a polybasic lysine-rich region at the C-terminal end of STIM1, and a region that includes the second and third of three predicted coiled-coil regions, variously named CAD (CRAC activation domain, residues 342–448; Park et al. 2009), SOAR (STIM1–Orai1 activating region, residues 344–442; Yuan et al. 2009), or CCb9 (coiled-coil domain containing region b9, residues 339–444; Kawasaki et al. 2009).

To examine any potential role of these regions in the cytosolic part of STIM1 that might be critical for ARC channel activation, we began by expressing an siRNA-resistant Lck-STIM1-C construct which was terminated at residue 448 (Lck-STIM1-Δ448), thereby removing the entire region C-terminal to the CAD domain, including the polybasic lysine-rich region at the C-terminal end of STIM1 (Fig. 2A). Measurements of arachidonic acid-activated currents in siRNA-treated HEK293 cells expressing the siRNA-resistant Lck-STIM1-Δ448 revealed markedly inwardly rectifying currents of 0.55 ± 0.03 pA pF−1 at −80 mV (n= 6), a value that was not significantly different from those recorded in the same cells expressing full-length Lck-STIM1-C (0.64 ± 0.10 pA pF−1, n= 6, P= 0.44; Fig. 2C and D). Based on these findings, we conclude that sequences distal to the end of the CAD domain in the cytosolic part of STIM1, including the C-terminal polybasic lysine-rich domain, appear to play no obvious functional role in the arachidonic acid-dependent activation of endogenous ARC channels by STIM1 in the plasma membrane. As such, these data differ from findings from studies on CRAC channel activation which have shown that deletion of the polybasic domain inhibits the ability of STIM1 to assemble into puncta on store depletion (Liou et al. 2007), although this can be restored by co-expression of Orai1 (Park et al. 2009; Walsh et al. 2010).

Figure 2. Examination of the cytosolic domains of STIM1 critical for the activation of ARC channels by arachidonic acid.

Figure 2

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A, diagrams illustrating the domain structure of the Lck-STIM1-C construct and the Δ448 and Δ341 deletion constructs. ‘CAD’ is the CRAC activation domain, ‘CC1–3’ are coiled-coil domains, ‘S/P’ is the serine-proline rich region, and ‘K’ is the C-terminal lysine-rich polybasic domain. B, amino acid sequence of the CAD domain indicating the two mutations (L373S and A376S) that have been reported to disrupt the coiled-coil structure of the CC2 to different degrees (see text for details). C, inward arachidonic acid-activated currents at −80 mV in cells treated with an siRNA targeting endogenous STIM1, and in the same siRNA-treated cells following expression of the Lck-STIM1-C construct (Lck), the Lck-STIM1-Δ448, the Lck-STIM1-Δ341, Lck-STIM1-373S and Lck-STIM1-373/6S constructs, as indicated. Values are means ± SEM, with n shown in parentheses. D, representative I–V curves for arachidonic acid-activated currents in the siRNA-treated cells expressing the Lck-STIM1-Δ448 construct (black symbols), and the Lck-STIM1-Δ341 construct (grey symbols). E, representative I–V curves for arachidonic acid-activated currents in the siRNA-treated cells expressing the Lck-STIM1-373S construct (black symbols), and the Lck-STIM1-373/6S construct (grey symbols).

We next examined the contribution of the above-mentioned CAD domain of STIM1 (residues 342–448) to ARC channel activation. Studies have shown that this region plays a key role in the activation of the CRAC channel, and that expression of this region alone is capable of resulting in the constitutive activation of CRAC channels (Kawasaki et al. 2009; Park et al. 2009; Yuan et al. 2009). We began by generating an siRNA-resistant Lck-STIM1-C construct in which the C-terminal region was terminated at residue 341 (Lck-STIM1-Δ341, Fig. 2A), thereby removing the entire CAD domain. Expression of this construct in siRNA-treated cells resulted in arachidonic acid-activated currents of only 0.19 ± 0.03 pA pF−1 at −80 mV (n= 5), a value that was not significantly different from the untransfected siRNA-treated cells (0.16 ± 0.02 pA pF−1, n= 6, P= 0.50) (Fig. 2C and D). Based on these data, we conclude that, just as with the CRAC channels, deletion of the CAD domain results in the loss of effective activation of the ARC channels.

