Bidirectional regulation of Ca~(2+) in exo–endocytosis coupling

正Vesicular exocytosis is a key process involved in neurotransmitter release, whereas vesicle recycling is critical for the homeostasis of plasma membrane structures and the maintenance of neurotransmission. Ca~(2+)regulates several fundamental cellular processes (Zhu et al., 2016; Xiong and Zhu, 2016). Especially, Ca~(2+)influx plays a dual role of triggering vesicular exocytosis and modulating compensatory endocytosis in neurons and endocrine cells. Vesicular exocytosis occurs either by Ca~(2+)-dependent or Ca~(2+)-independent mechanisms (Chai et al., 2017), and local Ca~(2+)signals beneath the plasma membrane plays a critical role in the determination of the fusion pore dilation and fusion     

GTP-bound Rab3A exhibits consecutive positive and negative roles during human sperm dense-core granule exocytosis

Exocytosis of mam maUan sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizingthe exocytotic response of streptolysin O-permeabiUzed human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranyigeranyiated, and active Rab3A elicited human sperm exocytosis perse. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino.terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.     

Cobalt ions enhance light responsiveness of carp cone horizontal cells in low calcium

Effects of cobalt ions (Co2+) on horizontal cells in low extracellular calcium were examined in isolated, superfused carp retinas. While 0.1mmol/L Co2+ completely suppressed both rod- and cone-driven horizontal cells in normal Ringer's solution, it enhanced light responses of cone horizontal cells in low (0.1mmol/L) calcium. The enhancement of the cone horizontal cell response by Co2+ was not caused by changes in light responsiveness of cone photoreceptors. Moreover, application of 50μmol/L IBMX, an inhibitor of phosphodiester enzyme, reduced the suppressive effect of 0.1 mmol/L Co2+ in normal Ringer's solution. In consequence, the above-described enhancement of the cone horizontal cell light responsiveness may be due to a depolarization of cones caused by low calcium, which increases the activity of voltage-dependent calcium channels at cone terminals.     

Glucose transporter 4 can be inserted in the membrane without exposing its catalytic site for photolabeling from the medium

Insulin stimulates the production of PI(3,4,5)P3 in muscle cells, and this is required to stimulate GLUT4 fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P3 to muscle cells recapitulates insulin's effects on GLUT4 fusion with the plasma membrane, but not glucose uptake. This study aims to explore the mechanism behind this difference. In L6-GLUT4myc muscle cells, the availability of the GLUT4 intracellular C-terminus and extracellular myc epitopes for immunoreactivity on plasma membrane lawns was detected with the corresponding antibody. The availability of the active site of GLUT4 from extracellular medium was assessed by affinity photolabeling with the cell impermeant compound Bio-LC-ATB-BMPA. 100nmol/L insulin and 10μmol/L PI(3,4,5)P3 caused myc signal gain on the plasma membrane lawns by 1.64-fold and 1.58-fold over basal, respectively. Insulin, but not PI(3,4,5)P3, increased photolabeling of GLUT4 and immunolabeling with C-terminus antibody by 2.47-fold and 2.04-fold over basal, respectively. Upon insulin stimulation, the C-terminus signal gain was greater than myc signal gain (2.04-fold vs. 1.64-fold over basal, respectively) in plasma membrane lawns. These results indicate that (i) PI(3,4,5)P3 does not make the active site of GLUT4 available from the extracellular surface despite causing GLUT4 fusion with the plasma membrane; (ii) the availability of the active site of GLUT4 from the extracellular medium and availability of the C-terminus from the cytosolic site are correlated; (iii) in addition to stimulating GLUT4 translocation, insulin stimulation displaces a protein which masks the GLUT4 C-terminus. We propose that a protein which masks the C-terminus also prevents the active site from being available for photolabelling and possibly glucose uptake after treatment with PI(3,4,5)P3.     

~ffects of endothelial microvesicles induced by A23187 on H9c2 cardiomyocytes

Objective To investigate the effects of endothelial microvesicles （EMVs） induced by calcium ionophore A23187 on H9c2 cardiomyocytes. Methods Human umbilical vein endothelial cells （HUVECs） were treated with 10 μmol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 Ilm latex beads and anti- PE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were co- cultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/Pl double staining. Results EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles （〈 1 μm） induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner （P〈0.05）. Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry. Conclusion Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.     

