C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes

Group IVA cytosolic phospholipase A₂ (cPLA₂α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA₂α binds zwitterionic lipids such as phosphatidylcholine in a Ca²⁺-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA₂α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA₂α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA₂α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA₂α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA₂α.     

Lactosylceramide Interacts with and Activates Cytosolic Phospholipase A2α

Lactosylceramide (LacCer) is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physiological processes. However, the direct targets of LacCer and cellular events mediated by LacCer are largely unknown. In this study, we examined the effect of LacCer on the release of arachidonic acid (AA) and the activity of cytosolic phospholipase A₂α (cPLA₂α). In CHO-W11A cells, treatment with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthase, reduced the glycosphingolipid level, and the release of AA induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or platelet-activating factor was inhibited. The addition of LacCer reversed the PPMP effect on the stimulus-induced AA release. Exogenous LacCer stimulated the release of AA, which was decreased by treatment with an inhibitor of cPLA₂α or silencing of the enzyme. Treatment of CHO-W11A cells with LacCer induced the translocation of full-length cPLA₂α and its C2 domain from the cytosol to the Golgi apparatus. LacCer also induced the translocation of the D43N mutant of cPLA₂α. Treatment of L929 cells with TNF-α induced LacCer generation and mediated the translocation of cPLA₂α and AA release, which was attenuated by treatment with PPMP. In vitro studies were then conducted to test whether LacCer interacts directly with cPLA₂α. Phosphatidylcholine vesicles containing LacCer increased cPLA₂α activity. LacCer bound to cPLA₂α and its C2 domain in a Ca²⁺-independent manner. Thus, we propose that LacCer is a direct activator of cPLA₂α.     

Effects of ceramide,ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A₂ (PLA₂) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca²⁺ binding regions in the C2 domain of α type cytosolic PLA₂ (cPLA₂α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA₂α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca²⁺ ionophore (A23187) -induced release of AA via cPLA₂α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca²⁺ nor the phosphorylation of cPLA₂α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA₂α that is independent of the Ca²⁺ signal and the PKC-ERK-mediated phosphorylation signal.     

Hyperforin induces Ca-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A2 activation through select phospholipid interactions

Here, we investigated the modulation of cytosolic phospholipase A₂ (cPLA₂)-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10 µM) versus unstimulated cells, which was blocked by cPLA₂α-inhibition, and induced translocation of cPLA₂ to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca²⁺ by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA₂ at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA₂ phosphorylation. However, neither AA release nor translocation of cPLA₂ was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA₂ in the presence of Ca²⁺. However, when Ca²⁺ was omitted, hyperforin caused a prominent increase in cPLA₂ activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional ¹H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA₂ activation by enabling its access towards select lipid membranes independent of Ca²⁺ ions. Such Ca²⁺- and phosphorylation-independent mechanism of cPLA₂ activation may apply also to other membrane-interfering molecules.     

Macrophage arachidonate release via both the cytosolic Ca2+-dependent and -independent phospholipases is necessary for cell spreading

We have observed that phospholipase A₂ (PLA₂) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA₂s in these processes. The PLA₂-specific inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca²⁺-independent PLA₂ (iPLA₂)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca²⁺-dependent phospholipase (cPLA₂)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. Cell spreading was normalized by the addition of AA to PLA₂-inhibited cells. AA release during spreading was also inhibited by Ca²⁺ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA₂. Inhibition of macrophage spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca²⁺ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA₂ (which appears to be constitutively active) and cPLA₂ (which appears to be stimulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA₂.     

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A₂ group IVA (cPLA₂α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA₂α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA₂α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E₂ production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA₂α inhibitor blocked HA-induced AA release and PGE₂ production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA₂α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE₂ production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE₂ production and COX2 expression were blocked in Tlr4^−/− and Myd88^−/− mice, but not in Cd44^−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA₂α/COX2^high and COX1/ALOX15/ALOX5/LTA4H^low gene and PGE₂/PGD₂/15-HETE^high and LXA₄^low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.     

