The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

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.     

Emerging perspectives in store-operated Ca2+ entry: roles of Orai, Stim and TRP

Depletion of intracellular Ca2+ stores induces Ca2+ influx across the plasma membrane through store-operated channels (SOCs). This store-operated Ca2+ influx is important for the replenishment of the Ca2+ stores, and is also involved in many signaling processes by virtue of the ability of intracellular Ca2+ to act as a second messenger. For many years, the molecular identities of particular SOCs, as well as the signaling mechanisms by which these channels are activated, have been elusive. Recently, however, the mammalian proteins STIM1 and Orai1 were shown to be necessary for the activation of store-operated Ca2+ entry in a variety of mammalian cells. Here we present molecular, pharmacological, and electrophysiological properties of SOCs, with particular focus on the roles that STIM1 and Orai1 may play in the signaling processes that regulate various pathways of store-operated entry.     

Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.     

Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries

Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (Ca2+L) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a Ca2+L) inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and Ca2+L channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2beta totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by Ca2+L channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of Ca2+L channels and lead to Ca2+ entry and vasoconstriction.     

A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

A novel mechanism for the store-operated calcium influx pathway

Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca(2+)-independent phospholipase A(2) (iPLA(2)) has an important role in the store-operated calcium influx pathway. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We also propose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA(2), resulting in activation of iPLA(2) and generation of lysophospholipids that in turn activate soc channels and capacitative calcium influx. Upon refilling of the stores and termination of CIF production, CaM rebinds to iPLA(2), inhibits it, and the activity of SOC channels and capacitative calcium influx is terminated.     

Store-operated Ca2+-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca2+ store in smooth muscle triggered a Ca2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca2+ ions. Two activation mechanisms have been identified which involve Ca2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of alpha-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

CIF and other mysteries of the store-operated Ca2+-entry pathway

The molecular mechanism of the store-operated Ca2+-entry (SOCE) pathway remains one of the most intriguing and long lasting mysteries of Ca2+ signaling. The elusive calcium influx factor (CIF) that is produced upon depletion of Ca2+ stores has attracted growing attention, triggered by new discoveries that filled the gap in the chain of reactions leading to activation of store-operated channels and Ca2+ entry. Ca2+-independent phospholipase A2 emerged as a target of CIF, and a major determinant of the SOCE mechanism. Here, we present our viewpoint on CIF and conformational-coupling models of SOCE from a historical perspective, trying to resolve some of the problem areas, and summarizing our present knowledge on how depletion of intracellular Ca2+ stores signals to plasma membrane channels to open and provide Ca2+ influx that is required for many important physiological functions.     

A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2

The coupling between receptor-mediated Ca2+ store release and the activation of "store-operated" Ca2+ entry channels is an important but so far poorly understood mechanism. The transient receptor potential (TRP) superfamily of channels contains several members that may serve the function of store-operated channels (SOCs). The 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2, is a recently described inhibitor of SOC activity in T-lymphocytes. We compared its action on SOC activation in a number of cell types and evaluated its modification of three specific TRP channels, canonical transient receptor potential 3 (TRPC3), TRPC5, and TRPV6, to throw light on any link between SOC and TRP channel function. Using HEK293 cells, DT40 B cells, and A7r5 smooth muscle cells, BTP2 blocked store-operated Ca2+ entry within 10 min with an IC50 of 0.1-0.3 microM. Store-operated Ca2+ entry induced by Ca2+ pump blockade or in response to muscarinic or B cell receptor activation was similarly sensitive to BTP2. Using the T3-65 clonal HEK293 cell line stably expressing TRPC3 channels, TRPC3-mediated Sr2+ entry activated by muscarinic receptors was also blocked by BTP2 with an IC50 of <0.3 microM. Importantly, direct activation of TRPC3 channels by diacylglycerol was also blocked by BTP2 (IC50 approximately 0.3 microM). BTP2 still blocked TRPC3 in medium with N-methyl-D-glucamine-chloride replacing Na+, indicating BTP2 did not block divalent cation entry by depolarization induced by activating monovalent cation entry channels. Whereas whole-cell carbachol-induced TRPC3 current was blocked by 3 microM BTP2, single TRPC3 channel recordings revealed persistent short openings suggesting BTP2 reduces the open probability of the channel rather than its pore properties. TRPC5 channels transiently expressed in HEK293 cells were blocked by BTP2 in the same range as TRPC3. However, function of the highly Ca(2+)-selective TRPV6 channel, with many channel properties akin to SOCs, was entirely unaffected by BTP2. The results indicate a strong functional link between the operation of expressed TRPC channels and endogenous SOC activity.     

STIM1, an essential and conserved component of store-operated Ca2+ channel function

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.     

A pyrazole derivative,YM-58483, potently inhibits store-operated sustained Ca2+ influx and IL-2 production in T lymphocytes

In nonexcitable cells, Ca(2+) entry is mediated predominantly through the store depletion-dependent Ca(2+) channels called store-operated Ca(2+) (SOC) or Ca(2+) release-activated Ca(2+) channels. YM-58483, a pyrazole derivative, inhibited an anti-CD3 mAb-induced sustained Ca(2+) influx in acute T cell leukemia, Jurkat cells. But it did not affect an anti-CD3 mAb-induced transient intracellular Ca(2+) increase in Ca(2+)-free medium, nor anti-CD3 mAb-induced phosphorylation of phospholipase Cgamma1. It was suggested that YM-58483 inhibited Ca(2+) influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca(2+) influx with an IC(50) value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca(2+) influx through SOC channels compared with voltage-operated Ca(2+) channels, while econazole inhibited both SOC channels and voltage-operated Ca(2+) channels with an equivalent range of IC(50) values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED(50) of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca(2+) entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca(2+) entry mechanisms through SOC/CRAC channels and for identification of putative Ca(2+) channel genes.     