Magnesium permeation through mechanosensitive channels： single-current measurements

Compelling evidence shows that intracellular free magnesium [Mg^2＋]i may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg^2＋ influx in mammalian cells are poorly understood. Here, we show that mechanosensitive （MS） cationic channels activated by stretch are permeable for Mg^2＋ ions at different extracellular concentrations including physiological ones. Single-channel currents were recorded from cell-attached and inside-out patches on K562 leukaemia cells at various concentrations of MgCl2 when Mg^2＋ was the only available carrier of inward currents. At 2 mM Mg^2＋, inward mechanogated currents representing Mg^2＋ influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg^2＋ levels, only slight increase of single-channel currents and conductance occurred, implying that Mg^2＋ permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg^2＋, mean conductance values for inward currents carried by Mg^2＋ were rather similar, being equal to 6.8 ± 0.5 and 6.4 ± 0.5 pS, respectively. The estimation of the channel-selective permeability according to constant field equation is obviously limited due to saturation effects. We conclude that the detection of single currents is the main evidence for Mg^2＋ permeation through membrane channels activated by stretch. Our single-current measurements document Mg^2＋ influx through MS channels in the plasma membrane of leukaemia cells.     

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.     

Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

The response of electron transport mediated by active NADPH dehydrogenase complexes to heat stress in the cyanobacterium Synechocystis 6803

The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem I (PSI) and photosystem II (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactiva-tion mutants △ndhB (M55) and △ndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETRPSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55 ℃ for 10 min significantly inhibited PSII-driven ETR (ETRPSII) in the WT, M55 and D1/D2 cells, and the ex-tent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETRPSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETRPSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETRPSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and medi-umcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETRPSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.     

Human recombinant B7-H3 expressed in E. coli enhances T lymphocyte proliferation and IL-10 secretion in vitro

To explore the biofunctions of human B7-H3 on activated T lymphocyte, the gene of human B7-H3 encoding the extracellular region (IgV-like and IgC-like domains) was obtained by RT-PCR from human lung cells and subcloned into the prokaryotic expression vector pGEX-5X-3 to express glutathione S-transferase (GST) fusion protein. A 49 kD fusion protein (named as GST/hB7-H3 hereafter) was induced by IPTG and purified by standard methods reported in prokaryotic system. In the presence of the first signal imitated by anti-CD3 monoclonal antibody, T lymphocyte proliferation was observed by incubating purified T cells with soluble GST/hB7-H3 fusion protein by MTT assay. The concentrations of IFN-γ and IL-10 in the supernatants of T cells were determined by ELISA. The results showed that the GST/hB7-H3 protein produced in bacteria had modest biological activities to proliferate the T lymphocyte and enhance IFN-γ as well as IL-10 secretion.     

Alpha-latrotoxin modulates the secretory machinery via receptor-mediated activation of protein kinase C

The hypothesis whether alpha-latrotoxin (LTX) could directly regulate the secretory machinery was tested in pancreatic beta cells using combined techniques of membrane capacitance (Cm) measurement and Ca2+ uncaging. Employing ramp increase in [Ca2+]i to stimulate exocytosis, we found that LTX lowers the Ca2+ threshold required for exocytosis without affecting the size of the readily releasable pool (RRP). The burst component of exocytosis in response to step-like [Ca2+]i increase generated by flash photolysis of caged Ca2+ was also speeded up by LTX treatment. LTX increased the maximum rate of exocytosis compared with control responses with similar postflash [Ca2+]i and shifted the Ca2+ dependence of the exocytotic machinery toward lower Ca2+ concentrations. LTXN4C, a LTX mutant which cannot form membrane pores or penetrate through the plasma membrane but has similar affinity for the receptors as the wild-type LTX, mimicked the effect of LTX. Moreover, the effects of both LTX and LTXN4C) were independent of intracellular or extracellular Ca2+ but required extracellular Mg2+. Our data propose that LTX, by binding to the membrane receptors, sensitizes the fusion machinery to Ca2+ and, hence, may permit release at low [Ca2+]i level. This sensitization is mediated by activation of protein kinase C.     

