Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca2+ release-activated Ca2+ (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca2+ sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233–474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca2+ stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca2+ stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.Store-operated Ca Protein–protein interactions play a pivotal role in the cellular signalling network. Monitoring protein-protein interactions in living cells is extremely useful for elucidating the dynamics and mechanisms of biological processes. In the past few decades, numerous methods 2+ influx is mainly via store-operated Ca2+ channels (SOCs) that play important roles in the control of gene expression, cell growth and differentiation, secretion, Ca2+ homeostasis, and apoptosis 2+ release-activated Ca2+ current (ICRAC). As the key subunit of CRAC channels, ER-resident protein STIM1 controls the opening of these channels. STIM1 is distributed throughout the ER; depletion of Ca2+ stores triggers the redistribution of STIM1 to sites of close apposition between the ER and the plasma membrane. This redistribution recruits Orai1 to STIM1 puncta, whereby the interaction of STIM1 and Orai1 can lead the opening of CRAC channels for Ca2+ influx to ER. Therefore, STIM1 is an ER Ca2+ sensor and is essential for the recruitment of Orai1 to puncta and activation of SOCs2+ binding protein, CRACR2A that could regulate store-operated Ca2+ entry (SOCE) 2+, thereby enhancing CRAC channel activity. As intracellular Ca2+ levels rise, CRACR2A binds Ca2+ and triggers SOCE inactivation by dissociating from the Orai–STIM complex In electrically non-excitable cells, Ca2+ and that it exists only in a monomeric form in the resting state in vitro 2+-replete conditions (resting cells) because both endogenous and overexpressed STIM1–STIM1 and STIM1–STIM2 complexes can be readily co-immunoprecipitated from store-replete cell lysates2+ sensing. The C-terminus of STIM1 contains two coiled-coil regions that overlap with an ezrin-radixin-moesin (ERM)-like domain, followed by a serine/proline and a lysine-rich region. The functions of the C- and N- terminal domains of STIM1 need further investigation.Previous studies suggest that STIM1 dimerises or oligomerises in the absence of CaIn the current study, BiFC was utilised for the first time to identify and visualise STIM1 in the living cells in resting state. STIM1 was found in an oligomeric form in cells in a resting state. The STIM1 oligomer (BiFC-STIM1) was identical to wild-type STIM1 in its morphological characteristics, dynamics, response to drugs and function. We have also confirmed that the key domain for the interaction between STIM1s in resting cells is not the SAM motif but the C-terminus (residues 233–474).2. Cells were transfected with Lipofectamine 2000 (Invitrogen) as per the manufacturer’s instructions. Briefly, HEK293T cells were plated onto 30-mm round glass coverslips in a 6-well plate. The following day, cells were transfected with various plasmids according to the experimental protocol. Six hours later, the medium bathing the cells was replaced with complete DMEM, and the cells were maintained in culture overnight. After 24 h, the experiments were carried out on the transfected cells plated onto 30 mm round glass coverslips mounted in a Teflon chamber.HEK293T(ATCC) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% heat inactivated foetal bovine serum, 50 U/ml penicillin, and 50 mg/ml streptomycin. HEK293T cells were maintained at 37°C in a humidified incubator set at 5% COmCherry-STIM1 was obtained from the laboratory of Richard S. Lewis, Stanford University, and the pHluorin-STIM1 from Pingyong Xu, Chinese Academy of Sciences. Full-length human Orai1 was amplified by PCR from a human placenta cDNA library (Invitrogen) and subcloned into pEYFP-N1 or pECFP-N1 plasmid. BiFC vectors VN173-ST1, VC155-ST1, VN173-ΔSAM, VC155-ΔSAM, VN173-ΔC, VC155-ΔC  were constructed by the method previously described 2, 1 MgCl2, 8 glucose, and 10 Hepes . For the Ca2+-free Ringer’s solution, CaCl2 was replaced with 1 mM EGTA and 2 mM MgCl2. Stock solutions of thapsigargin (TG) and 2-APB were prepared in Me2SO at a concentration of 1 mM. Fluo-4-AM was purchased from Invitrogen. Unless otherwise specified, all of the reagents and chemicals were from Sigma-Aldrich.In confocal imaging experiments, we used standard extracellular Ringer’s solution containing the following (in mM): 150 NaCl, 5 KCl, 1.8 CaCl○C. Following incubation, blots were washed and secondary HRP-conjugated antibodies (Jackson Immuno-Resaerch) were added in 5% milk for 1 h. Blots were visualized using SuperSignal ECL (Pierce).HEK293T cells was homogenized in NP40 buffer . Cell lysates from cultures were sonicated and protein concentrations were determined. An equal amount of protein (30 µg/30 µl/lane) was separated on SDS-PAGE 10% gels. Proteins transferred to nitrocellulose were blocked in 5% non-fat dry milk in PBS for 30 min then incubated in primary antibodies in 3% BSA/PBS overnight at 4The experiments were performed using an Olympus FV500 laser scanning confocal microscopy system . Coverslips containing cells were placed in a perfusion chamber on the stage of an inverted Olympus I×70 microscope. Data acquisition was performed in the sequential line mode for the best spatiotemporal