Jill L. Thompson

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.

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. However, the possible effects of STIM on other modes of receptor‐activated Ca2+ entry have not been examined. Here we show that STIM1 also regulates the arachidonic‐acid‐regulated Ca2+‐selective (ARC) channels – receptor‐activated Ca2+ entry channels whose activation is entirely independent of store depletion. Regulation of the ARC channels by STIM1 does not involve dissociation of Ca2+ from the EF‐hand, or any translocation of STIM1. Instead, a critical role of STIM1 resident in the plasma membrane is indicated. Thus, exposure of intact cells to an antibody targeting the extracellular N‐terminal domain of STIM1 inhibits ARC channel activity without significantly affecting the store‐operated channels. A similar specific inhibition of the ARC channels is seen in cells expressing a STIM1 construct in which the N‐linked glycosylation sites essential for the constitutive cell surface expression of STIM1, were mutated. We conclude that, in contrast to store‐operated channels, regulation of ARC channels by STIM1 depends exclusively on the pool of STIM1 constitutively residing in the plasma membrane. These data demonstrate that STIM1 is a more universal regulator of Ca2+ entry pathways than previously thought, and appears to have multiple modes of action.

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

Agonist‐activated Ca2+ 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 Ca2+‐release‐activated Ca2+ (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 Ca2+ (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.

Reciprocal Regulation of Capacitative and Arachidonate-regulated Noncapacitative Ca2+ Entry Pathways

Receptor-activated Ca2+ entry is usually thought to occur via capacitative or store-operated Ca2+ channels. However, at physiological levels of stimulation, where Ca2+ store depletion is only transient and/or partial, evidence has suggested that an arachidonic acid-dependent noncapacitative Ca2+ entry is responsible. Recently, we have described a novel arachidonate-regulated Ca2+-selective (ARC) conductance that is entirely distinct from store-operated conductances in the same cell. We now show that these ARC channels are indeed specifically activated by low agonist concentrations and provide the predominant route of Ca2+entry under these conditions. We further demonstrate that sustained elevations in cytosolic Ca2+, such as those resulting from activation of store-operated Ca2+ entry by high agonist concentrations, inhibit the ARC channels. This explains earlier failures to detect the presence of this noncapacitative pathway in experiments where store-operated entry had already been fully activated. The result is that the respective activities of ARC and store-operated Ca2+ channels display a unique reciprocal regulation that is related to the specific nature of the [Ca2+] i signals generated at different agonist concentrations. Importantly, these data show that at physiologically relevant levels of stimulation, it is the noncapacitative ARC channels that provide the predominant route for the agonist-activated entry of Ca2+.

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

The activation of Ca2+ entry is a critical component of agonist‐induced cytosolic Ca2+ 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 Ca2+‐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.

Discriminating between Capacitative and Arachidonate-activated Ca2+ Entry Pathways in HEK293 Cells

We have recently questioned whether the capacitative or store-operated model for receptor-activated Ca2+ entry can account for the influx of Ca2+ seen at low agonist concentrations, such a those typically producing [Ca2+] i oscillations. Instead, we have identified an arachidonic acid-regulated, noncapacitative Ca2+ entry mechanism that appears to be specifically responsible for the receptor-activated entry of Ca2+ under these conditions. However, it is unclear whether these two systems reflect the activity of distinct entry pathways or simply different mechanisms of regulating a common pathway. We therefore used the known selectivity of the Ca2+-stimulated type VIII adenylyl cyclase for Ca2+ entry occurring via the capacitative pathway (Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996)J. Biol. Chem. 271, 12438–12444) to attempt to discriminate between these two entry mechanisms in HEK293 cells. Consistent with the earlier reports, we found that thapsigargin induced an approximate 3-fold increase in adenylyl cyclase activity that was unrelated to global changes in [Ca2+] i or to the release of Ca2+ from internal stores but was specifically dependent on the induced capacitative entry of Ca2+. In marked contrast, the arachidonate-induced entry of Ca2+ completely failed to affect adenylyl cyclase activity despite producing a substantially greater rate of entry than that induced by thapsigargin. These data demonstrate that the arachidonate-activated entry of Ca2+occurs via an entirely distinct influx pathway.