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

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

Cross-talk with Ca(2+) influx does not underlie the role of extracellular signal-regulated kinases in cytotoxic T lymphocyte lytic granule exocytosis

One important mechanism cytotoxic T lymphocytes use to kill target cells is exocytosis of lytic granules that contain cytotoxic agents such as perforin and granzyme. Ca(2+) influx and activation of protein kinase C have been known for many years to be key signals for granule exocytosis. Recent work has suggested that activation of extracellular signal-regulated kinases (ERK), members of the mitogen-activated protein kinase (MAP kinase) family, may be a third required signal. We surmised that the involvement of ERK in lytic granule exocytosis could be mediated through cross-talk with Ca(2+) influx, rather than constituting an independent signal. We tested this idea using TALL-104 human leukemic CTLs as a model system and discovered the following. 1) ERK inhibition caused a modest decrease in the amplitude of increases in intracellular Ca(2+) concentration, but this effect cannot account for the profound inhibition of granule exocytosis. 2) Ca(2+) influx can activate ERK in TALL-104 cells, but this effect does not contribute to ERK activation stimulated by solid phase anti-CD3 monoclonal antibodies. We conclude that cross-talk between ERK signaling and Ca(2+) does not mediate the role of ERK in CTL lytic granule exocytosis.     

The sea anemone toxin Bc2 induces continuous or transient exocytosis, in the presence of sustained levels of high cytosolic Ca2+ in chromaffin cells

We have isolated and characterized a new excitatory toxin from the venom of the sea anemone Bunodosoma caissarum, named Bc2. We investigated the mechanism of action of the toxin on Ca(2+)-regulated exocytosis in single bovine adrenal chromaffin cells, monitoring simultaneously fura-2 fluorescence measurements and electrochemical recordings using a carbon fiber microelectrode. Bc2 induced quantal release of catecholamines in a calcium-dependent manner. This release was associated with a sustained rise in cytosolic Ca(2+) and displayed two different patterns of response: a continuous discharge of prolonged duration that changed to a transient burst as the toxin concentration (or incubation time) increased. Continuous secretion was dependent on the activity of native voltage-dependent Ca(2+) channels and showed a pattern similar to that of alpha-latrotoxin; however, its kinetics adjusted better to that of continuous cell depolarization with high K(+) concentration. In contrast, transient secretion was independent of Ca(2+) entry through native voltage-dependent Ca(2+) channels and showed inhibition of late vesicle fusion that was accompanied by "freezing" of F-actin disassembly. These new features make Bc2 a promising new tool for studying the machinery of neurotransmitter release.     

Extracellular ATP stimulates exocytosis via localized Ca(2+) release from acidic stores in rat pancreatic beta cells

Three different methods, membrane capacitance (C(m)) measurement, amperometry and FM dye labeling were used to investigate the role of extracellular ATP in insulin secretion from rat pancreatic beta cells. We found that extracellular application of ATP mobilized intracellular Ca(2+) stores and synchronously triggered vigorous exocytosis. No influence of ATP on the readily releasable pool of vesicles was observed, which argues against a direct modulation of the secretory machinery at a level downstream of Ca(2+) elevation. The stimulatory effects of ATP were greatly reduced by intracellular perfusion of BAPTA but not EGTA, suggesting a close spatial association of fusion sites with intracellular Ca(2+) releasing sites. ATP-induced Ca(2+) transients and exocytosis were not blocked by thapsigargin (TG), by a ryanodine receptor antagonist or by dissipation of pH in acidic stores by monensin alone, but they were greatly attenuated by IP(3) receptor inhibition as well as ionomycin plus monensin, suggesting involvement of IP(3)-sensitive acidic Ca(2+) stores. Taken together, our data suggest that extracellular ATP triggers exocytosis by mobilizing spatially limited acidic Ca(2+) stores through IP(3) receptors. This mechanism may explain how insulin secretion from the pancreas is coordinated through diffusible ATP that is co-released with insulin.     

RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca(2+) channels

Ca(2+) influx through voltage-gated channels initiates the exocytotic fusion of synaptic vesicles to the plasma membrane. Here we show that RIM binding proteins (RBPs), which associate with Ca(2+) channels in hair cells, photoreceptors, and neurons, interact with alpha(1D) (L type) and alpha(1B) (N type) Ca(2+) channel subunits. RBPs contain three Src homology 3 domains that bind to proline-rich motifs in alpha(1) subunits and Rab3-interacting molecules (RIMs). Overexpression in PC12 cells of fusion proteins that suppress the interactions of RBPs with RIMs and alpha(1) augments the exocytosis triggered by depolarization. RBPs may regulate the strength of synaptic transmission by creating a functional link between the synaptic-vesicle tethering apparatus, which includes RIMs and Rab3, and the fusion machinery, which includes Ca(2+) channels and the SNARE complex.     