reliability. The sample area was scanned at resolution of 512×512 pixels. The eCFP and eYFP were excited at 458 and 514 nm, respectively. Photobleaching experiments were performed using the same microscope with a 10 mW, 514 nm line of an argon laser. A 15 s pulse was used to locally photobleach ≈98% of the YFP intensity. All of the experiments were performed at 22°C–25°C.To determine FRET efficiencies, we used the acceptor photobleaching method. In the photobleaching experiments, a 15 s pulse of high-intensity laser at 514 nm was applied to bleach the YFP signal acceptor in a whole cell. A series of pre-bleaching and post-bleaching CFP (donor) fluorescence intensities were recorded. If any interactions leading to energy transfer were present in the cell, photobleaching of the acceptor would lead to an increase in donor fluorescence because it would no longer be quenched by the acceptor. The energy transfer efficiency (E) was calculated from the equation: pre and Fpost stand for the fluorescence intensity of the donor before and after acceptor photobleaching, respectively.Here, F2+]i was measured using a dual-wavelength excitation (340/380 nm) photometry system on an inverted microscope  equipped with a polychromatic xenon light source . The emission was collected at 510 nm with a photodiode controlled by the TILL photometry system and X-Chart extension of Pulse software . Ca2+ fluctuations are reported as the ratio of fluorescence emission at the two excitation wavelengths. Cells transfected alone with eYFP-STIM1 or cotransfected with VN173-ST1 and VC155-ST1 were chosen based on their fluorescence when excited at 514 nm.Cells were loaded with 2 µM fura-2/AM (Invitrogen) at 37°C for 30 min in standard Ringer’s solution. i elevation (0.79±0.07) in cells expressing eYFP-STIM1 .In order to verify the function of BiFC-STIM1 protein, we used Ca2+ depletion activates the CRAC channel remains elusive CRAC-signalling pathway. It is now clear that STIM1 is a Ca2+ sensor located in the ER membrane 2+ selective channels in the plasma membrane 2+ store depletion, STIM1 redistributes to form puncta close to the plasma membrane and activates ORAI1 channels that manifest the biophysical fingerprint of endogenous CRAC channels Although research in the past 20 years has provided a detailed electrophysiological and pharmacological characterisation of CRAC channels, the precise molecular mechanism by which ER CaE. coli, and the effects of Ca2+ binding on its conformation were studied in vitro 2+, the EF-hand/SAM domain exists as a monomer that has a compact alpha-helical structure. In contrast, when Ca2+ is depleted, the EF hand/SAM domain changes to a less compact conformation that promotes aggregation into dimers and oligomers. Repletion of Ca2+ caused these aggregates to revert back to monomers, demonstrating the reversibility of this Ca2+-dependent conformational change. However, other reports indicate that STIM1–STIM1 and STIM1–STIM2 interactions do occur in Ca2+ store-replete cells because both endogenous and overexpressed STIM1–STIM1 and STIM1–STIM2 complexes can be readily co-immunoprecipitated from store-replete cell lysates In previous studies, a sequence containing only the EF-hand and SAM domain of STIM1 was recombinantly expressed in 2+ store depletion induced BiFC-STIM1 to relocate to ER-PM junctions and form puncta. 2-APB, a reliable blocker of SOCE, prevented and reversed the relocalisation of BiFC-STIM1 to puncta near the plasma membrane. Our results also demonstrate that both the functional and pharmacological properties of BiFC-STIM1 were not altered compared with those of wild-type STIM1. These data indicate that BiFC-STIM1 is identical to wild-type STIM1 in morphological characteristics, dynamics, drug properties and functions. In addition, to further examine the state of STIM1 in resting cells, we used acceptor-photobleaching-based FRET to analyse the interactions of STIM1 in resting cells. HEK 293T cells coexpressing CFP/YFP-tagged STIM1 exhibited comparably higher FRET efficiency in the resting state, which suggests that the interaction between STIM1s exist in resting cells. Thus, using direct visualisation of STIM1 molecule interactions, we demonstrate for the first time that STIM1 exists in an oligomeric form in living cells at rest. Although the functional significance of the oligomeric form of STIM1 in resting cells remains unclear and needs further investigation, we propose that the oligomeric structure of STIM1 in the resting state is required for its rapid transition from lower order oligomers (such as dimers) to higher order oligomers upon Ca2+ store depletion. In addition, the oligomeric state of STIM1 in resting cells may be important for its rapid constitutive movement along microtubules We utilised the technique of bimolecular fluorescence complementation (BiFC) to identify and visualise the state of STIM1 molecules in living cells in the resting state. If VN173-ST1 interacts with VC155-ST1, the interaction would bring VN173 and VC155 into close proximity and an intact Venus molecule would be reconstituted allowing direct visualisation of the oligomeric state of STIM1. In our experiments, coexpression of VN173-ST1 and VC155-ST1 led to strong fluorescence emission, suggesting the presence of STIM1 oligomer (BiFC-STIM1), possibly as a dimer. We found that in resting cells, BiFC-STIM1 exhibited a fibrillar distribution similar to