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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

STIM1 and the noncapacitative ARC channels.

Our understanding of the nature and regulation of receptor-activated Ca(2+) entry in nonexcitable cells has recently undergone a radical change that began with the identification of the stromal interacting molecule proteins (e.g., STIM1) as playing a critical role in the regulation of the capacitative, or store-operated, Ca(2+) entry.

STIM1 regulates Ca2+ entry via arachidonate-regulated Ca2+-selective (ARC) channels without store depletion or translocation to the plasma membrane.

Recent studies have indicated a critical role for STIM (stromal interacting molecule) proteins in the regulation of the store-operated mode of receptor-activated Ca2+ entry. Current models emphasize the role of STIM located in the endoplasmic reticulum membrane, where a Ca2+-binding EF-hand domain within the N-terminal of the protein lies within the lumen and is thought to represent the sensor for the depletion of intracellular Ca2+ stores. Dissociation of Ca2+ from this domain induces the aggregation of STIM to regions of the ER immediately adjacent to the plasma membrane where it acts to regulate the activity of store-operated Ca2+ channels.

Arachidonate-regulated Ca2+-selective (ARC) channel activity is modulated by phosphorylation and involves an A-kinase anchoring protein.

In many non-excitable cells, the predominant mode of agonist-activated Ca(2+) entry switches from the arachidonic acid-regulated Ca(2+) (ARC) channels at low agonist concentrations, to store-operated channels at high concentrations. Underlying this process is the inhibition of the ARC channels by a calcineurin-mediated dephosphorylation, which inhibits the ability of arachidonic acid to activate the channels. Following such a dephosphorylation, we found that restoration of the sensitivity of the ARC channels to arachidonic acid, as well as to low concentrations of carbachol, was specifically dependent on protein kinase A (PKA) activity.

Agonist activation of arachidonate-regulated Ca2+-selective (ARC) channels in murine parotid and pancreatic acinar cells.

ARC channels (arachidonate-regulated Ca(2+)-selective channels) are a novel type of highly Ca(2+)-selective channel that are specifically activated by low concentrations of agonist-induced arachidonic acid. This activation occurs in the absence of any depletion of internal Ca(2+) stores (i.e.

ARC channels: a novel pathway for receptor-activated calcium entry.

In many nonexcitable cells, stimulation with low agonist concentrations specifically activates Ca2+ entry via arachidonic acid-regulated, highly Ca2+-selective ARC channels. Only at high agonist concentrations are the more widely studied store-operated channels activated, producing sustained elevated cytosolic Ca2+ concentration signals. These signals activate calcineurin, which in turn inhibits the ARC channels, resulting in a "reciprocal regulation" of these two distinct Ca2+-entry pathways that may have important functional implications for the cell.

Calcineurin directs the reciprocal regulation of calcium entry pathways in nonexcitable cells.

The reciprocal regulation of noncapacitative and capacitative (or store-operated) Ca2+ entry in nonexcitable cells (Mignen, O., Thompson, J. L.

Ca2+ selectivity and fatty acid specificity of the noncapacitative, arachidonate-regulated Ca2+ (ARC) channels.

The arachidonate-regulated, Ca(2+)-selective ARC channels represent a novel receptor-activated pathway for the entry of Ca(2+) in nonexcitable cells that is entirely separate from the widely studied store-operated, Ca(2+) release-activated Ca(2+) channels. Activation of ARC channels occurs specifically at the low agonist concentrations typically associated with oscillatory Ca(2+) signals and appears to provide the predominant mode of Ca(2+) entry under these conditions (Mignen, O., Thompson, J.