Effect of synaptosomal cytosolic [3H]GABA pool depletion on secretory ability of alpha-latrotoxin

alpha-Latrotoxin, a presynaptic neurotoxin from the venom of Latrodectus mactans tredecimguttatus, induces massive [3H]GABA release from rat brain synaptosomes as a result of interaction with either Ca(2+)-dependent (neurexin 1 alpha or Ca(2+)-independent (latrophilin) membrane receptor. The main aim of the study was to elucidate whether the binding of alpha-latrotoxin to different types of receptors led to [3H]GABA secretion from one pool or in each case the source of neurotransmitter differs: in the presence of Ca2+ exocytosis is induced, while in the absence of Ca(2+)--outflow by mobile membrane GABA transporter from cytoplasm. We examined the effect of the depletion of cytosolic [3H]GABA pool by competitive inhibitors of the GABA transporter (nipecotic acid and 2,4-diaminobutyric acid) on the alpha-latrotoxin-stimulated neurotransmitter release. We also compared the influence of these agents on neurosecretion, evoked by depolarization with that evoked by alpha-latrotoxin. Depolarization was stimulated by 4-aminopyridine in the Ca(2+)-containing saline and high KCl in Ca(2+)-free medium. In synaptosomes treated with nipecotic acid unstimulated [3H]GABA release was significantly augmented and high KCl-evoked Ca(2+)-independent [3H]GABA release was essentially inhibited. But under the same conditions neurosecretion stimulated by alpha-latrotoxin greatly raised with respect to the control response. The similar results were obtained with the synaptosomes treated with 2,4-diaminobutyric acid. Another way to determine which of GABA pool is the target of alpha-latrotoxin action lay in analysis of the toxin effects on the preliminary depolarized synaptosomes. alpha-Latrotoxin influence was diminished by the preceding depolarization by 4-aminopyridine in Ca2+ presence. But after the high KCl stimulation effect of alpha-latrotoxin didn't change. These data suggest that alpha-latrotoxin triggers neurotransmitter release from synaptic vesicles via exocytosis. We suppose that the type of membrane receptor does not determine the mechanism of GABA release evoked by the toxin.     

Alpha-latrotoxin and its receptors CIRL (latrophilin) and neurexin 1 alpha mediate effects on secretion through multiple mechanisms

Alpha-Latrotoxin and its plasma membrane receptors cause a number of distinct effects in secretory cells. First, by tethering alpha-latrotoxin to the plasma membrane, CIRL/latrophilin and neurexin 1 alpha facilitate alpha-latrotoxin-induced channel formation. The stimulation of secretion by alpha-latrotoxin in neuroendocrine cells is a consequence of Ca(2+) influx through these alpha-latrotoxin-induced channels. In addition to channel formation, alpha-latrotoxin enhances secretion in permeabilized cells through interaction with the plasma membrane receptor CIRL/latrophilin. Finally, overexpression of CIRL/latrophilin inhibits Ca(2+)-dependent secretion in permeabilized chromaffin cells in the absence of alpha-latrotoxin. This effect represents a 'constitutive' action of the G-protein coupled receptor to specifically inhibit an ATP-dependent priming step in the secretory pathway. The effect suggests that the receptor may have an important modulatory role in synaptic transmission.     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.     

Ca(2+)- and glycoconjugates-dependent prey capture in the heliozoon Actinophrys sol

Exocytosis of extrusomes, secretory granules found in protozoa, is involved in prey capture by the heliozoon Actinophrys sol. Here, we show that extracellular Ca(2+) is necessary for exocytosis and prey capture in A. sol. We found that A. sol could not capture prey cells in a Ca(2+)-free solution. L-type Ca(2+) channel blockers and a calmodulin antagonist also inhibited the capture of prey. These results suggest that Ca(2+) influx via L-type Ca(2+) channels plays a crucial role in exocytosis in A. sol. Concanavalin A (Con A) also inhibited prey capture, and the inhibition was relieved by the addition of its hapten sugar, alpha-mannoside, suggesting that Con A-binding glycoconjugates are implicated in exocytosis of extrusomes and the adhesion of prey cells.