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.

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.

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

The ARC channel is a small conductance, highly Ca²⁺-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 Ca²⁺ 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 Ca²⁺ entry during the oscillatory signals generated at low agonist concentrations.

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.

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 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.

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.

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.

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.

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.

A mathematical model of fluid secretion from a parotid acinar cell.

Salivary fluid secretion is crucial for preventing problems such as dryness of mouth, difficulty with mastication and swallowing, as well as oral pain and dental cavities. Fluid flow is driven primarily by the transepithelial movement of chloride and sodium ions into the parotid acinus lumen. The activation of Cl(-) channels is calcium dependent, with the average elevated calcium concentration during calcium oscillations increasing the conductance of the channels, leading to an outflow of Cl(-).

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.

A critical analysis of carbonic anhydrase function, respiratory gas exchange, and the acid-base control of secretion in the rectal gland of Squalus acanthias.

We compared in vivo responses of rectal gland secretion to carbonic anhydrase (CA) inhibition (10(-4) mol l(-1) acetazolamide) in volume-loaded dogfish with in vitro responses in an isolated-perfused gland stimulated with 5 x 10(-6) mol l(-1) forskolin and removed from systemic influences. We also measured respiratory gas exchange in the perfused gland, described the acid-base status of the secreted fluid, and determined the relative importance of various extracellular and intracellular acid-base parameters in controlling rectal gland secretion in vitro. In vivo, acetazolamide inhibited Cl(-) secretion and decreased pHi in the rectal gland, but interpretation was confounded by an accompanying systemic respiratory acidosis, which would also have contributed to the inhibition.

Control of rectal gland secretion by blood acid-base status in the intact dogfish shark (Squalus acanthias).

In order to address the possible role of blood acid-base status in controlling the rectal gland, dogfish were fitted with indwelling arterial catheters for blood sampling and rectal gland catheters for secretion collection. In intact, unanaesthetized animals, isosmotic volume loading with 500 mmol L-1 NaCl at a rate of 15 mL kg-1 h-1 produced a brisk, stable rectal gland secretion flow of about 4 mL kg-1 h-1. Secretion composition (500 mmol L-1 Na+ and Cl-; 5 mmol L-1 K+; <1 mmol L-1 Ca2+, Mg2+, SO(4)2-, or phosphate) was almost identical to that of the infusate with a pH of about 7.