A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca²⁺-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca²⁺ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca²⁺ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca²⁺ is quenched to or below resting intracellular Ca²⁺ concentration ([Ca²⁺]_e [Ca²⁺]_i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca²⁺]_e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca²⁺]_i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca²⁺ mobilization from stores (providing close to threshold Ca²⁺ levels) and Ca²⁺ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca²⁺ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.     

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Rotavirus infection modifies Ca²⁺ homeostasis, provoking an increase in Ca²⁺ permeation, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), and total Ca²⁺ pools and a decrease in Ca²⁺ response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca²⁺ and the amount of Ca²⁺ sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca²⁺ pools were evaluated as ⁴⁵Ca²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca²⁺ permeability and ⁴⁵Ca²⁺ uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca²⁺ homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased ⁴⁵Ca²⁺ uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca²⁺.     

Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δ strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K⁺ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K⁺ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K⁺ with Rb⁺, Cs⁺, or Na⁺ only slightly modulated the magnitude of the inward current, whereas replacement with Li⁺ reduced the inward current by approximately 50%, and tetraethylammonium (TEA⁺) and choline were relatively impermeant. The inward current was blocked by extracellular Ca²⁺ and Mg²⁺ with apparent K_is (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K⁺ increased the magnitude of the inward current independently of the electrochemical driving force for K⁺ influx, consistent with regulation of the inward current by cytosolic K⁺. Uptake of ⁸⁶Rb⁺ by intact trk1Δ trk2Δ cells was inhibited by extracellular Ca²⁺ with a K_i within the range observed for the inward current. Furthermore, increasing extracellular Ca²⁺ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.     

Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca²⁺) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca²⁺ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca²⁺-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca²⁺ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca²⁺ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca²⁺ pool in untreated cells when extracellular Ca²⁺ is low. However, low extracellular Ca²⁺ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca²⁺ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca²⁺ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca²⁺-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca²⁺]_cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca²⁺]_cyt and inhibition of growth that was similar to the effect of the Ca²⁺ channel blocker La³⁺ and the Ca²⁺ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca²⁺ channel blockers verapamil and nifedipine did not induce a decrease in [Ca²⁺]_cyt in these cells and also failed to inhibit growth. Al and La³⁺, but not verapamil or nifedipine, reduced the rate of Mn²⁺ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca²⁺- and Mn²⁺-permeable channels. These results suggest that Al may act to block Ca²⁺ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.     

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel

Increasing evidence suggests that changes in cytosolic Ca²⁺ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca²⁺ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca²⁺-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca²⁺ dependent. CDPK exhibits a Ca²⁺-induced electrophoretic mobility shift and its Ca²⁺-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca²⁺ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca²⁺-dependent protein phosphatase calcineurin enhances the Ca²⁺-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K⁺ channel KAT1 protein in a Ca²⁺-dependent manner. These results suggest that CDPK may be an important component of Ca²⁺ signaling in guard cells.     

Local Control of Excitation-Contraction Coupling in Human Embryonic Stem Cell-Derived Cardiomyocytes

We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca²⁺ influx via voltage-gated L-type Ca²⁺ channels activates Ca²⁺ release from the sarcoplasmic reticulum (SR) via a local control mechanism in hESC-CMs and hFVMs. Field-stimulated, whole-cell [Ca²⁺]_i transients in hESC-CMs required Ca²⁺ entry through L-type Ca²⁺ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca²⁺ or treatment with diltiazem, an L-type channel inhibitor. Ca²⁺ release from the SR also contributes to the [Ca²⁺]_i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca²⁺ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca²⁺ currents (I _Ca) and corresponding whole-cell [Ca²⁺]_i transients in hESC-CMs and hFVMs, and the amplitude of both I _Ca and the [Ca²⁺]_i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca²⁺]_i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca²⁺ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca²⁺ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca²⁺ release by the I _Ca during EC coupling develops early in human cardiomyocytes.     