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

ARC channels (arachidonate‐regulated Ca2+‐selective channels) are a novel type of highly Ca2+‐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 Ca2+ stores (i.e. they are ‘non‐capacitative’). Previous studies in HEK293 cells have shown that these channels provide the predominant pathway for the entry of Ca2+ seen at low agonist concentrations where oscillatory [Ca2+]i signals are typically produced. In contrast, activation of the more widely studied store‐operated Ca2+ channels (e.g. CRAC channels) is only seen at higher agonist concentrations where sustained ‘plateau‐type’[Ca2+]i responses are observed. We have now demonstrated the presence of ARC channels in both parotid and pancreatic acinar cells and shown that, again, they are specifically activated by the low concentrations of appropriate agonists (carbachol in the parotid, and both carbachol and cholecystokinin in the pancreas) that are associated with oscillatory [Ca2+]i signals in these cells. Uncoupling the receptor‐mediated activation of cytosolic phospholipase A2 (cPLA2) with isotetrandrine reduces the activation of the ARC channels by carbachol and, correspondingly, markedly inhibits the [Ca2+]i signals induced by low carbachol concentrations, whilst those signals seen at high agonist concentrations are essentially unaffected. Interestingly, in the pancreatic acinar cells, activation by cholecystokinin induces a current through the ARC channels that is only approximately 60% of that seen with carbachol. This is consistent with previous reports indicating that carbachol‐induced [Ca2+]i signals in these cells are much more dependent on Ca2+ entry than are the cholecystokinin‐induced responses.

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., and Shuttleworth, T. J. (2001) J. Biol. Chem. 276, 35676–35683) represents a switching between two distinct Ca2+-selective channels: the noncapacitative arachidonate-regulated Ca2+ channels (ARC channels) and the store-operated Ca2+ channels (SOC channels). This switch is directly associated with the change from oscillatory to sustained Ca2+ signals as agonist concentrations increase and involves a Ca2+-dependent inhibition of the ARC channels. Here we show that this process is mediated via a calcineurin-dependent inhibition of the noncapacitative ARC channels. Pharmacological and molecular inhibition of calcineurin activity (using cyclosporin or the FK506 analogue ascomycin, and a transfected C-terminal domain of the calcineurin inhibitory protein CAIN, respectively) results in a complete reversal of the Ca2+-dependent inhibition of the ARC channels. Agonist concentrations that result in oscillatory Ca2+ signals and specifically activate Ca2+ entry through the ARC channels fail to increase calcineurin activity. However, agonist concentrations that activate the store-operated Ca2+ channels and produce prolonged increases in cytosolic Ca2+ concentrations increase calcineurin activity. Thus, calcineurin is the key mediator of the reciprocal regulation of these co-existing channels, allowing each to play a unique and non-overlapping role in Ca2+ signaling.

External TEA Block of Shaker K+ Channels Is Coupled to the Movement of K+ Ions within the Selectivity Filter

Recent molecular dynamic simulations and electrostatic calculations suggested that the external TEA binding site in K+ channels is outside the membrane electric field. However, it has been known for some time that external TEA block of Shaker K+ channels is voltage dependent. To reconcile these two results, we reexamined the voltage dependence of block of Shaker K+ channels by external TEA. We found that the voltage dependence of TEA block all but disappeared in solutions in which K+ ions were replaced by Rb+. These and other results with various concentrations of internal K+ and Rb+ ions suggest that the external TEA binding site is not within the membrane electric field and that the voltage dependence of TEA block in K+ solutions arises through a coupling with the movement of K+ ions through part of the membrane electric field. Our results suggest that external TEA block is coupled to two opposing voltage-dependent movements of K+ ions in the pore: (a) an inward shift of the average position of ions in the selectivity filter equivalent to a single ion moving ∼37% into the pore from the external surface; and (b) a movement of internal K+ ions into a vestibule binding site located ∼13% into the membrane electric field measured from the internal surface. The minimal voltage dependence of external TEA block in Rb+ solutions results from a minimal occupancy of the vestibule site by Rb+ ions and because the energy profile of the selectivity filter favors a more inward distribution of Rb+ occupancy.

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 Ca2+ release-activated Ca2+ (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 Ca2+ 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 Ca2+ entry recorded in the heterozygous carriers.