Cyclic GMP modulates store-operated calcium entry inducing phosphatidylserine translocation at the surface of megakaryocytic cells

When subjected to stimulation, cells from the vascular compartment show a spontaneous collapse of the plasma membrane phospholipid asymmetry and phosphatidylserine is exposed at the external leaflet. Thus, phosphatidylserine externalization is essential for normal hemostasis and phagocytosis. The mechanism governing the migration of phosphatidylserine to the exoplasmic leaflet is not yet fully understood. We have proposed that store-operated calcium entry (SOCE) constitutes a key step of this process. Here, interaction of [Ca(2+)](i), cAMP and cGMP pathways and phosphatidylserine exposure was examined in human megakaryocytic cells. The membrane permeable cAMP and cGMP analogues, pCPT-cAMP and pCPT-cGMP, enhanced the Ca(2+) signal induced by ionophore and SOCE. Responses to pCPT-cAMP and pCPT-cGMP were independent of protein kinase A, protein kinase G (PKG) or ERK pathways. Inhibition of small G-proteins reduced or abolished the increase of [Ca(2+)](i) induced by pCPT-cAMP or pCPT-cGMP, respectively. pCPT-cGMP but not pCPT-cAMP enhanced the ability of cells to expose phosphatidylserine. This effect was not prevented by the inhibition of PKG or small G-proteins. These results show the differential role of cyclic nucleotides in the Ca(2+)-dependent membrane remodeling. Hence, pCPT-cGMP is another regulatory element for the completion of SOCE-induced phosphatidylserine transmembrane redistribution in HEL cells through a mechanism implicating small G-proteins.     

Free cholesterol alters lipid raft structure and function regulating neutrophil Ca2+ entry and respiratory burst: correlations with calcium channel raft trafficking

Recent studies associate cholesterol excess and atherosclerosis with inflammation. The link between these processes is not understood, but cholesterol is an important component of lipid rafts. Rafts are thought to concentrate membrane signaling molecules and thus regulate cell signaling through G protein-coupled pathways. We used methyl beta-cyclodextrin to deplete cholesterol from polymorphonuclear neutrophil (PMN) rafts and thus study the effects of raft disruption on G protein-coupled Ca(2+) mobilization. Methyl beta-cyclodextrin had no effect on Ca(2+) store depletion by the G protein-coupled agonists platelet-activating factor or fMLP, but abolished agonist-stimulated Ca(2+) entry. Free cholesterol at very low concentrations regulated Ca(2+) entry into PMN via nonspecific Ca(2+) channels in a biphasic fashion. The specificity of cholesterol regulation for Ca(2+) entry was confirmed using thapsigargin studies. Responses to cholesterol appear physiologic because they regulate respiratory burst in a proportional biphasic fashion. Investigating further, we found that free cholesterol accumulated in PMN lipid raft fractions, promoting formation and polarization of membrane rafts. Finally, the transient receptor potential calcium channel protein TRPC1 redistributed to raft fractions in response to cholesterol. The uniformly biphasic relationships between cholesterol availability, Ca(2+) signaling and respiratory burst suggest that Ca(2+) influx and PMN activation are regulated by the quantitative relationships between cholesterol and other environmental lipid raft components. The association between symptomatic cholesterol excess and inflammation may therefore in part reflect free cholesterol- dependent changes in lipid raft structure that regulate immune cell Ca(2+) entry. Ca(2+) entry-dependent responses in other cell types may also reflect cholesterol bioavailability and lipid incorporation into rafts.     

Relation between phosphatidylserine exposure and store-operated Ca(2+) entry in stimulated cells

A significant increase in intracellular Ca(2+) is required to trigger the remodeling of the cell plasma membrane. Scott syndrome is an extremely rare inherited disorder of the transmembrane migration of phosphatidylserine toward the exoplasmic leaflet in blood cells. We have recently reported a reduced capacitative Ca(2+) entry in Scott cells [Martínez et al. (1999) Biochemistry 38, 10092-10098]. We have investigated here the links between defective phosphatidylserine exposure and Ca(2+) signaling in Scott cells by focusing on the Ca(2+) entry following the emptying of intracellular stores. After depletion of caffeine- or thapsigargin-sensitive stores, Ca(2+) entry was lower in Scott compared to control lymphoblasts. However, the simultaneous depletion of both types of stores restored a normal Ca(2+) influx across the plasma membrane in Scott cells and phosphatidylserine externalization ability was improved concomitantly with capacitative Ca(2+) entry. These observations point to the essential role of capacitative Ca(2+) entry in the control of phosphatidylserine exposure of stimulated cells.     

