I(ARC), a novel arachidonate-regulated, noncapacitative Ca(2+) entry channel

Along with the inositol trisphosphate-induced release of stored Ca(2+), a receptor-enhanced entry of Ca(2+) is a critical component of intracellular Ca(2+) signals generated by agonists acting at receptors coupled to the activation of phospholipase C. Although the simple emptying of the intracellular Ca(2+) stores is known to be capable of activating Ca(2+) entry via the so-called "capacitative" mechanism, recent evidence suggests that Ca(2+) entry at physiological agonist concentrations, where oscillatory Ca(2+) signals are typically observed, does not conform to such a model. Instead, a noncapacitative Ca(2+) entry pathway regulated by arachidonic acid appears to be responsible for Ca(2+) entry under these conditions. Using whole-cell patch clamp techniques we demonstrate that low concentrations of arachidonic acid activate a Ca(2+)-selective current that is superficially similar to the store-operated current I(CRAC), but which also demonstrates certain distinct features. We have named this novel current I(ARC) (for arachidonate-regulated calcium current). Importantly, I(ARC) can be readily activated in cells whose Ca(2+) stores have been maximally depleted. I(ARC) represents a novel Ca(2+) entry pathway that is entirely separate from those activated by store depletion and is specifically activated at physiological levels of stimulation.     

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.     

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. L., and Shuttleworth, T. J. (2001) J. Biol. Chem. 276, 35676-35683). In this study we demonstrate that ARC channels are present in a variety of different cell types including both cell lines and primary cells. Examination of their pharmacology revealed that currents through these channels are significantly inhibited by low concentrations (< 5 microm) of Gd(3+), are unaffected by 100 microm 2-aminoethyoxydiphenyl borane, and are not activated by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (100 microm). Their selectivity for Ca(2+) was assessed by determining the EC(50) for external Ca(2+) block of the monovalent currents observed in the absence of external divalent cations. The value obtained (150 nm) indicates that the Ca(2+) selectivity of ARC channels is extremely high. Examination of the ability of various fatty acids, including arachidonic acid, to activate the ARC channels demonstrated that activation does not reflect any nonspecific membrane fluidity or detergent effects, shows a high degree of specificity for arachidonic acid over other fatty acids (especially monounsaturated and saturated fatty acids), and is independent of any arachidonic acid metabolite. Moreover, studies using the charged analogue arachidonyl coenzyme A demonstrate that activation of the ARC channels reflects an action of the fatty acid specifically at the internal face of the plasma membrane. Whether this involves a direct action of arachidonic acid on the channel protein itself or an action on some intermediary molecule is, at present, unclear.     

All-or-none activation of CRAC channels by agonist elicits graded responses in populations of mast cells

In nonexcitable cells, receptor stimulation evokes Ca(2+) release from the endoplasmic reticulum stores followed by Ca(2+) influx through store-operated Ca(2+) channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca(2+) release-activated Ca(2+) (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca(2+)-dependent activation of protein kinase Calpha and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca(2+)-dependent phospholipase A(2) and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C(4). In cell population studies, ERK activation, arachidonic acid release, and leukotriene C(4) secretion were all graded with stimulus intensity. However, at a single cell level, Ca(2+) influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca(2+) influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca(2+)entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.     

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. As such, current models emphasize the role of STIM1 located in the endoplasmic reticulum membrane, where it senses the status of the intracellular Ca(2+) stores via a luminal N-terminal Ca(2+)-binding EF-hand domain. Dissociation of Ca(2+) from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca(2+) channels (e.g., CRAC channels). Thus, the specific dependence on store-depletion, and the role of the Ca(2+)-binding EF-hand domain in this process, are critical to all current models of the action of STIM1 on Ca(2+) entry. However, until recently, the effects of STIM1 on other modes of receptor-activated Ca(2+) entry have not been examined. Surprisingly, we found that STIM1 exerts similar, although not identical, actions on the arachidonic acid-regulated Ca(2+)-selective (ARC) channels-a widely expressed mode of agonist-activated Ca(2+) entry whose activation is completely independent of Ca(2+) store depletion. Regulation of the ARC channels by STIM1 is not only independent of store depletion, but also of the Ca(2+)-binding function of the EF-hand, and translocation of STIM1 to the plasma membrane. Instead, it is the pool of STIM1 that constitutively resides in the plasma membrane that is critical for the regulation of the ARC channels. Thus, ARC channel activity is selectively inhibited by exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1. Similarly, introducing mutations in STIM1 that prevent the N-linked glycosylation-dependent constitutive expression of the protein in the plasma membrane specifically inhibits the activity of the ARC channels without affecting the CRAC channels. These studies demonstrate that STIM1 is a far more universal regulator of Ca(2+) entry pathways than previously assumed, and has multiple, and entirely distinct, modes of action. Precisely how this same protein can act in such separate and specific ways on these different pathways of agonist-activated Ca(2+)entry remains an intriguing, yet currently unresolved, question.     