the distribution of wild-type GFP-STIM1. BiFC-STIM1 did not constitutively form puncta beneath the plasma membrane, which suggests that higher order oligomers rather than lower order oligomers (such as dimers) of STIM1 have the ability to migrate to ER-PM junctions to form aggregates. Ca2+-binding EF-hand motifs with a single sterile alpha-motif (SAM) domain on the luminal side of the ER. The EF hand motif of STIM1 is, as expected, responsible for Ca2+ sensing in the ER lumen 2+ store depletion-mediated puncta formation and does not result in constitutive STIM1 aggregation as puncta nor constitutive activation of the SOC channel 2+-dependent conformational transition of the luminal region. Beyond the single conserved transmembrane (TM) segment, STIM1 has two coiled-coil regions that overlap with an ezrin-radixin-moesin (ERM)-like domain, followed by a divergent region that contains multiple serine and proline residues (S/P rich) and lysine residues (K rich) in the cytoplasmic side. Previous studies indicate that the cytoplasmic domain mediates STIM1 aggregation and SOC activation. Three recent studies have independently identified a crucial Orai-recruiting and Orai-activating domain within the ERM domain of STIM1; it was named the CRAC-activating domain CAD (CAD) STIM1 contains two CaFigure S1Orai1 recruitment to puncta at the ER-PM junctions depends on binding to STIM1. Part A: mCh-STIM1 expressed alone in HEK293 cells redistributes from a diffuse ER distribution (Rest) to the cell periphery after Ca2+ store depletion with TG. After TG treatment, puncta are visible at the cell footprint. Part B: Orai1-eYFP expressed alone does not redistribute into puncta after Ca2+ store depletion. Part C: when expressed together, mCh-STIM1 and Orai1-eYFP form colocalised puncta after Ca2+ store depletion. Scale bars, 20 µm.(TIF)Click here for additional data file.


ATP released from damaged brain cells can activate ionotropic and metabotropic purinergic receptors, and act as a chemoattractant for microglia. Metabotropic P2Y receptors evoke a Ca2+ rise through release from intracellular Ca2+ stores and store-operated Ca2+ entry, and some have been implicated in microglial migration. This Ca2+ rise is expected to activate small-conductance Ca2+-dependent K+ (SK) channels, if present. We previously found that SK3 (KCa2.3) and KCa3.1 (SK4/IK1) are expressed in rat microglia and contribute to LPS-mediated activation and neurotoxicity. However, neither current has been studied by elevating Ca2+ during whole-cell recordings. We hypothesized that, rather than responding only to Ca2+, each channel type might be coupled to different receptor-mediated pathways. Here, our objective was to determine whether the channels are differentially activated by P2Y receptors, and, if so, whether they play differing roles. We used primary rat microglia and a rat microglial cell line (MLS-9) in which riluzole robustly activates both SK3 and KCa3.1 currents. Using electrophysiological, Ca2+ imaging and pharmacological approaches, we show selective functional coupling of KCa3.1 to UTP-mediated P2Y2 receptor activation. KCa3.1 current is activated by Ca2+ entry through Ca2+-release-activated Ca2+ (CRAC/Orai1) channels, and both CRAC/Orai1 and KCa3.1 channels facilitate refilling of Ca2+ stores. The Ca2+ dependence of KCa3.1 channel activation was skewed to abnormally high concentrations, and we present evidence for a close physical association of the two channel types. Finally, migration of primary rat microglia was stimulated by UTP and inhibited by blocking either KCa3.1 or CRAC/Orai1 channels. This is the first report of selective coupling of one type of SK channel to purinergic stimulation of microglia, transactivation of KCa3.1 channels by CRAC/Orai1, and coordinated roles for both channels in store refilling, Ca2+ signaling and microglial migration.Microglial activation involves Ca The SOCE pathway used by microglia for migration following P2Y receptor activation has not been identified. By combining molecular, biophysical and pharmacological approaches, we previously identified the Ca2+-release activated Ca2+ (CRAC) channel as a major SOCE pathway in primary rat microglia In the mature CNS, microglial cells with highly branched processes continually survey the local microenvironment and rapidly respond to stranger and danger signals 2+ in microglia is opening of SK  channels. We previously showed that SK4 (KCa3.1) 2+ influx through CRAC channels. However, the SK currents were not monitored, and roles of SK3 and KCa3.1 channels in regulating SOCE have not been examined in microglia. Recently, we discovered that both SK3 and KCa3.1 currents are reliably activated in the MLS-9 microglia cell line by the neuroprotective drug, riluzole, with little or no rise in intracellular Ca2+2+ levels 2+ to ∼1 µM, which is well above the normal EC50 values reported for native and heterologously expressed channels (see Discussion). Instead, our results on MLS-9 cells raise the possibility that SK3 and KCa3.1 channels in microglia require more than a simple elevation in Ca2+. If so, it is possible that the two channel types can selectively respond to different stimuli.An expected immediate response to elevated intracellular Ca2Y2 receptors in microglia elevate intracellular Ca2+ and activate SK3 and KCa3.1 channels, and if so, whether this requires Ca2+ entry through CRAC channels. Having found