Regulation of phosphatidylserine transbilayer redistribution by store-operated Ca2+ entry: role of actin cytoskeleton

The phosphatidylserine transmembrane redistribution at the cell surface is one of the early characteristics of cells undergoing apoptosis and also occurs in cells fulfilling a more specialized function, such as the phosphatidylserine-dependent procoagulant response of platelets after appropriate activation. Although an increase in cytoplasmic Ca2+ is essential to trigger the remodeling of the plasma membrane, little is known about intracellular signals leading to phosphatidylserine externalization. Here, the role of store-operated Ca2+ entry on phosphatidylserine exposure was investigated in human erythroleukemia HEL cells, a pluripotent lineage with megakaryoblastic properties. Ca2+ entry inhibitors (SKF-96365, LaCl(3), and miconazole) inhibited store-operated Ca2+ entry in A23187- or thapsigargin-stimulated cells and reduced the degree of phosphatidylserine externalization concomitantly, providing evidence for a close link between the two processes. In cells pretreated with cytochalasin D, an agent that disrupts the microfilament network of the cytoskeleton, store-operated Ca2+ entry and phosphatidylserine externalization at the cell surface were inhibited. In a context where most of the key actors remain to be identified, these results provide evidence for the implication of both store-operated Ca2+ entry and cytoskeleton architectural organization in the regulation of phosphatidylserine transbilayer migration.     

The cytosolic domains of Ca2+-sensitive adenylyl cyclases dictate their targeting to plasma membrane lipid rafts

Lipid rafts are specialized, cholesterol-rich domains of the plasma membrane that are enriched in certain signaling proteins, including Ca(2+)-sensitive adenylyl cyclases. This restrictive localization plays a key role in the regulation of the Ca(2+)-stimulable AC8 and the Ca(2+)-inhibitable AC6 by capacitative calcium entry. Interestingly, AC7, a Ca(2+)-insensitive AC, is found in the plasma membrane but is excluded from lipid rafts (Smith, K. E., Gu, C., Fagan, K. A., Hu, B., and Cooper, D. M. F. (2002) J. Biol. Chem. 277, 6025-6031). The mechanisms governing the specific membrane targeting of adenylyl cyclase isoforms remain unknown. To address this issue, a series of chimeras were produced between the raft-targeted AC5 and the non-raft-targeted AC7, involving switching of their major domains. The AC5-AC7 chimeras were expressed in HEK 293 cells and lipid rafts were isolated from the bulk plasma membrane by either detergent-based or non-detergent-based fractionation methods. Additionally, confocal imaging was used to investigate the precise cellular targeting of the chimeras. Surprisingly, the two tandem six-transmembrane domains of AC5 were not required for localization to lipid rafts. Rather, AC5 localization depended on the complete cytoplasmic loops (C1 and C2); constructs with mixed domains were either retained in the endoplasmic reticulum or degraded. Similar conclusions are drawn for the lipid raft localization of the Ca(2+)/calmodulin-stimulable AC8; again, the C1 and C2 domains are critical. Thus, protein-protein interactions may be more important than protein-lipid interactions in targeting these calcium-sensitive enzymes to lipid rafts.     