Regulation of store-operated calcium channels by the intermediary metabolite pyruvic acid

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses, including neurotransmitter release, muscle contraction, and cell growth and proliferation. A major route for Ca(2+) influx is through store-operated Ca(2+) channels. One important intracellular target for Ca(2+) entry through store-operated channels is the mitochondrion, which increases aerobic metabolism and ATP production after Ca(2+) uptake. Here, we reveal a novel feedback pathway whereby pyruvic acid, a critical rate-limiting substrate for mitochondrial respiration, increases store-operated entry by reducing inactivation of the channels. Importantly, the effects of pyruvic acid are manifest at physiologically relevant concentrations and membrane potentials. The reduction in the inactivation of calcium release-activated calcium (CRAC) channels by pyruvate is highly specific in that it is not mimicked by other intermediary metabolic acids, does not require its metabolism, is independent of its Ca(2+) buffering action, and does not involve mitochondrial Ca(2+) uptake or ATP production. These results reveal a new and direct link between intermediary metabolism and ion-channel gating and identify pyruvate as a potential signaling messenger linking energy demand to calcium-channel function.     

Discriminating between capacitative and arachidonate-activated Ca(2+) entry pathways in HEK293 cells.

We have recently questioned whether the capacitative or store-operated model for receptor-activated Ca(2+) entry can account for the influx of Ca(2+) seen at low agonist concentrations, such a those typically producing [Ca(2+)](i) oscillations. Instead, we have identified an arachidonic acid-regulated, noncapacitative Ca(2+) entry mechanism that appears to be specifically responsible for the receptor-activated entry of Ca(2+) 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 Ca(2+)-stimulated type VIII adenylyl cyclase for Ca(2+) 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 [Ca(2+)](i) or to the release of Ca(2+) from internal stores but was specifically dependent on the induced capacitative entry of Ca(2+). In marked contrast, the arachidonate-induced entry of Ca(2+) 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 Ca(2+) occurs via an entirely distinct influx pathway.     

Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake

Store-operated Ca(2+) channels, which are activated by the emptying of intracellular Ca(2+) stores, provide one major route for Ca(2+) influx. Under physiological conditions of weak intracellular Ca(2+) buffering, the ubiquitous Ca(2+) releasing messenger InsP(3) usually fails to activate any store-operated Ca(2+) entry unless mitochondria are maintained in an energized state. Mitochondria rapidly take up Ca(2+) that has been released by InsP(3), enabling stores to empty sufficiently for store-operated channels to activate. Here, we report a novel role for mitochondria in regulating store-operated channels under physiological conditions. Mitochondrial depolarization suppresses store-operated Ca(2+) influx independently of how stores are depleted. This role for mitochondria is unrelated to their actions on promoting InsP(3)-sensitive store depletion, can be distinguished from Ca(2+)-dependent inactivation of the store-operated channels and does not involve changes in intracellular ATP, oxidants, cytosolic acidification, nitric oxide or the permeability transition pore, but is suppressed when mitochondrial Ca(2+) uptake is impaired. Our results suggest that mitochondria may have a more fundamental role in regulating store-operated influx and raise the possibility of bidirectional Ca(2+)-dependent crosstalk between mitochondria and store-operated Ca(2+) channels.     

Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). The part played by Ca(2+) released from intracellular stores in the regulation of secretion is well established; however, the role of Ca(2+) influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (>or=10 microM) elicited an additional component of exocytosis despite reducing Ca(2+) influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (approximately 70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 microM) was potentiated (approximately 45%) by wortmannin. Exocytosis stimulated by 1 microM acetylcholine in the absence of extracellular Ca(2+) was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca(2+) entry through store-operated Ca(2+) channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd(3+) (2 microM), but not by inhibition of noncapacitative Ca(2+) entry by SB203580 (10 microM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca(2+) influx.     

Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+ channels

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca(2+) selective I(CRAC) and relatively Ca(2+) selective I(SOC), respectively. STIM1 has been proposed to communicate the Ca(2+) content of the intracellular Ca(2+) stores to the plasma membrane store-operated Ca(2+) channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca(2+) stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca(2+). In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca(2+) permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca(2+) entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca(2+) entry.     

Evidence for a receptor-activated Ca2+ entry pathway independent from Ca2) store depletion in endothelial cells

Ca(2+) entry in endothelial cells is a key signaling event as it prolongs the Ca(2+) signal activated by a receptor agonist, and thus allows an adequate production of a variety of compounds. The possible routes that lead to Ca(2+) entry in non-excitable cells include the receptor-activated Ca(2+) entry (RACE), which requires the presence of an agonist to be activated, and the store-operated Ca(2+) entry (SOCE) pathway, whose activation requires the depletion of the ER Ca(2+) store. However, the relative importance of these two influx pathways during physiological stimulation is not known. In the present study we experimentally differentiated these two types of influxes and determined under which circumstances they are activated. We show that La(3+) (at 10 microM) is a discriminating compound that efficiently blocks SOCE but is almost without effect on histamine-induced Ca(2+) entry (RACE). In line with this, histamine does not induce massive store depletion when performed in the presence of extracellular Ca(2+). In addition, inhibition of mitochondrial respiration significantly reduces SOCE but modestly affects RACE. Thus, agonist-induced Ca(2+) entry is insensitive to La(3+), and only modestly affected by mitochondrial depolarization. These data shows that agonist relies almost exclusively on RACE for sustained Ca(2+) signaling in endothelial cells.     

