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Store-operated Ca(2+) entry (SOCE) is the main Ca(2+) entry pathway of non-excitable cells. In the past decade, the activation of this entry has been unveiled, with STIM1, a protein of the endoplasmic reticulum able to sense the intraluminal Ca(2+) content, and Orai1, the pore-forming unit of the Ca(2+) release activated Ca(2+) (CRAC) channels. When Ca(2+) ions are released from the endoplasmic reticulum, STIM1 proteins oligomerize and directly interact with Orai1 proteins, allowing the opening of the CRAC channels and a massive Ca(2+) ion influx known as SOCE. As Ca(2+) is involved in various cellular processes, the discovery of new drugs acting on the SOCE should be of interest to control the cell activity. By testing analogs of 2-aminoethyl diphenylborinate (2-APB), a well known, though not so selective effector of the SOCE, we identified methoxy diethylborinate (MDEB), a molecule able to potentiate the SOCE in three leukocyte and two breast cancer cell lines by increasing the Ca(2+) influx amplitude. Unlike 2-APB, MDEB does not affect the Ca(2+) pumps or the Ca(2+) release from the endoplasmic reticulum. MDEB could therefore represent the first member of a new group of molecules, specifically able to potentiate SOCE. Although not toxic for non-activated Jurkat T cells, it could induce the apoptosis of phytohemagglutinin-stimulated cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

2-Aminoethyl diphenylborinate (2-APB) is a well-known effector of the store-operated Ca(2+) entry of several cell types such as immune cells, platelets and smooth muscle cells. 2-APB has a dual effect: potentiation at 1-5μM and inhibition at >30μM. Unfortunately, it is also able to modify the activity of other Ca(2+) transporters and, thus, cannot be used as a therapeutic tool to control the leukocyte activity in diseases like inflammation. Previously, we have shown that SOCE potentiation by 2-APB depends on the presence of the central boron-oxygen core (BOC) and that the phenyl groups determine the sensitivity of the molecule to inhibit and/or potentiate the SOCE. We hypothesized that by modifying the two phenyl groups of 2-APB, we could identify more efficient and specific analogues. In fact, the addition of methoxyl groups to one phenyl group greatly decreased the potentiation ability without any significant effect on the inhibition. Surprisingly, when the free rotation of the two phenyl groups was blocked by a new hydrocarbon bridge, the BOC was no longer able to potentiate. Furthermore, larger aryl groups than phenyl also impaired the activity of the BOC. Thus, the potentiation site in the Ca(2+) channel is not accessible by the BOC when the lateral groups are too large or unable to freely rotate. However, these molecules are potent inhibitors of store-operated calcium entry with affinities below 1μM, and they can block the activation of the Jurkat T cells. Thus, it is possible to characterize 2-APB analogues with different properties that could be the first step in the discovery of new immunomodulators. This article is part of a special issue entitled "Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Store-operated calcium entry (SOCE) is a key regulator in the activation of leukocytes. 3,5-Bistrifluoromethyl pyrazole (BTP) derivatives have been identified recently as inhibitors of T lymphocyte activation. The inhibitory effect of one of these compounds, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), appears to be a result of inhibition of SOC influx. Polymorphonuclear neutrophils provide effective protection against bacterial infection, but they are also involved in tissue damage during chronic inflammation. As for T lymphocytes, their activation relies on SOCE. We therefore investigated the effect of BTP2 on calcium homeostasis and functional responses of human neutrophils. BTP2 significantly inhibited the calcium influx after stimulation with thapsigargin or fMLF. This inhibition was seen after 5 min of incubation with 10 microM BTP2 and after 24 h with lower concentrations. With 24 h incubation, the effect appeared irreversible, as the removal of BTP2 3 h before the experiment did not reduce this inhibition in granulocyte-differentiated HL60 cells. In human neutrophils, BTP2 reduced superoxide anion production by 82% after 24 h of incubation. On the contrary, phagocytosis, intraphagosomal radical production, and bacterial killing by neutrophils were not reduced significantly, even after 24 h treatment with 10 microM BTP2. This work suggests that BTP2 could become an important tool to characterize calcium signaling in neutrophils. Furthermore, BTP2 or related compounds could constitute a new approach to the down-regulation of neutrophils in chronic inflammatory disease without compromising antibacterial host defense.

Ca2+-induced exocytosis in neuronal and neuroendocrine cells involves ATP-dependent steps believed to 'prime' vesicles for exocytosis. Primed, docked vesicles are released in response to Ca2+ influx through voltage-gated Ca2+ channels. Neutrophils, however, do not possess voltage-gated Ca2+ channels and appear to have no docked vesicles. Furthermore, neutrophils have several types of granules with markedly different Ca2+ requirements for exocytosis. These differential Ca2+ dependencies were used as a tool to investigate the ATP dependence of different granule populations. Here we demonstrate distinct ATP requirements for release of neutrophil granule populations, with respect to rate as well as amplitude. Intracellular ATP was depleted to various levels, and exocytosis was stimulated with different Ca2+ concentrations and measured with the patch-clamp capacitance technique or by detecting enzyme release. Primary granule exocytosis displayed a distinct ATP dependence with an apparent K(m) of approximately 80 microM ATP and no ATP-independent exocytosis. Release of secondary and tertiary granules displayed a more shallow ATP dependence (K(m) approximately 330 microM), and more than 50% of secondary and tertiary granules appeared not to need ATP at all for their release. Individual granules in human neutrophils have distinct ATP requirements for exocytosis, suggesting that the ATP-sensitive elements are localised to the granules. Primary granule exocytosis has a very low affinity for ATP. Furthermore, substantial ATP-independent exocytosis of secondary and tertiary granule occurs despite the absence of docked granules. These characteristics should help neutrophils to fulfil their bactericidal functions at poorly irrigated sites of infection with low glucose supply.

Phagolysosome fusion and granule exocytosis in neutrophils are calcium-dependent processes. The calcium requirements vary between granule types, suggesting the presence of different calcium sensors. The synaptotagmins, a family of calcium-binding proteins, previously shown to participate in vesicle fusion and vesicle recycling in excitable cells, are putative calcium-sensors of exocytosis in excitable cells. In this study, we show that synaptotagmin II is present in human neutrophils and may participate in phagocytic and in exocytotic processes. In protein extracts from human neutrophils, we identified synaptotagmin II by Western blot as an 80 kDa protein. Subcellular fractionation revealed that synaptotagmin II was associated with the specific granules. In fMLP-stimulated cells, synaptotagmin II translocated to the plasma membrane. This correlated with the upregulation of complement receptor 3 (CR 3), reflecting the translocation of specific granules to the cell surface. Synaptotagmin II also translocated to the phagosome after complement-mediated phagocytosis in the presence of calcium. LAMP-1 translocated in parallel but probably was located to another subcellular compartment than synaptotagmin II. Under calcium-reduced conditions, neither synaptotagmin II nor LAMP-1 translocated to the phagosome. We therefore suggest a role for synaptotagmin II as calcium-sensor during phagocytosis and secretion in neutrophils.