Epidermal growth factor activates store-operated Ca2+ channels through an inositol 1,4,5-trisphosphate-independent pathway in human glomerular mesangial cells

One of the fastest cellular responses following activation of epidermal growth factor receptor is an increase in intracellular Ca2+ concentration. This event is attributed to a transient Ca2+ release from internal stores and Ca2+ entry from extracellular compartment. Store-operated Ca2+ channels are defined the channels activated in response to store depletion. In the present study, we determined whether epidermal growth factor activated store-operated Ca2+ channels and further, whether depletion of internal Ca2+ stores was required for the epidermal growth factor-induced Ca2+ entry in human glomerular mesangial cells. We found that 100 nm epidermal growth factor activated a Ca2+-permeable channel that had identical biophysical and pharmacological properties to channels activated by 1 microm thapsigargin in human glomerular mesangial cells or A431 cells. The epidermal growth factor-induced Ca2+ currents were completely abolished by a selective phospho-lipase C inhibitor, U73122. However, xestospongin C, a specific inositol 1,4,5-trisphosphate receptor inhibitor, did not affect the membrane currents elicited by epidermal growth factor despite a slight reduction in background currents. Following emptying of internal Ca2+ stores by thapsigargin, epidermal growth factor still potentiated the Ca2+ currents as determined by the whole-cell patch configuration. Furthermore, epidermal growth factor failed to trigger measurable Ca2+ release from endoplasmic reticulum. However, another physiological agent linked to phospholipase C and inositol 1,4,5-trisphosphate cascade, angiotensin II, produced a striking Ca2+ transient. These results indicate that epidermal growth factor activates store-operated Ca2+ channels through an inositol 1,4,5-trisphosphate-independent, but phospholipase C-dependent, pathway in human glomerular mesangial cells.     

2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry

Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.     

Role of the actin cytoskeleton in store-mediated calcium entry in glioma C6 cells

The effects of actin cytoskeleton disruption by cytochalasin D and latrunculin A on Ca2+ signals evoked by ADP, UTP or thapsigargin were investigated in glioma C6 cells. Despite the profound alterations of the actin cytoskeleton architecture and cell morphology, ADP and UTP still produced cytosolic calcium elevation in this cell line. However, calcium mobilization from internal stores and Ca2+ influx through store-operated Ca2+ channels induced by ADP and UTP were strongly reduced. Cytochalasin D and latrunculin A also diminished extracellular Ca2+ influx in unstimulated glioma C6 cells previously incubated in Ca2+ free buffer. In contrast, the disruption of the actin cytoskeleton had no effect on thapsigargin-induced Ca2+ influx in this cell line. Both agonist- and thapsigargin-generated Ca2+ entry was significantly decreased by the blocker of store-operated Ca2+ channels, 2-aminoethoxydiphenylborate. The data reveal that two agonists and thapsigargin activate store-operated Ca2+ channels but the mechanism of activation seems to be different. While the agonists evoke a store-mediated Ca2+ entry that is dependent on the actin cytoskeleton, thapsigargin apparently activates an additional mechanism, which is independent of the disruption of the cytoskeleton.     

Two types of store-operated Ca2+ channels with different activation modes and molecular origin in LNCaP human prostate cancer epithelial cells

The one or more coupling mechanisms of store-operated channels (SOCs) to endoplasmic reticulum (ER) Ca2+ store depletion as well as the molecular identity of SOCs per se still remain a mystery. Here, we demonstrate the co-existence of two populations of molecular distinct endogenous SOCs in LNCaP prostate cancer epithelial cells, which are preferentially activated by either active inositol 1,4,5-trisphosphate (IP3)-mediated or passive thapsigargin-facilitated store depletion and have different ER store content sensitivity. The first population, called SOC(CC) (for "conformational coupling"), is characterized by preferential IP3 receptor-dependent mode of activation, as judged from sensitivity to cytoskeleton modifications, and dominant contribution of transient receptor potential (TRP) TRPC1 within it. The second one, called SOC(CIF) (for "calcium influx factor"), depends on Ca(2+)-independent phospholipase A2 for activation with probable CIF involvement and is mostly represented by TRPC4. The previously identified SOC constituent in LNCaP cells, TRPV6, seems to play equal role in both SOC populations. These results provide new insight into the nature of SOCs and their representation in the single cell type as well as permit reconciliation of current SOC activation hypotheses.     

Close functional coupling between Ca2+ release-activated Ca2+ channels, arachidonic acid release, and leukotriene C4 secretion

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types, particularly of hemopoietic origin, store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. However, little is known about the downstream consequences of CRAC channel activation. Here, we report that Ca2+ entry through CRAC channels stimulates arachidonic acid production, whereas Ca2+ release from the stores is ineffective even though the latter evokes a robust intracellular Ca2+ signal. We find that arachidonic acid released by Ca2+ entering through CRAC channels is used to synthesize the potent paracrine proinflammatory signal leukotriene C4 (LTC4). Both pharmacological inhibitors of CRAC channels and mitochondrial depolarization, which impairs CRAC channel activity, suppress arachidonic acid release and LTC4 secretion. Thus, arachidonic acid release is preferentially stimulated by elevated subplasmalemmal Ca2+ levels due to open CRAC channels, suggesting that the enzyme is located close to the CRAC channels. Our results also identify a novel role for CRAC channels, namely the activation of a downstream signal transduction pathway resulting in the secretion of LTC4. Finally, mitochondria are key determinants of the generation of both intracellular (arachidonic acid) and paracrine (LTC4) signals through their effects on CRAC channel activity.