Phosphorylation-mediated structural changes within the SOAR domain of stromal interaction molecule 1 enable specific activation of distinct Orai channels.

Low-conductance, highly calcium-selective channels formed by the Orai proteins exist as store-operated CRAC channels and store-independent, arachidonic acid-activated ARC channels. Both are activated by stromal interaction molecule 1 (STIM1), but CRAC channels are activated by STIM1 located in the endoplasmic reticulum membrane, whereas ARC channels are activated by the minor plasma membrane-associated pool of STIM1. Critically, maximally activated CRAC channel and ARC channel currents are completely additive within the same cell, and their selective activation results in their ability to each induce distinct cellular responses. We have previously shown that specific ARC channel activation requires a PKA-mediated phosphorylation of a single threonine residue (Thr389) within the cytoplasmic region of STIM1. Here, examination of the molecular basis of this phosphorylation-dependent activation revealed that phosphorylation of the Thr389 residue induces a significant structural change in the STIM1-Orai-activating region (SOAR) that interacts with the Orai proteins, and it is this change that determines the selective activation of the store-independent ARC channels versus the store-operated CRAC channels. In conclusion, our findings reveal the structural changes underlying the selective activation of STIM1-induced CRAC or ARC channels that determine the specific stimulation of these two functionally distinct Ca2+ entry pathways.

Anchoring protein AKAP79-mediated PKA phosphorylation of STIM1 determines selective activation of the ARC channel, a store-independent Orai channel.

KEY POINTS: Although both the calcium store-dependent CRAC channels and the store-independent ARC channels are regulated by the protein STIM1, CRAC channels are regulated by STIM1 in the endoplasmic reticulum, whilst ARC channels are regulated by the STIM1 constitutively resident in the plasma membrane. We now demonstrate that activation of the ARC channels, but not CRAC channels, is uniquely dependent on phosphorylation of a single residue (T389) in the extensive cytosolic domain of STIM1 by protein kinase A. We further demonstrate that the phosphorylation of the T389 residue by protein kinase A is mediated by the association of plasma membrane STIM1 with the scaffolding protein AKAP79. Together, these findings indicate that the phosphorylation status of this single residue in STIM1 represents a key molecular determinant of the relative activities of these two co-existing Ca(2+) entry channels that are known to play critical, but distinct, roles in modulating a variety of physiologically relevant activities. ABSTRACT: The low-conductance, highly calcium-selective channels encoded by the Orai family of proteins represent a major pathway for the agonist-induced entry of calcium associated with the generation and modulation of the key intracellular calcium signals that initiate and control a wide variety of physiologically important processes in cells. There are two distinct members of this channel family that co-exist endogenously in many cell types: the store-operated Ca(2+) release-activated CRAC channels and the store-independent arachidonic acid-regulated ARC channels. Although the activities of both channels are regulated by the stromal-interacting molecule-1 (STIM1) protein, two distinct pools of this protein are responsible, with the major pool of STIM1 in the endoplasmic reticulum membrane regulating CRAC channel activity, whilst the minor pool of plasma membrane STIM1 regulates ARC channel activity. We now show that a critical feature in determining this selective activation of the two channels is the phosphorylation status of a single threonine residue (T389) within the extensive (∼450 residue) cytosolic domain of STIM1. Specifically, protein kinase A (PKA)-mediated phosphorylation of T389 of STIM1 is necessary for effective activation of the ARC channels, whilst phosphorylation of the same residue actually inhibits the ability of STIM1 to activate the CRAC channels. We further demonstrate that the PKA-mediated phosphorylation of T389 occurs at the plasma membrane via the involvement of the anchoring protein AKAP79, which is constitutively associated with the pool of STIM1 in the plasma membrane. The novel mechanism we have described provides a means for the cell to precisely regulate the relative activities of these two channels to independently modulate the resulting intracellular calcium signals in a physiologically relevant manner.

Exploring the unique features of the ARC channel, a store-independent Orai channel.