In the activation of the CRAC channels, it is thought that the specific role of the CAD domain lies in the ability of the coiled-coil regions of this domain to form interactions with complementary predicted coiled-coil sequences in the cytosolic C-terminal portion of the Orai proteins that form the channel. Consistent with this, studies have shown that point mutations that disrupt the predicted coiled-coil structure of the CAD domain result in the loss of interactions between STIM1 and Orai and the failure to activate the channels (Frischauf et al. 2009). Interestingly, it has been demonstrated that, whilst a single point mutation (L373S) in the STIM1 coiled-coil region is sufficient to result in the loss of effective interaction with Orai1 and consequent CRAC channel activation, disruption of interactions with the C-terminal coiled-coil domains of Orai3 require a double mutation (L373S + A376S) in this region of CAD (Fig. 2B). Given the heteromeric (Orai1 + Orai3) nature of the endogenous ARC channels (Mignen et al. 2009), we examined the effect of these mutations in CAD on the ability of the Lck-STIM1-C construct to activate endogenous ARC channels. Expression of an siRNA-resistant Lck-STIM1-C construct bearing the single L373S mutation (Lck-STIM1-373S) in the siRNA-treated cells resulted in inwardly rectifying arachidonic acid-activated currents of 0.50 ± 0.05 pA pF−1 at −80 mV (n= 5), a value not significantly different from that recorded in the same cells expressing the normal Lck-STIM1-C construct (P= 0.28) (Fig. 2C and E). However, expression of the corresponding L373S + A376S double mutant Lck-STIM1-C (Lck-STIM1-373/6S) resulted in arachidonic acid-activated currents of only 0.23 ± 0.04 pA pF−1 at −80 mV (n= 6), a value that was not significantly different from that recorded in the untransfected siRNA-treated cells (P= 0.13; Fig. 2C and E). Interestingly, examination of the effect of these coiled-coil mutations on the distribution of the Lck-STIM1-C within the cell using green fluorescent protein (GFP)-tagged constructs revealed that, whilst the single Lck-STIM1-373S mutant displayed the predicted plasma membrane localization, the double mutant (Lck-STIM1-373/6S) was largely cytosolic (Supplemental Fig. S1, available online only). Although the underlying reason for the failure of this construct to efficiently target the plasma membrane, despite the presence of the Lck domain, is unclear, this finding is consistent with the inability of the latter to successfully support activation of the ARC channels. Overall, these data suggest that, whilst the CAD domain is critical for the effective STIM1-dependent activation of both CRAC and ARC channels, the molecular basis of this activation for the ARC channels differs from that for the CRAC channels.

Recently, more detailed information concerning the precise structural basis for the interactions between STIM1 and Orai1 has been revealed as a result of studies demonstrating the importance of a series of basic residues within the second coiled-coil of STIM1 (residues 382–387) for interactions with the Orai1 C-terminus (Calloway et al. 2010; Korzeniowski et al. 2010). Consistent with this, recent crystallographic studies of the structure of the CAD/SOAR region of STIM1 indicate that these basic residues line a V-shaped cleft that is thought to engage with acidic regions in the Orai1 C-terminus (Yang et al. 2012). Importantly, such a ‘cleft’ structure can only be formed by a dimer of STIM1 molecules (Yang et al. 2012), and several studies have indicated that such dimers indeed represent the normal resting state of cellular STIM1 (Williams et al. 2002; Baba et al. 2006; Covington et al. 2010; Yang et al. 2012). We therefore began by examining whether preventing Lck-STIM-C from dimerizing affected its ability to activate the ARC channels. To achieve this, we expressed a Lck-STIM1-C construct bearing three mutations (W430A, I433A and L436A) that recent structural studies have identified as being located at the dimer interface, and which effectively block dimer formation and the appearance of thapsigargin-induced store-operated Ca2+ entry (Yang et al. 2012). Following the notation of Yang et al. (2012), we named this construct the Lck-STIM1-C2A mutant (Fig. 3A). Expression of the siRNA-resistant Lck-STIM1-C2A construct in siRNA-treated cells resulted in the failure of arachidonic acid to induce significant ARC channel currents (mean inward current at −80 mV = 0.25 ± 0.03 pA pF−1, n= 6, a value not significantly different from that recorded in untransfected siRNA-treated cells, P= 0.26; Fig. 3B and C). Together, these data indicate that, just as with the activation of CRAC channels, dimerization of STIM1 is a necessary prerequisite for the effective activation of the ARC channels.

Figure 3. Effects of mutations that either impair dimerization (C2A) or eliminate the cluster of positively-charged residues (BRmut) in the Lck-STIM1-C construct.

Figure 3

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A, diagram illustrating the location of the ‘C2A’ and ‘BRmut’ mutations within the CAD domain of the Lck-STIM1-C construct. B, inward arachidonic acid-activated currents at −80 mV in cells treated with an siRNA targeting endogenous STIM1, and in the same siRNA-treated cells following expression of the Lck-STIM1-C construct (Lck), the Lck-STIM1-C2A mutant (C2A), and the Lck-STIM1-BRmut (BRmut) constructs, as indicated. Values are means ± SEM, with n shown in parentheses. C, representative I–V curves for arachidonic acid-activated currents in the siRNA-treated cells expressing the Lck-STIM1-C construct (red symbols), the Lck-STIM1-C2A construct (black symbols), and the Lck-STIM1-BRmut construct (grey symbols).