Involvement of a calcium-dependent dephosphorylation of BAD associated with the localization of Trpc-1 within lipid rafts in 7-ketocholesterol-induced THP-1 cell apoptosis

7-Ketocholesterol is a component of oxidized LDL, which plays a central role in atherosclerosis. It is a potent inducer of cell death towards a wide number of cells involved in atherosclerosis. In this study, it is reported that 7-ketocholesterol treatment induces an increase of cytosolic-free Ca(2+) in THP-1 monocytic cells. This increase is correlated with the induction of cytotoxicity as suggested from experiments using the Ca(2+) channel blockers verapamil and nifedipine. This 7-ketocholesterol-induced apoptosis appears to be associated with the dephosphorylation of serine 75 and serine 99 of the proapoptotic protein Bcl-2 antagonist of cell death (BAD). We demonstrated that this dephosphorylation results mainly from the activation of calcium-dependent phosphatase calcineurin by the oxysterol-induced increase in Ca(2+). Moreover, this Ca(2+) increase appears related to the incorporation of 7-ketocholesterol into lipid raft domains of the plasma membrane, followed by the translocation of transient receptor potential calcium channel 1, a component of the store operated Ca(2+) entry channel, to rafts.     

Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here)

Activation of phospholipase C by G-protein-coupled receptors results in release of intracellular Ca(2+) and activation of Ca(2+) channels in the plasma membrane. The intracellular release of Ca(2+) is signaled by the second messenger, inositol 1,4,5-trisphosphate. Ca(2+) entry involves signaling from depleted intracellular stores to plasma membrane Ca(2+) channels, a process referred to as capacitative calcium entry or store-operated calcium entry. The electrophysiological current associated with capacitative calcium entry is the calcium-release-activated calcium current, or I(crac). In the 20 years since the inception of the concept of capacitative calcium entry, a variety of activation mechanisms have been proposed, and there has been considerable interest in the possibility of transient receptor potential channels functioning as store-operated channels. However, in the past 2 years, two major players in both the signaling and permeation mechanisms for store-operated channels have been discovered: Stim1 (and possibly Stim2) and the Orai proteins. Activation of store-operated channels involves an endoplasmic reticulum Ca(2+) sensor called Stim1. Stim1 acts by redistributing within a small component of the endoplasmic reticulum, approaching the plasma membrane, but does not appear to translocate into the plasma membrane. Stim1, either directly or indirectly, signals to plasma membrane Orai proteins which constitute pore-forming subunits of store-operated channels.     

Role of STIM and Orai proteins in the store-operated calcium signaling pathway

Ca(2+) signals are universal among cells in regulating a spectrum of cellular responses. Phospholipase C-coupled receptors activate two components of Ca(2+) signals--rapid Ca(2+) release from ER stores, followed by slower Ca(2+) entry from outside the cell. The coupling process between ER and PM to mediate this "store-operated" Ca(2+) entry process remained until recently a molecular mystery. The recent discovery of the necessity for STIM1 and Orai proteins in this process has provided crucial information on the coupling mechanism between stores and PM Ca(2+) entry. STIM1 is a single spanning membrane protein with an unpaired Ca(2+) binding EF-hand and appears to function as the sensor of ER luminal Ca(2+), and, through redistribution in the ER, transduces information directly to the PM. Orai1 is a tetra-spanning PM protein and functions as the highly Ca(2+)-selective channel in the PM that is gated through interactions with the store-activated ER Ca(2+) sensor. Recent evidence shows the two proteins together are necessary and sufficient for the function of store-operated Ca(2+) entry. However, many questions arise about how and where the interactions of the STIM1 and Orai1 proteins occur within cells. Here we discuss recent information and ideas about the coupling between these proteins that leads to store-operated channel activation.     

Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and degranulation in a rat mast cell line

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.     

Ca2+ entry mediated by store depletion, S-nitrosylation, and TRP3 channels. Comparison of coupling and function