The discovery of the Orai proteins, and the identification of STIM1 as the molecule that regulates them, was based on their role in the agonist-activated store-operated entry of calcium via the CRAC channels. However, these same proteins are also essential components of the ARC channels responsible for a similar agonist-activated, but store-independent, arachidonic acid-regulated entry of calcium. The fact that these 2 biophysically similar calcium entry pathways frequently co-exist in the same cells suggests that they must each possess different features that allow them to function in distinct ways to regulate specific cellular activities. This review begins to address this question by describing recent findings characterizing the unique features of the ARC channels--their molecular composition, STIM1-dependent activation, and physiological activities--and the importance of defining such features for the accurate therapeutic targeting of these 2 Orai channel subtypes.

The ARC channel--an endogenous store-independent Orai channel.

Although Orai channels and their regulator stromal interacting molecule 1 (STIM1) were originally identified and described as the key components of the store-operated highly calcium-selective CRAC channels, it is now clear that these proteins are equally essential components of the agonist-activated, store-independent calcium entry pathway mediated by the arachidonic acid-regulated calcium-selective (ARC) channel. Correspondingly, ARC channels display biophysical properties that closely resemble those of CRAC channels but, whereas the latter is formed exclusively by Orai1 subunits, the ARC channel is formed by a combination of Orai1 and Orai3 subunits. Moreover, while STIM1 in the membrane of the endoplasmic reticulum is the critical sensor of intracellular calcium store depletion that results in the activation of the CRAC channels, it is the pool of STIM1 resident in the plasma membrane that regulates the activity of the store-independent ARC channels. Here, we describe the unique features of the ARC channels and their activation and discuss recent evidence indicating how these two coexisting, and biophysically very similar, Orai channels act to play entirely distinct roles in the regulation of various important cellular activities.

How many Orai's does it take to make a CRAC channel?

CRAC (Calcium Release-Activated Calcium) channels represent the primary pathway for so-called "store-operated calcium entry" - the cellular entry of calcium induced by depletion of intracellular calcium stores. These channels play a key role in diverse cellular activities, most noticeably in the differentiation and activation of Tcells, and in the response of mast cells to inflammatory signals. CRAC channels are formed by members of the recently discovered Orai protein family, with previous studies indicating that the functional channel is formed by a tetramer of Orai subunits. However, a recent report has shown that crystals obtained from the purified Drosophila Orai protein display a hexameric channel structure. Here, by comparing the biophysical properties of concatenated hexameric and tetrameric human Orai1 channels expressed in HEK293 cells, we show that the tetrameric channel displays the highly calcium-selective conductance properties consistent with endogenous CRAC channels, whilst the hexameric construct forms an essentially non-selective cation channel.

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

Currently, Orai proteins are known to encode two distinct agonist-activated, highly calcium-selective channels: the store-operated Ca(2+) release-activated Ca(2+) (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.

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel.

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.

Modelling the effects of calcium waves and oscillations on saliva secretion.

An understanding of Ca(2+) signalling in saliva-secreting acinar cells is important, as Ca(2+) is the second messenger linking stimulation of cells to production of saliva. Ca(2+) signals affect secretion via the ion channels located both apically and basolaterally in the cell. By approximating Ca(2+) waves with periodic functions on the apical and basolateral membranes, we isolate individual wave properties and investigate them for their effect on fluid secretion in a mathematical model of the acinar cell. Mean Ca(2+) concentration is found to be the most significant property in signalling secretion. Wave speed was found to encode a range of secretion rates. Ca(2+) oscillation frequency and amplitude had little effect on fluid secretion.

Orai3--the 'exceptional' Orai?

The field of agonist-activated Ca(2+) entry in non-excitable cells underwent a revolution some 5 years ago with the discovery of the Orai proteins as the essential pore-forming components of the low-conductance, highly Ca(2+)-selective CRAC channels whose activation is dependent on depletion of intracellular stores. Mammals possess three distinct Orai proteins (Orai1, 2 and 3) of which Orai3 is unique to this class, apparently evolving from Orai1. However, the sequence of Orai3 shows marked differences from that of Orai1, particularly in those regions of the protein outside of the essential pore-forming domains. Correspondingly, studies from several different groups have indicated that the inclusion of Orai3 is associated with the appearance of conductances that display unique features in their gating, selectivity, regulation and mode of activation. In this Topical Review, these features are discussed with the purpose of proposing that the evolutionary appearance of Orai3 in mammals, and the consequent development of conductances displaying novel properties - whether formed by Orai3 alone or in conjunction with the other Orai proteins - is associated with the specific role of this member of the Orai family in a unique range of distinct cellular activities.