Given this apparent necessity of STIM1 dimerization to allow activation of the ARC channels, we next examined whether the series of basic residues lining the V-shaped cleft that is formed by such dimers (see above) was also critical for the activation of the ARC channels. Following the approach used by Korzeniowski et al. (2010) we mutated the four lysines at positions 382 and 384–386 that lie within this putative ‘activation cleft’ of the Lck-STIM1-C construct to alanines and glycines (Fig. 3A). Addition of exogenous arachidonic acid (8 μm) to siRNA-treated cells expressing this basic region-mutated Lck-STIM1-C (Lck-STIM1-BRmut) resulted in inward currents (0.22 ± 0.02 pA pF−1 at −80 mV, n= 5) that were not significantly different from those in untransfected siRNA-treated cells (0.20 ± 0.03, n= 6, P= 0.68; Fig. 3B and C). Again, these data suggest that, just as with the store-operated activation of the CRAC channels, the cluster of basic residues that are thought to line the putative ‘activation cleft’ structure formed by a STIM1 dimer, are critical for the effective activation of ARC channels by arachidonic acid.

Taken together, the data obtained indicate that many of the essential components required for the store-operated activation of CRAC channels and the arachidonic acid-mediated activation of ARC channels are the same. Specifically, such activation is dependent on the presence of the coiled-coil regions within the CAD domain, on STIM1 dimerization, and on the formation of the putative ‘activation cleft’ and the positively charged residues therein.

Molecular mechanisms underlying the activation of ARC channels

The above indication of broad similarity in the essential domains and specific residues within the cytosolic part of the STIM1 molecule required for the activation of both CRAC and ARC channels raises the question of whether the actual underlying mechanism of activation might also show such similarities. In this context, important new details of the molecular and structural processes involved in the activation of the CRAC channels by STIM1 have been revealed in recent studies using a variety of approaches (Korzeniowski et al. 2010; Muik et al. 2011; Yang et al. 2012). Among these is the finding that a short sequence within the first coiled-coil region of STIM1 (residues 302–322) appears to play a key role in preventing the inappropriate activation of the CRAC channels in the absence of store depletion. This autoinhibitory sequence apparently forms a helical structure containing a number of acidic residues, including a cluster of four glutamic acids within the last five C-terminal residues (Korzeniowski et al. 2010). Deletion of most of this sequence (residues 310–337), or simple mutation of the four C-terminal glutamic acids (E318–E320 and E322) to alanines, results in the constitutive activation of calcium entry via the CRAC channels in the absence of store depletion (Korzeniowski et al. 2010; Yang et al. 2012). Although the precise basis of this autoinhibitory function is currently uncertain, it seems that either the oligomerization of STIM1 following store depletion, or the incorporation of the above mutations, induces conformational changes in the cytosolic regions of STIM1 that disrupt this inhibitory action, thereby enabling the STIM1 oligomers to interact with an appropriate complementary sequence in the C-terminal part of Orai1 (Korzeniowski et al. 2010; Muik et al. 2011; Yang et al. 2012).

Based on these findings, we investigated whether making the same specific mutations within the Lck-STIM-C construct identified by Korzeniowski et al. (2010) might also induce the constitutive activation of the ARC channels (see Fig. 4A). Expression of an siRNA-resistant Lck-STIM1-C construct bearing these mutations (designated here as Lck-STIM1-4EA) in siRNA-treated cells failed to result in the appearance of any significant constitutive currents (mean inward currents at −80 mV = 0.15 ± 0.02 pA pF−1, n= 6, for the cells expressing Lck-STIM1-4EA versus 0.18 ± 0.03, n= 6, for the untransfected siRNA-treated cells, P= 0.42; Fig. 4B and C). It should be noted that this absence of any constitutive currents indicates that the endogenous CRAC channels are also not activated, a finding that would appear to contradict the above reports showing that expression of STIM1 bearing these mutations induces the constitutive activation of CRAC channels. However, in our studies these mutations were incorporated into the Lck-STIM1-C construct which, as we have previously shown, is incapable of supporting the activation of CRAC channels, even after store depletion. More surprising, however, was the finding that subsequent addition of exogenous arachidonic acid (8 μm) also failed to induce any significant increase in measured currents (mean inward currents at −80 mV: 0.23 ± 0.05 pA pF−1, n= 4) compared to those recorded above without addition of arachidonic acid (P= 0.16; Fig. 4B and C). Importantly, this failure of the Lck-STIM1-4EA construct to support the arachidonic acid-induced activation of the ARC channels was not due to any lack of expression or failure to target correctly to the plasma membrane as expression of this construct revealed a clear and pronounced enhanced (e)GFP signal at the plasma membrane, essentially identical to that seen with the wild-type Lck-STIM1-C construct (Fig. 4D). Thus, it appears that these acidic region mutations that have been shown to result in the constitutive activation of the CRAC channels not only fail to induce the same effect with ARC channels, but also prevent the ability of arachidonic acid to activate these channels.