that only KCa3.1 channels were activated, and that CRAC channels were involved, we next asked whether KCa3.1 channels selectively control store-operated Ca2+ entry and store refilling. This was the case. Finally, we asked whether P2Y2 receptors increase microglia migration through mechanisms that require CRAC and the selective activation of KCa3.1. Again, we found this to be the case.This study was designed to address three overall questions. First, we asked whether metabotropic P2). The resulting suspension of non-adherent microglia was centrifuged , the pellet was re-suspended in MEM with reduced serum (2% FBS). Under these conditions, we have found that the microglial cells are a relatively non-activated state Animals were used in strict accordance with the guidelines established by the Canadian Council on Animal Care, and was approved by the Animal Care Committee of the University Health Network (AUP #914). Primary cultures of rat microglia (≥98% pure) were prepared from brains harvested from 1–2 day old Sprague Dawley pups  Essentially pure microglia cultures were prepared according to our standard protocols + channels − channels 4 cells/coverslip for Ca2+ imaging and patch-clamp analysis. An important advantage of using MLS-9 cells is that they lack three currents that can interfere with isolating Ca2+-activated K+ currents. Primary rat microglia have an inward-rectifier K+ current at negative membrane potentials Our laboratory derived the MLS-9 cell line many years ago by treating pure cultures of rat microglia with colony stimulating factor-1 for several weeks. Individual cell colonies were harvested and used to establish continuous cell lines, of which we named one, MLS-9 2Y receptor blocker, suramin, the Orai1/CRAC blockers 2-APB and BTP2, and the KCa3.1 blocker, TRAM-34. Uridine 5′-triphosphate  and the SK1-SK3 blocker, apamin, were dissolved in double distilled water. All stock solutions were aliquoted and stored at –20°C until used.Chemicals were from Sigma-Aldrich, unless otherwise indicated. Stock solutions of several antagonists were made with DMSO; i.e., the P4 cells per 15 mm diameter coverslip) were incubated at room temperature with 3.5 µg/ml Fura-2AM (Invitrogen) for 45 min in the dark. A coverslip was then mounted in a 300 µl volume perfusion chamber , containing the same bath solution as for patch-clamping (see below). The effects of ion channel blockers  on UTP-evoked calcium signals were assessed on different batches of cells from separate coverslips. Images were acquired at room temperature using a Nikon Diaphot inverted microscope, Retiga-EX camera , and Northern Eclipse image acquisition software . A Lambda DG-4 Ultra High Speed Wavelength Switcher  was used to alternately acquire images at 340 and 380 nm excitation wavelengths. Images were acquired every 4 s, and the excitation shutter was closed between acquisitions to prevent photobleaching. The intracellular free Ca2+ concentration was calculated from the standard equation 2+-free bath solution with 2 µM ionomycin and 3 mM MnCl2 was perfused in to obtain the minimum 340∶380 ratio.The Fura-2 imaging methods were the same as we recently described 4/coverslip), mounted in the same perfusion chamber as for Ca2+ imaging. The cells were superfused with an extracellular (bath) solution containing the following (in mM): 125 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 5 glucose, and 10 HEPES, adjusted to pH 7.4 (with NaOH) and to ∼300 mOsm with sucrose. For Ca2+-free bath solution, CaCl2 was omitted and 1 mM EGTA was added. Bath solutions were exchanged using a gravity-driven perfusion system flowing at 1.5–2 ml/min and all recordings were made at room temperature. Whole-cell recordings were made with pipettes pulled from thin-walled borosilicate glass  to a resistance of 5–8 MΩ using a Narishige puller . Pipettes were filled with a solution (intracellular) containing (in mM): 100 K-aspartate, 40 KCl, 1 MgCl2, 2 MgATP, and 10 HEPES, pH adjusted to 7.2 (with KOH), 280 mOsm/kgH2O. Either 1 EGTA+0.5 CaCl2 or 1 BAPTA+0.45 CaCl2 (where noted) was used to buffer the initial internal free Ca2+ to ∼120 nM . We chose low buffer concentrations (1 mM) to allow the purinergic receptor agonist, UTP, to transiently elevate intracellular Ca2+. Recordings were made with an Axon Multiclamp 700A amplifier, compensated on-line for capacitance and series resistance. Patch-clamp data were filtered at 5 kHz, and acquired and digitized using a Digidata 1322A board with pClamp software. Junction potentials were reduced by using agar bridges made with bath solution, and were calculated with the utility in pCLAMP. After correction, all reported voltages were about 5 mV more negative than reported in the text and figures.MLS-9 cells were plated on 15 mm diameter coverslips  were seeded on the upper inserts of 24-well TranswellTM Migration Chambers , which contained uncoated filters with open 8 µm-diameter holes. Microglia were bathed in MEM with 2% FBS and allowed to adhere for ∼1 h prior to adding compounds. When used, UTP was added to the lower well to act as a chemoattractant, and each antagonist was added to the upper well. The chamber was incubated . To quantify the transmigration of microglia to the underside of each filter, we first removed the remaining cells from the upper side, using a Q-tipTM. The filters were fixed , rinsed 3× in PBS, stained with 0.5% crystal violet (1 