STIM and Orai proteins and the non-capacitative ARC channels.

The ARC channel is a small conductance, highly Ca²âº-selective ion channel whose activation is specifically dependent on low concentrations of arachidonic acid acting at an intracellular site. They are widely distributed in diverse cell types where they provide an alternative, store-independent pathway for agonist-activated Ca²âº entry. Although biophysically similar to the store-operated CRAC channels, these two conductances function under distinct conditions of agonist stimulation, with the ARC channels providing the predominant route of Ca²âº entry during the oscillatory signals generated at low agonist concentrations. Despite these differences in function, like the CRAC channel, activation of the ARC channels is dependent on STIM1, but it is the pool of STIM1 that constitutively resides in the plasma membrane that is responsible. Similarly, both channels are formed by Orai proteins but, whilst the CRAC channel pore is a tetrameric assembly of Orai1 subunits, the ARC channel pore is formed by a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. There is increasing evidence that the activity of these channels plays a critical role in a variety of different cellular activities.

Orai channel-dependent activation of phospholipase C-δ: a novel mechanism for the effects of calcium entry on calcium oscillations.

The frequency of oscillatory Ca(2+) signals is a major determinant in the selective activation of discrete downstream responses in non-excitable cells. An important modulator of this oscillation frequency is known to be the rate of agonist-activated Ca(2+) entry. However precisely how this is achieved and the respective roles of store-operated versus store-independent Ca(2+) entry pathways in achieving this are unclear. Here, we examine the possibility that a direct stimulation of a phospholipase C (PLC) by the entering Ca(2+) can induce a modulation of Ca(2+) oscillation frequency, and examine the roles of the endogenous store-operated and store-independent Orai channels (CRAC and ARC channels, respectively) in such a mechanism. Using the decline in the magnitude of currents through expressed PIP(2)-dependent Kir2.1 channels as a sensitive assay for PLC activity, we show that simple global increases in Ca(2+) concentrations over the physiological range do not significantly affect PLC activity. Similarly, maximal activation of endogenous CRAC channels also fails to affect PLC activity. In contrast, equivalent activation of endogenous ARC channels resulted in a 10-fold increase in the measured rate of PIP(2) depletion. Further experiments show that this effect is strictly dependent on the Ca(2+) entering via these channels, rather than the gating of the channels or the arachidonic acid used to activate them, and that it reflects the activation of a PLCδ by local Ca(2+) concentrations immediately adjacent to the active channels. Finally, based on the effects of expression of either a dominant-negative mutant Orai3 that is an essential component of the ARC channel, or a catalytically compromised mutant PLCδ, it was shown that this specific action of the store-independent ARC channel-mediated Ca(2+) entry on PLCδ has a significant impact on the oscillation frequency of the Ca(2+) signals activated by low concentrations of agonist.

The N-terminal domain of Orai3 determines selectivity for activation of the store-independent ARC channel by arachidonic acid.

Although highly selective Ca²(+) entry pathways play a critical role in agonist-activated Ca²(+) signals in non-excitable cells, only with the recent discovery of the Orai proteins have the first insights into the molecular nature of these pathways been possible. To date, just two such highly Ca²(+)-selective "Orai channels" have been identified in native cells - the store-operated CRAC channels and the store-independent, arachidonic acid-activated ARC channels. Studies have shown that the functional CRAC channel pore is formed by a tetrameric arrangement of Orai1 subunits, whilst a heteropentamer of three Orai1 subunits and two Orai3 subunits forms the functional ARC channel pore. Importantly, this inclusion of Orai3 subunits in the ARC channel structure has been shown to play a specific role in determining the selectivity of these channels for activation by arachidonic acid. Using an approach based on the expression of various concatenated constructs, we examined the basis for this Orai3-dependent effect on selectivity for arachidonic acid. We show that, whilst heteropentamers containing only one Orai3 subunit are sensitive to arachidonic acid, specific selectivity for activation by this fatty acid is only achieved on inclusion of the second Orai3 subunit in the pentamer. Further studies identified the cytosolic N-terminal domain of Orai3 as the region specifically responsible for this switch in selectivity. Substitution of just this domain into an otherwise complete single Orai1 subunit within a concatenated 31111 pentamer is sufficient to change the resulting channel from one that is predominantly store-operated, to one that is exclusively activated by arachidonic acid.