Figure 4. Effects of mutations that induce the reported active, or ‘extended’, conformation of STIM1.

Figure 4

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A, diagram illustrating the location of the ‘4EA’ mutations within the first coiled-coil region of the Lck-STIM1-C construct. B, inward arachidonic acid-activated currents at −80 mV in cells treated with an siRNA targeting endogenous STIM1, and in the same siRNA-treated cells following expression of the Lck-STIM1-C construct (Lck), the Lck-STIM1-4EA mutant construct (4EA), and the same construct after addition of 8 μm exogenous arachidonic acid (+AA), as indicated. Values are means ± SEM, with n shown in parentheses. C, representative I–V curves for currents in the siRNA-treated cells expressing the Lck-STIM1-C construct (red symbols), and the Lck-STIM1-4EA construct (black symbols), and the Lck-STIM1-4EA construct following addition of 8 μm arachidonic acid (grey symbols). D, representative image of cells expressing the Lck-STIM1-4EA construct bearing a C-terminal eGFP tag. Scale bar is 5 μm.

Interactions between the ARC channels and plasma membrane STIM1

One possible explanation for the above difference in the effect of the acidic region mutations in STIM1 on its ability to activate CRAC channels versus ARC channels could relate to the distinct location and/or orientation of the STIM1 protein, relative to the channels with which it interacts. Thus, activation of the CRAC channels is specifically dependent on STIM1 located in an opposing membrane (the ER). This implies that the relevant cytosolic portions of STIM1 must extend across the gap between these two membranes and interact in an ‘anti-parallel’ orientation with the Orai1 subunits of the channel. Interestingly, studies by Muik et al. (2011) using a novel intramolecular FRET sensor based on the cytosolic coiled-coil regions of STIM1, have demonstrated the possibility of just such a conformational change. Thus, incorporation in the sensor sequence of the same acidic-region mutations that result in the constitutive activation of the CRAC channels (see above) resulted in a change in the sensor construct from a condensed to an extended configuration (Muik et al. 2011). However, such an extended conformation might be inappropriate for effective activation of the ARC channels, given that this involves interactions between STIM1 and Orai molecules arranged in a parallel orientation within the same membrane (the plasma membrane). Such marked differences in the physical arrangement and orientation of STIM1 and the relevant Orai proteins in these two cases clearly raise the possibility that the activation of these two channels might involve rather distinct mechanisms. For example, such an arrangement raises the possibility that STIM1 and the ARC channel may exist in some form of pre-assembled complex that simply requires arachidonic acid to induce some conformational change that results in the activation of the channel. Alternatively, STIM1 and the channel might exist as spatially separate entities, and the action of arachidonic acid is to induce their physical interaction.

To assess these possibilities, we began by examining potential endogenous interactions between STIM1 and Orai3, and the effects of arachidonic acid on such interactions, using a co-immunoprecipitation assay. In this, we chose to specifically examine potential interactions with Orai3 as we have already shown that such interaction is uniquely essential for the successful activation of ARC channels (see above). Endogenous Orai3, immunoprecipitated from cell lysates obtained from untransfected cells using a C-terminal antibody, was run on a gel which was then probed with an antibody to STIM1. The data obtained indicated that these proteins co-immunoprecipitated under resting conditions (Fig. 5A). The specificity of this interaction was confirmed by the absence of any detectible STIM1 band in Orai3 immunoprecipitates obtained from lysates treated with the relevant C-terminal peptide antigen prior to immunoprecipitation (Fig. 5A). A similar co-immunoprecipitation between Orai3 and STIM1 was seen using an alternative Orai3 antibody that was directed to a region in the second extracellular loop of the protein (Supplemental Fig. S2A). As a negative control for these data, we compared the ability of Orai3 to co-immunoprecipitate with expressed wild-type STIM1 versus an expressed N-glycosylation mutant STIM1 (N131Q/N171Q). Previous studies have shown that glycosylation at these two sites is essential for the expression of STIM1 at the cell surface (Williams et al. 2002), and that the transfection of STIM1 bearing these two mutations selectively reduces ARC channel currents to negligible values (Mignen et al. 2007). Consistent with the proposed association between Orai3 and, specifically, STIM1 in the plasma membrane, co-immunoprecipitation of STIM1 with Orai3 was essentially eliminated in cells expressing the N131Q/N171Q-mutant STIM1 (Supplemental Fig. S2B). Finally, to examine whether the level of co-immunoprecipitation was affected by activation of the ARC channels, we repeated the above experiment using the C-terminal Orai3 antibody and cells stably expressing STIM1, and examined whether the observed co-immunoprecipitation was affected by incubation with exogenous arachidonic acid (8 μm for 5 min). No obvious change in the apparent level of co-immunoprecipitated STIM1 and Orai3 could be detected following such exposure to arachidonic acid (Fig. 5A). Although this approach is quantitatively limited, the data obtained suggest a potential constitutive association between STIM1 and Orai3 that is unaffected by activation of the ARC channels. Consistent with this, examination of cells co-expressing an enhanced (e)GFP-tagged Lck-STIM1-C construct and an mCherry-tagged Orai3 construct revealed that these proteins co-localized in the plasma membrane (Fig. 5B).