min), quickly rinsed, and allowed to air dry. Microglia that had migrated to the underside of each filter were viewed at 20× with an Olympus CK2 Phase Contrast Inverted microscope . Cells were counted in 5 fields of view per filter.Cultured primary rat microglia . Values of p<0.05 were taken as statistically significant.All data are expressed as mean ± SEM. For analysis of drug effects on currents, Ca2+ rise, with an initial rapid increase and a more slowly decaying phase  for each cell. For this cell, Ca2+ rapidly increased to ∼4 µM, spontaneously declined, and then returned to baseline after UTP was washed out (2+ level was 5.3±1.2 µM (n = 19). After UTP was washed out, Ca2+ decreased to ∼85 nM, which is similar to our previous results for MLS-9 cells The metabotropic purinergic receptor agonist, UTP, induced a biphasic Cang phase . The Ca2shed out . On aver2+-activated K+ channels. Both SK3 (KCa2.3) and KCa3.1 currents are reliably activated (by riluzole) + Nernst potential (−85 mV) with the bath and pipette solutions used. The UTP-evoked K+ current was transient   for the Ca2+ decay was 56±2 s (n = 21) and 68±12 s (n = 4) for the current  in trans-activating KCa3.1.The MLS-9 microglia cell line has several of the same ion channels as primary rat microglia, and we have extensively used these cells. MLS-9 cells have important advantages for studying small-conductance Caransient , and its n = 23) . Instead current . This tecal data  show tha2+ level (5.3 µM); therefore, we next examined the Ca2+-dependence of current activation without UTP. Whole-cell recordings were established with pipette solutions containing 2.5, 5.2, 8.0, 10.9 or 15.3 µM free Ca2+. The bath contained apamin to preclude any possible contribution of SK3. The example cell shows activation of KCa3.1 current with 10.9 µM intracellular Ca2+ (2+ dose-response curve was constructed by plotting current density (pA/pF) versus intracellular free Ca2+ , and there was no current at 1.1 µM Ca2+.The peak of the UTP-response reached a high Calar Ca2+ , a gradular Ca2+ . The curlar Ca2+ . A Ca2+ ree Ca2+ . It inclree Ca2+  and 0.1 3-mediated release of Ca2+ from intracellular stores, followed by store-operated Ca2+ entry and store refilling 2Y receptor-mediated biphasic rise in Ca2+  channel 2+ influx through CRAC channels is needed to activate KCa3.1 current, we used 50 µM 2-APB   to the bath. With Ca2+-free bath solution, 2-APB further reduced the peak amplitude  and area under the curve (to 145±15), consistent with its inhibition of Ca2+ release from stores.We next asked whether CRAC and KCa3.1 channels contribute to the Canchanged . The samµM 2-APB , as expe+ entry)  but did as added . Compare2+ rise was substantial (2+ (to 3.9±1.8% of the control level) or in the presence of 2-APB (14.1±4.1%), BTP2 (17.5±5.4%) or TRAM-34 (17.7±4.9%) in the bath. The response with BTP2 was essentially identical to that in the Ca2+ free bath, as expected for normal Ca2+ release from stores without influx. When Ca2+ was omitted from the bath, the already negligible second peak was unchanged by 2-APB or BTP2 (not shown). Again, apamin had no effect. Together, these data support the hypothesis that, following P2Y receptor activation, Ca2+ influx through CRAC/Orai1 channels replenishes the depleted stores, and this is facilitated by KCa3.1 (but not SK3) channel activity.To more directly analyze the role of CRAC and KCa3.1 channels in store refilling, we applied a second UTP treatment after a 5 min recovery period  than for EGTA (2.7×106 M−1s−1) 2+ (120 nM), the baseline current in standard bath was small, and UTP activated a KCa3.1 current  than with EGTA in the pipette solution  proteins in MLS-9 cells . Orai1 s2Y2 receptor. Most notably, transmigration was strongly inhibited by the CRAC/Orai1 channel blockers, 2-APB (reduced by 84%) and BTP2 (reduced by 76%). In addition, there was a 55% inhibition by the KCa3.1 blocker, TRAM-34. The SK1–SK3 blocker, apamin, did not reduce migration. This pharmacological profile exactly parallels that of the UTP-induced Ca2+ signaling and activation of KCa3.1 channels  have been identified in microglia  strongly activates P2Y2 and P2Y4 receptors, but only weakly activates P2Y62Y2, inhibits P2Y 6 by ∼30%, and does not inhibit P2Y42Y receptors elevate Ca2+ through IP3-mediated release from internal stores and influx through store-operated Ca2+ entry (SOCE). Here, UTP evoked a biphasic Ca2+ rise, with a rapid transient  followed by a smaller plateau phase .Microglial activation is multi-faceted, with diverse Ca2+ stores, rapid replenishment is important to prepare the cell for another stimulus; e.g., in cells with Ca2+ oscillations and during migration  channel is activated following store depletion (by thapsigargin) 2+ plateau phase and the refilling of stores were mediated by Ca2+ influx through CRAC channels. Both processes were nearly abolished by omitting extracellular Ca2+, by the channel blocker, 2-APB and, importantly, by BTP2 at a concentration that is selective for CRAC/Orai1 channels Following depletion of Ca2+ influx, a hyperpolarizing conductance is required. In microglia, depolarization reduced the Ca2+ rise after UTP stimulation 2+ rise and refilling of stores. A reciprocal relation was seen, wherein KCa3.1 activation required Ca2+ influx and this was mediated by CRAC/Orai1 channels. Based on this functional coupling, we anticipated that KCa3.1 and Orai1 