A dynamic model of saliva secretion.

We construct a mathematical model of the parotid acinar cell with the aim of investigating how the distribution of K(+) and Cl(-) channels affects saliva production. Secretion of fluid is initiated by Ca(2+) signals acting on Ca(2+) dependent K(+) and Cl(-) channels. The opening of these channels facilitates the movement of Cl(-) ions into the lumen which water follows by osmosis. We use recent results into both the release of Ca(2+) from internal stores via the inositol (1,4,5)-trisphosphate receptor (IP(3)R) and IP(3) dynamics to create a physiologically realistic Ca(2+) model which is able to recreate important experimentally observed behaviours seen in parotid acinar cells. We formulate an equivalent electrical circuit diagram for the movement of ions responsible for water flow which enables us to calculate and include distinct apical and basal membrane potentials to the model. We show that maximum saliva production occurs when a small amount of K(+) conductance is located at the apical membrane, with the majority in the basal membrane. The maximum fluid output is found to coincide with a minimum in the apical membrane potential. The traditional model whereby all Cl(-) channels are located in the apical membrane is shown to be the most efficient Cl(-) channel distribution.

The molecular architecture of the arachidonate-regulated Ca2+-selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunits.

The activation of Ca(2+) entry is a critical component of agonist-induced cytosolic Ca(2+) signals in non-excitable cells. Although a variety of different channels may be involved in such entry, the recent identification of the STIM and Orai proteins has focused attention on the channels in which these proteins play a key role. To date, two distinct highly Ca(2+)-selective STIM1-regulated and Orai-based channels have been identified - the store-operated CRAC channels and the store-independent arachidonic acid activated ARC channels. In contrast to the CRAC channels, where the channel pore is composed of only Orai1 subunits, both Orai1 and Orai3 subunits are essential components of the ARC channel pore. Using an approach involving the co-expression of a dominant-negative Orai1 monomer along with different preassembled concatenated Orai1 constructs, we recently demonstrated that the functional CRAC channel pore is formed by a homotetrameric assembly of Orai1 subunits. Here, we use a similar approach to demonstrate that the functional ARC channel pore is a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. Expression of concatenated pentameric constructs with this stoichiometry results in the appearance of large currents that display all the key biophysical and pharmacological features of the endogenous ARC channels. They also replicate the essential regulatory characteristics of native ARC channels including specific activation by low concentrations of arachidonic acid, complete independence of store depletion, and an absolute requirement for the pool of STIM1 that constitutively resides in the plasma membrane.

Arachidonic acid, ARC channels, and Orai proteins.

A critical role for arachidonic acid in the regulation of calcium entry during agonist activation of calcium signals has become increasingly apparent in numerous studies over the past 10 years or so. In particular, low concentrations of this fatty acid, generated as a result of physiologically relevant activation of appropriate receptors, induces the activation of a unique, highly calcium-selective conductance now known as the ARC channel. Activation of this channel is specifically dependent on arachidonic acid acting at the intracellular surface of the membrane, and is entirely independent of any depletion of internal calcium stores. Importantly, a specific role of this channel in modulating the frequency of oscillatory calcium signals in various cell types has been described. Recent studies, subsequent to the discovery of STIM1 and the Orai proteins and their role in the store-operated CRAC channels, have revealed that these same proteins are also integral components of the ARC channels and their activation. However, unlike the CRAC channels, activation of the ARC channels depends on the pool of STIM1 that is constitutively resident in the plasma membrane, and the pore of these channels is comprised of both Orai1 and Orai3 subunits. The clear implication is that CRAC channels and ARC channels are closely related, but have evolved to play unique roles in the modulation of calcium signals-largely as a result of their entirely distinct modes of activation. Given this, although the precise details of how arachidonic acid acts to activate the channels remain unclear, it seems likely that the specific molecular features of these channels that distinguish them from the CRAC channels--namely Orai3 and/or plasma membrane STIM1--will be involved.

The Orai1 severe combined immune deficiency mutation and calcium release-activated Ca2+ channel function in the heterozygous condition.