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. SOC-mediated Ca(2+) entry is complex and may reflect more than one type of channel and coupling mechanism. To assess such possible divergence the function and coupling of SOCs was compared with two other distinct yet related Ca(2+) entry mechanisms. SOC coupling in DDT(1)MF-2 smooth muscle cells was prevented by the permeant inositol 1,4,5-trisphosphate (InsP(3)) receptor blockers, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C. In contrast, Ca(2+) entry induced by S-nitrosylation and potentiated by store depletion (Ma, H-T., Favre, C. J., Patterson, R. L., Stone, M. R., and Gill, D. L. (1999) J. Biol. Chem. 274, 35318-35324) was unaffected by 2-APB, suggesting that this entry mechanism is independent of InsP(3) receptors. The cycloalkyl lactamimide, MDL-12, 330A (MDL), prevented SOC activation (IC(50) 10 micrometer) and similarly completely blocked S-nitrosylation-mediated Ca(2+) entry. Ca(2+) entry mediated by the TRP3 channel stably expressed in HEK293 cells was activated by phospholipase C-coupled receptors but independent of Ca(2+) store depletion (Ma, H.-T., Patterson, R. L., van Rossum, D. B., Birnbaumer, L., Mikoshiba, K., and Gill, D. L. (2000) Science 287, 1647-1651). Receptor-induced TRP3 activation was 2-APB-sensitive and fully blocked by MDL. Direct stimulation of TRP3 channels by the permeant diacylglycerol derivative, 1-oleoyl-2-acetyl-sn-glycerol, was not blocked by 2-APB, but was again prevented by MDL. The results indicate that although the activation and coupling processes for each of the three entry mechanisms are distinct, sensitivity to MDL is a feature shared by all three mechanisms, suggesting there may be a common structural feature in the channels themselves or an associated regulatory component.     

Interaction of STIM1 with endogenously expressed human canonical TRP1 upon depletion of intracellular Ca2+ stores

STIM1 (stromal interaction molecule 1) has recently been proposed to communicate the intracellular Ca(2+) stores with the plasma membrane to mediate store-operated Ca(2+) entry. Here we describe for the first time that Ca(2+) store depletion stimulates rapid STIM1 surface expression and association with endogenously expressed human canonical TRP1 (hTRPC1) independently of rises in cytosolic free Ca(2+) concentration. These events require the support of the actin cytoskeleton in human platelets, as reported for the coupling between type II inositol 1,4,5-trisphosphate receptor in the Ca(2+) stores and hTRPC1 in the plasma membrane, which has been suggested to underlie the activation of store-operated Ca(2+) entry in these cells. Electrotransjection of cells with anti-STIM1 antibody, directed toward the N-terminal sequence that includes the Ca(2+)-binding region, prevented the migration of STIM1 toward the plasma membrane, the interaction between STIM1 and hTRPC1, the coupling between hTRPC1 and type II inositol 1,4,5-trisphosphate receptor, and reduced store-operated Ca(2+) entry. These findings provide evidence for a role of STIM1 in the activation of store-operated Ca(2+) entry probably acting as a Ca(2+) sensor.     

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.     

Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane

Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca(2+) release-activated Ca(2+) (CRAC) channel activation, but it is unclear whether redistribution is rapid enough to play a causal role. Furthermore, the location of STIM1 puncta is uncertain, with recent reports supporting retention in the ER as well as insertion into the plasma membrane (PM). Using total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording from single Jurkat cells, we show that STIM1 puncta form several seconds before CRAC channels open, supporting a causal role in channel activation. Fluorescence quenching and electron microscopy analysis reveal that puncta correspond to STIM1 accumulation in discrete subregions of junctional ER located 10-25 nm from the PM, without detectable insertion of STIM1 into the PM. Roughly one third of these ER-PM contacts form in response to store depletion. These studies identify an ER structure underlying store-operated Ca(2+) entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.     

Orai1 and STIM reconstitute store-operated calcium channel function

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca(2+) entry (SOCE) in human embryonic kidney 293 cells and the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca(2+)entry by up to 103-fold. This entry is entirely store-dependent since the same coexpression causes no measurable store-independent Ca(2+) entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (store-independent) Ca(2+) entry. STIM proteins are known to mediate Ca(2+) store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca(2+) entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.     

Cutting edge: optical microspectrophotometry supports the existence of gel phase lipid rafts at the lamellipodium of neutrophils: apparent role in calcium signaling

Although much progress has been made in elucidating the biochemical properties of lipid rafts, there has been less success in identifying these structures within living cell membranes, which has led to some concern regarding their existence. One difficulty in analyzing lipid rafts using optical microscopy is their small size. We now test the existence of lipid rafts in polarized neutrophils, which redistribute lipid raft markers into comparatively large lamellipodia. Optical microspectrophotometry of Laurdan-labeled neutrophils revealed a large blue shift at lamellipodia relative to cell bodies. This blue shift disappeared after exposure to methyl-beta-cyclodextrin (m beta CD), which disrupts lipid rafts. The Ca(2+) channel transient receptor potential-like channel-1, a lipid raft marker, traffics to lamellipodia, but redistributes uniformly about cells after exposure to m beta CD. This is accompanied by disruption of Ca(2+) waves normally initiated at lamellipodia. Thus, m beta CD-sensitive lipid-ordered domains are present at and participate in signaling from the lamellipodia of living neutrophils.     