proteins would be in close proximity, and immunostaining showed that both were prevalent and widespread throughout MLS-9 cells. We recently reported a similar Orai1 distribution in primary rat microglia To maintain the driving force for optimal Ca2+, P2Y2 receptors can evoke Ca2+-activated K+ currents in some other cell types. For instance, UTP-stimulation of P2Y2 receptors activated KCa3.1 channels when exogenously co-expressed in Xenopus oocytes 2+ channels is not known. Activation of KCa3.1 by nearby CRAC/Orai1 channels in microglia might reflect a specific coupling in non-excitable cells that do not express or use voltage-dependent Ca2+ channels (VDCCs). In endothelial cells, opening of TRPV4 channels evoked local Ca2+ “sparklets” that activated nearby small- and intermediate-conductance channels that were presumed to be KCa2.3 and KCa3.1 2+ channels (VDCCs), N-methyl d-aspartate (NMDA) receptors, and nicotinic acetylcholine receptors Not surprisingly, by elevating intracellular Cain vivo and in vitro , and blocking Ca2+ entry through CRAC/Orai1 channels inhibited podosome formation and migration Purinergic receptors mediate numerous microglial functions. ATP is considered an important trigger for responses to acute injury, and it increases microglial motility and migration in vitro . The foc2+ in the intracellular (pipette) solution. The EC50 was 7.6 µM Ca2+ and no current was seen at 2.5 µM Ca2+. The UTP-evoked Ca2+ rise peaked at 5.3 µM in the whole cell but is undoubtedly higher adjacent to open CRAC channels. While a range of EC50 values has been reported for activating KCa3.1 channels, they are well below 1 µM Ca2+  was activated by UTP, and channel activation was remarkably insensitive to simply elevating Ca2+ binding to calmodulin, which is bound to the CaM-binding domain (CaMBD) in the proximal C-terminus of each channel monomer 2+; two each in the C- and N-lobes, which are connected by a flexible linker region 2+-dependent gating of SK channels and this produces a Hill coefficient of ∼4 in the Ca2+-dose-response curve 2+ sensitivity in this study reflects fewer functional Ca2+ binding sites. The selective activation of KCa3.1 but not SK3 is intriguing because we recently found that the neuroprotective drug, riluzole, reliably activates both channels in MLS-9 cells 2+ dependence to the left, thereby increasing the probability of channel opening at lower Ca2+ levels. Riluzole reduced the EC50 from 470 to 112 nM Ca2+ for expressed rat SK2 2+2+ for human SK3 and KCa3.1 channels 2+ alone did not activate the currents (present study), and riluzole elevated Ca2+ only to 200 nM 2Y2 receptor activation affects an unidentified accessory molecule that aids in channel activation.SK and KCa3.1 channels are tetramers and are activated by Ca2+ sensitivity is affected by CaM phosphorylation by CK2 protein kinase and PP2A protein phosphatase, which bind to the channels 2+-sensitivity of KCa3.1 channels. While some studies show modulation by ATP in the intracellular solution, there is some conflicting data. The EC50 for channel activation in transfected oocytes was 490 nM Ca2+ without ATP and 320 nM with ATP d was 1.35 µM without ATP and 0.66 µM with ATP d  in our study of KCa3.1 in microglia. KCa3.1 regulation might be cell-type specific. For instance, our early study suggested that CaMKII regulates the endogenous KCa3.1 channel in T cells but not the expressed channel in CHO cells 2+ sensitivity Xenopus oocytes (transfected) and in the T84 cell line (non-transfected) but not in transfected HEK cells 2+ sensitivity 2+ dependence to account for the low sensitivity of the microglial KCa3.1 channel. AMP-activated protein kinase (AMPK) inhibits the current and directly binds to the distal C-terminus of the KCa3.1 protein 3P phosphatase, myotubularin-related protein 6 (MTMR6), interacts with KCa3.1 and inhibits its function by dephosphorylating PI3P near the channel For SK2 and SK3 channels, Ca2+ affinity 2+ sensitivities. Most relevant is the recent discovery of an SK2 variant with three extra amino acids in the CaMBD 2+ sensitivity, with an EC50 of 1 µM rather than ∼300 nM, but no change in the Hill coefficient. Three KCa3.1 variants have been found in rat colon: KCNN4a, KCNN4b and KCNN4c encode proteins of 425, 424, and 395 amino acids, respectively 2+ sensitivities were not examined. A dominant-negative variant found in lymphoid tissues lacks the N-terminus, and because it suppresses normal membrane KCa3.1 expression 2+.A recent study found that CaM-CaMBD interactions change the CaM conformation and increase its Cain vitro and decreased retinal ganglion cell degeneration in vivo2+ sensitivity, and coupling to specific receptors and channels.This work has broader implications because KCa3.1 channels are expressed in numerous cell types (mainly non-excitable), and have been implicated in a range of cell functions, as extensively reviewed in recent years