As further evidence for such an association in intact cells, we used a bimolecular fluorescence complementation (BiFC) approach. For this, we used a Lck-STIM1-C construct bearing a C-terminal tag comprising residues 1–158 of enhanced (e)YFP, and an Orai3 construct bearing the complementary eYFP sequence (residues 159–238) as an N-terminal tag (Hynes et al. 2008). Lck-STIM-C was chosen for this assay, rather than full-length STIM1, in order to avoid any possible contribution from STIM1 located in the ER membrane. Co-expression of these constructs resulted in a clear plasma membrane-located fluorescence signal (Fig. 5C), again suggesting the presence of a constitutive association between these two proteins. Of course, the above assays each have their own limitations. For example, co-immunoprecipitation assays may simply reflect an association that only occurred following disruption of the cells, and the co-localization of fluorescent-tagged constructs merely indicates that they are located within the same pixel (0.2 μm). In addition, a BiFC signal may be generated simply as a result of the intrinsic affinity of the two eYFP ‘halves’ forcing the apparent association of the two tagged proteins. Moreover, the BiFC technique is not suitable for assessing any time-dependent effects, such as might be induced on addition of arachidonic acid.

Because of this, we turned to a FRET-based assay for assessing potential interactions between Lck-STIM1-C and Orai3, and the affects of arachidonic acid on any such interaction. Co-expression of the Lck-STIM1-C construct bearing a C-terminal Cerulean tag (Lck-STIM1-Cer), together with an Orai3 construct N-terminally tagged with Venus (Ven-Orai3), resulted in a clear FRET signal specifically associated with the plasma membrane (Fig. 5D, with a mean E% value of 14.9 ± 1.3 (n= 29) consistent with a constitutive close association of these two proteins in the membrane. As a control for these data, we examined FRET signals between the Lck-STIM1-Cer construct and a Venus-tagged Orai3 construct bearing a double coiled-coil mutation L285S/L292S that results in the inability of the Orai3 to interact with the C-terminus of STIM1 (Frischauf et al. 2009). The data obtained showed that although co-expression of these two constructs resulted in clear plasma membrane localization, they failed to result in any significant FRET signal (mean E%= 0.04 ± 0.01, n= 17; see Supplemental Fig. S3). An important point to note is that, for proteins localized to the plasma membrane, measured FRET values may be influenced by clustering of the tagged proteins in specific lipid domains. Consequently, we examined the effects of co-expression of the Ven-Orai3 construct with a C-terminally Cerulean-tagged version of the Lck-STIM1-C dimerization-mutant construct (Lck-STIM1-C2A-Cer). As discussed above, the mutations introduced in this construct prevent STIM1 from forming dimers (Yang et al. 2012), and result in the loss of significant ARC channel activity (see Fig. 3B). Therefore, this should act as a negative control for the above data as, although this construct should be spatially distributed in a similar manner to wild-type Lck-STIM1-C, its inability to dimerize would be expected to significantly impair its ability to interact with Orai3. Consistent with this, cells co-expressing Lck-STIM1-C2A-Cer and Ven-Orai3 displayed markedly reduced FRET signals (Fig. 5D) resulting in a mean E% of only 5.1 ± 1.3 (n= 15).

We next compared these findings with those obtained with the Lck-STIM1-4EA construct that contains the four acidic region mutations. As we have shown above, expression of this construct results in the inability to support either constitutive ARC channel activity or arachidonic acid-induced activity (Fig. 4B). Although we have already shown that this construct displays a predominantly plasma membrane localization when expressed alone (Fig. 4D), we now wished to determine whether its inability to support activation of the ARC channels might reflect a failure of the Lck-STIM1-4EA construct to actually associate with Orai3 in the membrane. Examination of FRET signals in cells co-expressing Ven-Orai3 with a C-terminally Cerulean-tagged Lck-STIM1-4EA (Lck-STIM1-4EA-Cer) construct revealed pronounced, membrane-associated FRET signals (mean E%= 24.3 ± 3.7, n= 15; Fig. 5D). Based on these data, we conclude that the failure of the Lck-STIM1-4EA mutant to support any activation of the ARC channels, whether constitutive or arachidonic acid activated, is not due to any obvious loss of interactions with Orai3 in the plasma membrane.