Ca(2+) entry activated by S-nitrosylation. Relationship to store-operated ca(2+) entry

The coupling between Ca(2+) pools and store-operated Ca(2+) entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca(2+) entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca(2+) pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T. , and Gill, D. L. (1998) J. Biol. Chem. 273, 30855-30858). In DDT(1)MF-2 smooth muscle cells and DC-3F fibroblasts, Ca(2+) entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca(2+) removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca(2+) entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca(2+) entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel by S-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba(2+) entry and sensitivity to blockade by La(3+) were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca(2+) entry.     

Stimulation of eryptosis by anti-A IgG antibodies.

Anti-A IgG antibodies have previously been shown to stimulate Ca(2+) entry into red blood cells. Increased cytosolic free Ca(2+) concentration is known to trigger eryptosis, i.e. suicidal erythrocyte death, characterized by exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. The present experiments have been performed to explore whether anti-A IgGs trigger phosphatidylserine exposure of erythrocytes. Phosphatidylserine exposure was estimated from annexin-V binding as determined in FACS analysis. Exposure to anti-A IgGs (0.5 microg/ml) indeed significantly increased annexin-V binding in erythrocytes with blood group A, but not in erythrocytes with blood group 0. According to Fluo3 fluorescence, anti-A IgGs increased cytosolic Ca(2+) concentration. Whole cell patch clamp recordings revealed the activation of a Ca(2+)-permeable cation channel following treatment with anti-A-IgGs. Annexin-V binding following anti-A IgG exposure was blunted by Ca(2+) removal while anti-A IgG-stimulated cation channel activity was not dependent on extracellular Ca(2+). Osmotic shock (exposure of erythrocytes to 850 mOsm) increased annexin binding, an effect further enhanced by exposure to anti-A IgGs. In conclusion, anti-A IgGs activate erythrocyte cation channels leading to Ca(2+) entry and subsequent erythrocyte cell membrane scrambling. The effect most likely contributes to the elimination of erythrocytes following an immune reaction against the A antigen.     

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.     

Cloning and functional expression of human short TRP7, a candidate protein for store-operated Ca2+ influx.

The regulation and control of plasma membrane Ca(2+) fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca(2+) influx. This increased Ca(2+) entry was blocked by inhibitors of capacitative calcium entry such as La(3+) and Gd(3+). Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca(2+) entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca(2+) influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.     

Lipid rafts function in Ca2+ signaling responsible for activation of sperm motility and chemotaxis in the ascidian Ciona intestinalis

Lipid rafts are specialized membrane microdomains that function as signaling platforms across plasma membranes of many animal and plant cells. Although there are several studies implicating the role of lipid rafts in capacitation of mammalian sperm, the function of these structures in sperm motility activation and chemotaxis remains unknown. In the ascidian Ciona intestinalis, egg-derived sperm activating- and attracting-factor (SAAF) induces both activation of sperm motility and sperm chemotaxis to the egg. Here we found that a lipid raft disrupter, methyl-β-cyclodextrin (MCD), inhibited both SAAF-induced sperm motility activation and chemotaxis. MCD inhibited both SAAF-promoted synthesis of intracellular cyclic AMP and sperm motility induced by ionophore-mediated Ca(2+) entry, but not that induced by valinomycin-mediated hyperpolarization. Ca(2+)-imaging revealed that lipid raft disruption inhibited Ca(2+) influx upon activation of sperm motility. The Ca(2+)-activated adenylyl cyclase was clearly inhibited by MCD in isolated lipid rafts. The results suggest that sperm lipid rafts function in signaling upstream of cAMP synthesis, most likely in SAAF-induced Ca(2+) influx, and are required for Ca(2+)-dependent pathways underlying activation and chemotaxis in Ciona sperm.