Biotic and abiotic stimuli induce Ca2+ signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca2+ signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca2+ signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca2+ imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca2+ sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca2+-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca2+ signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca2+ dynamics in intact plants. We describe a simple method to image Ca2+ signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca2+ signals. It is shown that Ca2+ signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.Calcium (Ca Transgenic expression of fluorescence resonance energy transfer (FRET)-based fluorescent Ca2+ reporters such as the popular yellow cameleon (YC) or of the bioluminescent aequorin has permitted non-invasive monitoring of free Ca2+ levels and enabled real-time imaging of Ca2+ levels in different cell-types and organisms, including plants   and aequorin  we describe a simple method that only requires a cooled-CCD camera to visualize Ca2+ signals in plant leaves as, for example, Ca2+ waves propagating along leaf veins of intact plants after imposing a salt stress to roots of these plants. It is concluded that G5A reporter is an interesting alternative to aequorin.Here, (i) we applied G5A in Arabidopsis, (ii) we show that, in comparison to aequorin, G5A enhances G5A construct , for transformation of Arabidopsis thaliana ecotype Col-0 by the floral dip method  were used and compared to transgenic plants expressing aequorin in the cytoplasm  obtained from Prof. Marc Knight .The pGWB502Ω-Aeq and G5A plants were surface-sterilized and placed on half-strength Murashige and Skoog plate medium supplemented with sucrose 1% (w/v) and with hygromycin (15 μg/mL)(G5A) or kanamycin (50 μg/mL)(Aeq), and stratified at 4°C for 2 days in the dark. Seedlings were subsequently grown in a growth chamber at 22°C with a 70% relative humidity, in long-day conditions (150 μE/m2/s light for 16 h a day) for 7 days. These 7-day old seedlings were either used directly or further grown in soil under short day conditions (200 μE/m2/s light for 8 h a day) for 3–7 weeks, as indicated.Seeds from In vivo reconstitution of functional aequorin was performed by incubating 7 day-old seedlings for 4 h at 22°C in the dark with a 2.5 μM aqueous solution of coelenterazine HCP (Interchim). For imaging, coelenterazine-treated seedlings were placed within a dark chamber over a gelosed layer (water with 1% agar) in large Petri dishes. A cooled-CCD camera (Hamamatsu 4880-30), fitted at the top of the chamber, collected photons. Sequential image acquisition was carried out using the Hipic 5.1.0 software with an exposure time per image in the 15–60 s range . Images were analyzed in ImageJ  tubes filled with water through a hole in the cap of the tube to separate leaves from roots. Plants were left for 1 h in the dark at room temperature for recovery. Images of the rosette were acquired as described above for seedlings, with a 30 s integration time. NaCl at a final concentration of 200 mM was injected with a remotely controlled syringe at the root level after 25 min of acquisition.Mature leaves were excised from 6–7-week old plants grown as described above and incubated at 22°C in the dark for 4 h in a 2.5 μM aqueous solution of coelenterazine HCP. The treated excised leaf was then transferred to a dark chamber under the cooled-CCD camera for G5A imaging as described above with 15 s exposure time by frame. For salt stress, 100 μL of 200 mM NaCl was pipetted onto the excised leaf petiole  of each velocity value was plotted with Matlab® software (R2006a).All images were analyzed in ImageJ. Shading correction of all images was performed by subtraction of a dark field image (acquisition without sample) acquired with the same exposure time. Backgrounds of each image were normalized by subtraction of ROIs of non-plant pixels. ROIs of plant pixels were then quantified and the average values are plotted over the time. Background noise after chlorophyll fluorescence decay was determined by imaging light emission from wild-type plant leaves and then subtracted for data obtained from Soluble protein was extracted from 50 pooled seedlings . Wells of 96-well plates were filled with 50 μL of different Ca2+ solutions, of which the free Ca2+ concentration was estimated by MaxChelator Software (http://www.stanford.edu/~cpatton/downloads.htm). To start probe calibration, 50 μL of either G5A or Aeq buffer was dispensed into each well and maximum light emitted per second (L) was measured. In a second step, 100 μL of a 2 M CaCl2 solution was dispensed into each well for discharging the remaining reconstituted G5A or aequorin reporters. Light emitted at this time  allowed the total amount of functional Ca2+ reporter to be estimated. All light measurements were made with a plate-spectrophotometer Victor2 (Perkin Elmer). Collected photons were integrated over 1 s lapses during 180 s. Results are expressed as the ratio ±SE of maximum light over total light .Soluble proteins were extracted from d Mazars  and wered Mazars . So-callG5A construct was expected to yield a broad constitutive expression pattern of the G5A probe. This was checked in a Ca2+-independent manner by direct excitation, at 488 nm, of the GFP moiety of the chimera probe  was estimated at 1.48 ± 0.14.An anti-aequorin polyclonal antibody  was used to evaluate the amount of G5A and aequorin proteins in the soluble protein fraction Figure . This anG5A or Aeq plants. Equal amounts of G5A and aequorin reporters were used for in vitro calibration curves. Data are expressed as maximum light emitted per second (denoted “L”)  ratio  and 0.888 ± 0.015 (R2 = 0.987) for G5A and aequorin respectively . This difference is, however, negligible compared to signal after injection