Having demonstrated the apparent constitutive association of Lck-STIM1-C and Orai3 in the plasma membrane, we next examined whether the degree of association between these constructs is affected by the addition of arachidonic acid. For this, we used a time-dependent ‘Live FRET’ analysis protocol (see Methods) with the same fluorescently tagged constructs used above. Values of the plasma membrane-localized FRET signal were recorded at 10 s intervals for 50 s prior to addition of arachidonic acid (8 μm), followed by the subsequent 250 s after such addition, a time period considered appropriate because maximal activation of ARC channel currents following addition of arachidonic acid is typically achieved within 70–100 s (Mignen et al. 2003b, 2008). All values were normalized to the mean value obtained for the 50 s period prior to addition of arachidonic acid. A series of experiments using this analysis in cells expressing the Lck-STIM1-Cer and Ven-Orai3 constructs failed to reveal any significant change in the mean FRET value at any time period after arachidonic acid addition (Fig. 6A). For example, the largest recorded change occurred at 200 s after addition of arachidonic acid, where the mean FRET value was 1.023 ± 0.013 (n= 17, P= 0.39). Based on these data, we conclude that activation of the channels by arachidonic acid does not appear to be associated with any significant increase in the FRET signal between the Lck-STIM1 and the Orai3 subunits.

Figure 6. FRET signals between Orai3 and Lck-STIM1-C, and the effects of ARC channel activation.

Figure 6

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A, the normalized ‘Live FRET’ signal obtained in cells co-expressing the Lck-STIM1-C constructs bearing a C-terminal Cerulean tag and the Orai3 construct N-terminally tagged with Venus. Exogenous arachidonic acid (8 μm) was added as indicated (arrow). All values (means ± SEM, n= 17) were normalized to the mean value recorded for the 50 s prior to addition of arachidonic acid (AA; dashed line). B, as in A above, except that the cells used were HEK cells stably expressing the m3 muscarinic receptor, and carbachol (CCh; 10 μm) was added as indicated. Values are mean ± SEM, n= 18.

Finally, we also examined whether the degree of association between Lck-STIM1-C and Orai3 might be affected by a more physiologically relevant activation of the ARC channels involving agonist-induced stimulation of an appropriate membrane receptor. For this, we used a HEK cell-line stably expressing the muscarinic m3 receptor (m3-HEK cells). We have previously shown that transient expression of the Lck-STIM1-C construct in these m3-HEK cells allows the exclusive activation of ARC channel activity on stimulation of the muscarinic receptors with carbachol (Thompson & Shuttleworth, 2012). Correspondingly, we examined the effect of muscarinic receptor activation on the FRET signals generated as a result of the co-expression of the Lck-STIM1-Cer and Ven-Orai3 constructs, using the same time-dependent analysis protocol described above. Previous studies have shown that such stimulation of this muscarinic receptor-mediated pathway results in a significantly more rapid activation of the ARC channels, with maximal currents being achieved within 40 s (Thompson & Shuttleworth, 2012). Consequently, in this analysis plasma membrane-localized FRET signals were recorded at 5 s intervals for 40 s prior to addition of carbachol (10 μm), followed by the subsequent 150 s after such addition. As before, all values were normalized to the mean value obtained for the period prior to addition of arachidonic acid. Consistent with our findings above, FRET signals prior to addition of carbachol were pronounced in the plasma membrane under resting conditions. However, similar to the data obtained with activation by exogenous arachidonic acid, stimulation of the muscarinic receptors with carbachol failed to significantly change the recorded FRET signal (Fig. 6B), with the largest recorded change occurring at 20 s after addition of carbachol, where the mean FRET value was 1.025 ± 0.009 (n= 18, P= 0.07). As with the above data obtained using exogenous arachidonic acid as the activator, we conclude that physiological stimulation of the channels by expressed muscarinic receptors is not associated with any significant increase in the FRET signal between the Lck-STIM1 and Orai3.