Ca2+-independent PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta

During an agonist stimulation of endothelial cells, the sustained Ca2+ entry occurring through store-operated channels has been shown to significantly contribute to smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). However, the mechanisms linking Ca2+ stores depletion to the opening of such channels are still elusive. We have used Ca2+ and tension measurements in intact aortic strips to investigate the role of the Ca2+-independent isoform of phospholipase A2 (iPLA2) in endothelial store-operated Ca2+ entry and endothelium-dependent relaxation of smooth muscle. We provide evidence that iPLA2 is involved in the activation of endothelial store-operated Ca2+ entry when Ca2+ stores are artificially depleted. We also show that the sustained store-operated Ca2+ entry occurring during physiological stimulation of endothelial cells with the circulating hormone ATP is due to iPLA2 activation and significantly contributes to the amplitude and duration of ATP-induced endothelium-dependent relaxation. Consistently, both iPLA2 metabolites arachidonic acid and lysophosphatidylcholine were found to stimulate Ca2+ entry in native endothelial cells. However, only the latter triggered endothelium-dependent relaxation through NO release, suggesting that lysophosphatidylcholine produced by iPLA2 upon Ca2+ stores depletion may act as an intracellular messenger that stimulates store-operated Ca2+ entry and subsequent NO production in endothelial cells. Finally, we found that ACh-induced endothelium relaxation also depends on iPLA2 activation, suggesting that the iPLA2-dependent control of endothelial store-operated Ca2+ entry is a key physiological mechanism regulating arterial tone.     

Permeation of monovalent cations through the non-capacitative arachidonate-regulated Ca2+ channels in HEK293 cells. Comparison with endogenous store-operated channels

In a manner similar to voltage-gated Ca(2+) channels and Ca(2+) release-activated Ca(2+) (CRAC) channels, the recently identified arachidonate-regulated Ca(2+) (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca(2+) (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca(2+) current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg(2+). Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca(2+) channels and store-operated Ca(2+) channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances.     

Large store-operated calcium selective currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1

The molecular nature of store-operated Ca(2+)-selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca(2+)-selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca(2+) entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca(2+) entry and Ca(2+)-selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca(2+)-selective store-operated Ca(2+) entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca(2+) entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca(2+) sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle.     

Ca(2+) entry into primary cultured pig coronary smooth muscle cells after previous store depletion by repetitive P2Y purinoceptor stimulation

Store-operated Ca(2+) entry, stimulated by depletion of intracellular Ca(2+) pools, has not been fully elucidated in vascular smooth muscle cells of pig coronary arteries. Therefore, [Ca(2+)](i) was measured in cultured cells derived from extramural pig coronary arteries using the Fura-2/AM fluorometry. Divalent cation entry was visualized with the Fura-2 Mn(2+)-quenching technique. Ca(2+) stores were depleted either by repetitive stimulation of P2Y purinoceptors with ATP (10 micromol/L), or by the sarcoendoplasmic Ca(2+)-ATPase inhibitor 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ; 1 micromol/L) in Ca(2+)-free medium (EGTA 1 mmol/L). Addition of Ca(2+)(1 mmol/L) induced refilling of ATP-sensitive Ca(2+) stores and an increase in [Ca(2+)](i) in the presence of BHQ. Both could be significantly diminished by Ni(2+)(5 and 1mmol/L), La(3+)(10 micromol/L), Gd(3+)(10 micromol/L), and Mg(2+)(5.1 mmol/L). In contrast to the BHQ-mediated rise in [Ca(2+)](i), refilling of ATP-depleted stores was affected by neither flufenamate (0.1 mmol/L), nor by nitrendipine, nifedipine, and nisoldipine (each 1 micromol/L). The data suggest that after store depletion in pig coronary smooth muscle cells ATP and BHQ may converge on a common, Ni(2+)-, La(3+)-, Gd(3+)-, and Mg(2+)- sensitive Ca(2+) entry pathway, i.e. on a store-operated Ca(2+) entry. An additional contribution of the Na(+)/Ca(2+) exchanger cannot be excluded. Flufenamate-sensitive non-selective cation channels and dihydropyridine-sensitive L-type Ca(2+) channels are not involved in refilling of Ca(2+) stores after previous depletion by repetitive P2Y purinoceptor stimulation. The store-operated Ca(2+) entry in-between repetitive purinoceptor stimulation, i.e. in the absence of the agonist, may be responsible for the maintenance of agonist-induced rhythmic Ca(2+) responses.     

Suppression of TRPC3 leads to disappearance of store-operated channels and formation of a new type of store-independent channels in A431 cells

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.