The V Antigen of Yersinia pestis Regulates Yop Vectorial Targeting as Well as Yop Secretion through Effects on YopB and LcrG

Yersinia pestis expresses a set of secreted proteins called Yops and the bifunctional LcrV, which has both regulatory and antihost functions. Yops and LcrV expression and the activity of the type III mechanism for their secretion are coordinately regulated by environmental signals such as Ca²⁺ concentration and eukaryotic cell contact. In vitro, Yops and LcrV are secreted into the culture medium in the absence of Ca²⁺ as part of the low-Ca²⁺ response (LCR). The LCR is induced in a tissue culture model by contact with eukaryotic cells that results in Yop translocation into cells and subsequent cytotoxicity. The secretion mechanism is believed to indirectly regulate expression of lcrV and yop operons by controlling the intracellular concentration of a secreted negative regulator. LcrG, a secretion-regulatory protein, is thought to block secretion of Yops and LcrV, possibly at the inner face of the inner membrane. A recent model proposes that when the LCR is induced, the increased expression of LcrV yields an excess of LcrV relative to LcrG, and this is sufficient for LcrV to bind LcrG and unblock secretion. To test this LcrG titration model, LcrG and LcrV were expressed alone or together in a newly constructed lcrG deletion strain, a ΔlcrG2 mutant, of Y. pestis that produces low levels of LcrV and constitutively expresses and secretes Yops. Overexpression of LcrG in this mutant background was able to block secretion and depress expression of Yops in the presence of Ca²⁺ and to dramatically decrease Yop expression and secretion in growth medium lacking Ca²⁺. Overexpression of both LcrG and LcrV in the ΔlcrG2 strain restored wild-type levels of Yop expression and Ca²⁺ control of Yop secretion. Surprisingly, when HeLa cells were infected with the ΔlcrG2 strain, no cytotoxicity was apparent and translocation of Yops was abolished. This correlated with an altered distribution of YopB as measured by accessibility to trypsin. These effects were not due to the absence of LcrG, because they were alleviated by restoration of LcrV expression and secretion alone. LcrV itself was found to enter HeLa cells in a nonpolarized manner. These studies supported the LcrG titration model of LcrV’s regulatory effect at the level of Yop secretion and revealed a further role of LcrV in the deployment of YopB, which in turn is essential for the vectorial translocation of Yops into eukaryotic cells.     

Characterization of a Membrane Calcium Pathway Induced by Rotavirus Infection in Cultured Cells

Some viruses induce changes in membrane permeability during infection. We have shown previously that the porcine strain of rotavirus, OSU, induced an increase in the permeability to Na⁺, K⁺, and Ca²⁺ during replication in MA104 cells. In this work, we have characterized the divalent cation entry pathway by measuring intracellular Ca²⁺ in fura-2-loaded MA104 and HT29 cells in suspension. The permeability to Ca²⁺ and other cations was evaluated by the change of the intracellular concentration following an extracellular cation pulse. Rotavirus infection induced an increase in permeability to Ca²⁺, Ba²⁺, Sr²⁺, Mn²⁺, and Co²⁺. The rate of cation entry decreased over time as the intracellular concentration increased during the first 20 s. This indicates that regulatory mechanisms, including channel inactivation, are triggered. La³⁺ did not enter the cell and blocked the entry of the divalent cations in a dose-dependent manner. Metoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibited the entry of Ca²⁺ in virus-infected MA104 and HT29 cells. The results suggest that rotavirus infection of cultured cells activates a cation channel rather than nonspecific permeation through the plasma membrane. This activation involves the synthesis of viral proteins through mechanisms yet unknown. The increase in intracellular Ca²⁺ induced by the activation of this channel may be related to the increase in cytoplasmic and endoplasmic reticulum Ca²⁺ pools required for virus maturation and cell death.