Discussion

Several points arise from the data obtained. First, many of the components of STIM1 that appear to be critical for its interaction with the Orai channels are shared by both ARC and CRAC channels. Thus, the CAD region of STIM1, the cluster of basic residues within the V-shaped cleft structure formed by STIM1 dimers, and the consequent requirement for STIM1 to be in a dimerized state under resting conditions, are essential components for the inter-molecular interactions with, and activation of, both ARC channels and CRAC channels. Despite this, certain differences are also apparent. For example, we could not detect any effect of deletion of those parts of the STIM1 molecule lying C-terminal to the CAD domain, a region that includes a cluster of several serine and proline residues (residues 600–629), as well as the C-terminal polybasic lysine-rich sequence. In contrast, it has been reported that deletion of the lysine-rich domain results in a profound inhibition of CRAC channel activation (Huang et al. 2006), although this effect is reversed by overexpression of Orai1 (Li et al. 2007; Park et al. 2009). Interestingly, the invertebrate STIMs (e.g. Drosophila, C. elegans) do not possess this domain, yet are able to fully activate CRAC channels upon store depletion, either in their native cellular environment (Roos et al. 2005) or when exogenously expressed in mammalian cells (Lorin-Nebel et al. 2007). Consequently, evidence indicates that the main function of this region is not directly related to the activation of channel activity, but rather to facilitate the targeting and clustering of STIM1 in the ER following store depletion to the regions close to the plasma membrane where it can interact with the CRAC channels (Park et al. 2009), an effect that is thought to involve the binding to negatively charged phospholipids in the inner face of the plasma membrane (Liou et al. 2007; Korzeniowski et al. 2009; Walsh et al. 2010; Calloway et al. 2011). Consequently, our finding that removal of this domain failed to result in any detectible effect on ARC channel activity could simply reflect the fact that the Lck-STIM1-C construct is already anchored to the inner face of the plasma membrane via the Lck domain. However, other possibilities exist, as will be discussed below.

An additional distinction revealed in these studies is that the ability to activate the ARC channels appears to critically depend upon interactions of the CAD domain with the Orai3 subunits within the heteromeric channel. It is possible that this effect is simply a reflection of the much stronger predicted coiled-coil nature of this region for Orai3 compared to that of Orai1 (∼18-fold higher, Frischauf et al. 2009), and the apparent constitutive association of the plasma membrane STIM1 with the channel as indicated by the FRET experiments (see below). Of course, this does not preclude possible interactions with Orai1 subunits in the assembled channel, only that such interactions are not required for STIM1 to associate with the channel. Whatever the precise basis, any possible implications of the apparent constitutive STIM1–ARC channel relationship for the actual gating of the channel remain to be determined.

Despite the many similarities in the specific regions of the STIM1 cytosolic domain that appear critical for the activation of both CRAC and ARC channels, the data obtained indicate that the actual activation process differs significantly in these two related channel types (see Fig. 7). This is particularly revealed by procedures or mutations that have been shown to result in the transition of STIM1 in the ER membrane into an ‘activated state’, specifically the loss of calcium from the EF-hand, or the introduction of select mutations within the acidic autoinhibitory region. Both of these result in the constitutive activation of CRAC channels, but have very different effects on the ARC channels. Thus, it would be predicted that the removal of calcium from the EF-hand of STIM1 in the plasma membrane should induce STIM1 to assume such an activated state. However, this does not result in the appearance of any significant ARC channel activity, which only occurs after the addition of arachidonic acid. More curious is the fact that the constructs bearing the acidic-region mutations – which, as noted above, have also been shown to result in STIM1 assuming a constitutively active conformation for CRAC channel function – not only fail to induce any constitutive activation of the ARC channels, but also fail to allow their activation by exogenous arachidonic acid. Critically, not only do these findings for the activation of the ARC channels differ markedly from their effects on CRAC channel activation, they also appear to be internally inconsistent. Thus, although both apparently failed to induce STIM1 into a constitutively active state, the removal of calcium from the extracellular EF-hand in the divalent-free experiments did allow the activation of the ARC channels following addition of arachidonic acid. In contrast, the inclusion of the acidic region mutations prevented any activation of the ARC channels, even by exogenous arachidonic acid. One possible explanation for these differences could be that the acidic region mutations result in the expression of STIM1 in a unique conformation that actually interferes with its ability to interact with the channel, specifically when both are in the same membrane. However, this suggestion would seem to be contradicted by the finding of pronounced plasma membrane FRET signals recorded between Orai3 and the Lck-STIM1-C construct bearing the acidic region mutations (Fig. 5D), suggesting that both are constitutively associated with the channel. Alternatively, it is possible that the loss of calcium from the EF-hand of plasma membrane STIM1 by exposure to a divalent-free extracellular medium does not result in STIM1 assuming an active configuration and that this, at least for the ARC channels, is only achieved on addition of arachidonic acid. Another possibility is that the failure of the acidic region mutant Lck-STIM1-C construct to support any activity of the ARC channels may indicate that this specific set of mutations is acting in some other way that prevents the activation of the ARC channels, even after addition of arachidonic acid. Presumably, any such effect must be independent of the action of these mutations on the conformation of STIM1. Precisely what this inhibitory action might be, and why it should apply exclusively to the ARC channels, remains unclear. An additional point arises from these findings. As already discussed, we would predict that simply exposing cells to a divalent-free extracellular medium should result in the loss of calcium from the EF-hand of STIM1 in the plasma membrane. It is well-established that this loss of calcium from the EF-hand of STIM1, at least that portion of the STIM1 population that is resident in the ER membrane, is the specific initial step in the normal activation of CRAC channels, yet no such activation of these channels was detected in our experiments. Interestingly, this failure to activate the CRAC channels by simple exposure to a divalent-free external solution was also reported in an earlier study by Bakowski & Parekh (2002). The indication, therefore, is that the pool of STIM1 that resides in the plasma membrane is incapable of activating the CRAC channels. How such ‘discrimination’ between the activities of these two pools of cellular STIM1 and their effects on the different endogenous Orai channel types is achieved, remains an interesting question.