Homozygous expression of Orai1 bearing the R91W mutation results in the complete abrogation of currents through the store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, resulting in a form of hereditary severe combined immune deficiency (SCID) syndrome (Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S. H., Tanasa, B., Hogan, P. G., Lewis, R. S., Daly, M., and Rao, A. (2006) Nature 441, 179-185). Although heterozygous carriers of the mutation show no clinical symptoms of immunodeficiency, store-operated Ca(2+) entry in their T cells is impaired, suggesting a gene-dosage effect of the mutation. We have recently demonstrated that the functional CRAC channel pore is composed of a tetrameric assembly of Orai1 subunits (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2008) J. Physiol. 586, 419-425). Therefore, to directly quantify the effect of the SCID mutant in the heterozygous situation, we generated a series of concatenated tetramers of Orai1 that included different numbers and arrangements of the R91W Orai1 subunits. The data obtained show that inclusion of increasing numbers of mutant subunits results in a graded reduction in CRAC channel currents and that this effect is independent of the spatial arrangement or order of the mutant subunits in the tetramer. Macroscopic biophysical properties of the channels were unchanged by inclusion of the mutant subunits, although the rate at which the current activates on store depletion was slowed. We conclude that incorporation of R91W mutant Orai1 subunits in the CRAC channel pore affects the overall magnitude of its conductance and that this effect is related solely to the number of mutant subunits incorporated. Predictions based on the tetrameric channel structure indicate that the graded effect of incorporation of SCID mutant subunits into such an assembly is quantitatively consistent with the previously demonstrated impaired effects on Ca(2+) entry recorded in the heterozygous carriers.

Orai1 subunit stoichiometry of the mammalian CRAC channel pore.

Agonist-activated Ca2+ entry plays a critical role in Ca2+ signalling in non-excitable cells. One mode of such entry is activated as a consequence of the depletion of intracellular Ca2+ stores. This depletion is sensed by the protein STIM1 in the endoplasmic reticulum, which then translocates to regions close to the plasma membrane where it induces the activation of store-operated conductances. The most thoroughly studied of these conductances are the Ca2+ release-activated Ca2+ (CRAC) channels, and recent studies have identified the protein Orai1 as comprising the essential pore-forming subunit of these channels. Although evidence suggests that Orai1 can assemble as homomultimers, whether this assembly is necessary for the formation of functional CRAC channels and, if so, their relevant stoichiometry is unknown. To examine this, we have used an approach involving the expression of preassembled tandem Orai1 multimers comprising different numbers of subunits into cells stably overexpressing STIM1, followed by the recording of maximally activated CRAC channel currents. In each case, any necessity for recruitment of additional Orai1 units to these preassembled multimers in order to form functional channels was evaluated by coexpression with a dominant-negative Orai1 mutant. In this way we were able to demonstrate, for the first time, that the functional CRAC channel pore is formed by a tetrameric assembly of Orai1 subunits.

Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels.

Agonist-activated Ca(2+) signals in non-excitable cells are profoundly influenced by calcium entry via both store-operated and store-independent conductances. Recent studies have demonstrated that STIM1 plays a key role in the activation of store-operated conductances including the Ca(2+)-release-activated Ca(2+) (CRAC) channels, and that Orai1 comprises the pore-forming component of these channels. We recently demonstrated that STIM1 also regulates the activity of the store-independent, arachidonic acid-regulated Ca(2+) (ARC) channels, but does so in a manner entirely distinct from its regulation of the CRAC channels. This shared ability to be regulated by STIM1, together with their similar biophysical properties, suggested that these two distinct conductances may be molecularly related. Here, we report that whilst the levels of Orai1 alone determine the magnitude of the CRAC channel currents, both Orai1 and the closely related Orai3 are critical for the corresponding currents through ARC channels. Thus, in cells stably expressing STIM1, overexpression of Orai1 increases both CRAC and ARC channel currents. Whilst similar overexpression of Orai3 alone has no effect, ARC channel currents are specifically increased by expression of Orai3 in cells stably expressing Orai1. Moreover, expression of a dominant-negative mutant Orai3, either alone or in cells expressing wild-type Orai1, profoundly and specifically reduces currents through the ARC channels without affecting those through the CRAC channels, and siRNA-mediated knockdown of either Orai1 or Orai3 markedly inhibits ARC channel currents. Importantly, our data also show that the precise effects observed critically depend on which of the three proteins necessary for effective ARC channel activity (STIM1, Orai1 and Orai3) are rate limiting under the specific conditions employed.