Figure 7. Diagram summarizing the differences identified in the mechanism of activation of CRAC and ARC channels by STIM1.

Figure 7

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AA, arachidonic acid; ER, endoplasmic reticulum; PM, plasma membrane.

Despite the above uncertainties, the data obtained indicate that arachidonic acid is not required to drive the association of STIM1 with the channel but, rather, is acting on STIM1 and the channel within a preassembled complex. This is supported by data obtained from a variety of approaches, including co-immunoprecipitation, co-localization, bimolecular fluorescence complementation (BiFC), and FRET analyses, all of which indicate that the plasma membrane STIM1 is constitutively associated with the channel, or at least with the Orai3 subunits within the channel, even under unstimulated conditions. Moreover, the addition of either arachidonic acid, which is required to induce the activation of the channel, or the muscarinic agonist carbachol, failed to significantly affect the measured FRET signal. This activation of a preformed assembly between the channel and plasma membrane STIM1 represents a clear difference from the STIM1-mediated activation of the CRAC channels where oligomerization and interaction with the channel are spatially and temporally distinct events in the activation process, and can be clearly identified as such (Luik et al. 2008; Muik et al. 2008). Given this obvious distinction, it seems that the actions of arachidonic acid are more likely to be associated with the actual gating of the channel. Unfortunately, although evidence suggests that gating involves interactions between STIM1 and domains or residues within the N-terminal region of the Orai proteins (Park et al. 2009), precisely how STIM1 acts to induce such gating is far from clear, either for the CRAC channels or for the ARC channels. Also unclear is precisely why the interactions between STIM1 and Orai1 are, in themselves, sufficient to induce gating of the CRAC channels, whilst addition of arachidonic acid is required to induce gating of the ARC channels despite being in an apparently pre-associated complex with STIM1. With regard to the overall functional effects of these differences on the channel activities, perhaps the highly dynamic and essentially labile nature of the physical and spatial interactions between STIM1 and Orai that underlie the activation of the CRAC channels are ‘duplicated’ by the equally labile nature of the actual activation signal, namely arachidonic acid, and its actions on the constitutively assembled complex of plasma membrane STIM1 and Orai proteins in the ARC channel. Finally, it is also important to note that, as yet, we cannot assume that the effect of arachidonic acid on the STIM1–channel complex is direct. For example, it may involve additional components such as scaffolding proteins, or even physical changes in the membrane properties induced by this fatty acid. Examination of such ideas will require further studies to unravel the precise roles of arachidonic acid and plasma membrane STIM1 in the actual gating of the channel. However, if confirmed, the identified key distinctions in the underlying activities of these two channels will undoubtedly influence our thinking regarding potential mechanisms for activation of the ARC channels, and will also help determine the types of experimental approaches that may be used to investigate the processes involved.

Acknowledgments

The authors thank Dr David Yule of the Department of Pharmacology and Physiology, University of Rochester Medical Center for providing access to the Olympus Fluoview 1000 multiphoton/confocal laser scanning microscope system.

Glossary

ARC channel

arachidonate-regulated Ca2+ channel

BiFC assay

bimolecular fluorescence complementation assay

CAD

CRAC activation domain

CFP

cyan fluorescent protein

CRAC channel

Ca2+ release-activated Ca2+ channel

E%

FRET efficiency

eGFP/eYFP

enhanced GFP/YFP

ER

endoplasmic reticulum

FRET

fluorescence resonance energy transfer

GFP

green fluorescent protein

Lck

N-terminal sequence based on the Src homology domain of Lck kinase

siRNA

small interfering RNA

STIM1

stromal interacting molecule 1

YFP

yellow fluorescent protein

Additional information

Competing interests

None.

Author contributions

J.L.T. and T.J.S. both contributed to the conception, design, analysis and interpretation of the data. T.J.S. drafted the manuscript, with assistance from J.L.T. Both authors have approved the final version.

Funding

This work was supported by National Institutes of Health Grant GM040457 to T.J.S.

Supplementary material

Supplemental material

tjp0591-3507-SD1.docx (743.1KB, docx)
tjp0591-3507-SD2.suppl (43B, suppl)

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Supplementary Materials

tjp0591-3507-SD1.docx (743.1KB, docx)
tjp0591-3507-SD2.suppl (43B, suppl)

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