of free Ca2+. At the basal level of cytosolic free Ca2+ (0.1 μM), maximum light level was increased to 6.61 × 103 ± 0.32 × 103 and 1.72 × 103 ± 0.29 × 103 RLU for G5A and aequorin respectively. At 1 μM free Ca2+, light levels increased up to 90.14 × 103 ± 7.44 × 103 and 20.25 × 103 ± 1.65 × 103 RLU for G5A and aequorin respectively. Data expressed as signal/noise ratio for the two reporters  is presented in Figure 2+ levels increased in the petioles. Elevated calcium levels then propagated to the rest of the leaves. It is interesting to note that the Ca2+ responses of mature and young leaves differed in terms of kinetics. Mature leaves responded by an initial rapid, transient, Ca2+ peak  with a maximum at 12–13 min. The young leaves displayed a single rapid Ca2+ transient peak  and the Ca2+ wave had a maximum at 19–20 RLU/30 s  and later in the mesophyll tissue. Quantification of increase in free Ca2+ from the entire leaf shows that salt stress induced two different peaks of Ca2+, which correspond to these two successive episodes of Ca2+ increase, firstly in the basal part of the leaf, subsequently in the rest of the leaf  to travel through a 57 mm-long leaf. Thus, in this example the average velocity was 0.181 mm/s. A further analysis was performed on the primary vein of this leaf  show that Ca2+ signal velocities along different veins  are presented in Table In this example (representative of five independent leaves), elevation of free Cas Figure . Two of s Figure . Analyses Figure . Higher s Figure . Details2+-signaling reporter in plant tissues  may be used to follow various stresses. These experimental conditions allowed the collected information to be ascribed to a given cell-type, without spatial localization of the measured signals.Since the successful use of aequorin as a CaAequoria victoria jellyfish, a naturally evolved BRET phenomenon between aequorin and GFP occurs. Several artificial proteins assembling aequorin with GFP-derived proteins have been engineered to mimic the natural BRET observed in Aequoria victoria. Performance of these artificial Ca2+ reporters depends on the linker motif placed in between the BRET partner proteins  5-fold amplification of light in plants expressing G5A is similar to the 5.7-fold increase in the light signal resulting from BRET between luciferin and GFP in Renilla reniformis  have major absorption peaks between 469 and 490 nm  is attracting increasing interest in the plant biology community  with an acceptably high signal/noise ratio. Under the present experimental conditions, integration time for a frame was well under 1 min: depending on amplitude of the Ca2+ signal this integration time ranged from 30 s  is one of the interesting aspects which could be investigated. Velocity values observed for these Ca2+ signals are of the same order of magnitude than those of “fast” electrical signals  reported to travel leaf tissues in Arabidopsis (Favre et al., 2+ and electrical signaling (Król et al., 2+ channels were reported to play a role in leaf to leaf signaling after wounding (Mousavi et al., 2+ with electrophysiological recording of electrical signals might be a powerful method to decipher the molecular basis of electrical signaling in plants upon different types of stress, including wounding.We consider that G5A reporter opens exciting perspectives for the study of cell-to-cell communication in plants. The physiological meaning of Ca2+ elevation in intact plant leaves, making this probe a promising addition in the toolbox of plant cell physiology. Ca2+ imaging in intact plant leaves with widely affordable imaging equipment has the potential to boost the investigation of Ca2+ signaling in plants.In conclusion, G5A allows to image free CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Odors are rarely composed of a single compound, but rather contain a large and complex variety of chemical components. Often, these mixtures are perceived as having unique qualities that can be quite different than the combination of their components. In many cases, a majority of the components of a mixture cannot be individually identified. This synthetic processing of odor information suggests that individual component representations of the mixture must interact somewhere along the olfactory pathway. The anatomical nature of sensory neuron input into segregated glomeruli with the bulb suggests that initial input of odor information into the bulb is analytic. However, a large network of interneurons within the olfactory bulb could allow for mixture interactions via mechanisms such as lateral inhibition. Currently in mammals, it is unclear if postsynaptic mitral/tufted cell glomerular mixture responses reflect the analytical mixture input, or provide the initial basis for synthetic processing with the olfactory system. To address this, olfactory bulb glomerular binary mixture representations were compared to representations of each component using transgenic mice expressing the calcium indicator G-CaMP2 in olfactory bulb mitral/tufted cells. Overall, dorsal surface mixture representations showed little mixture interaction and often appeared as a simple combination of the component representations. Based on this, it is concluded that dorsal surface glomerular mixture representations remain largely analytical with nearly all component information preserved. Olfaction is considered a synthetic sense, with odors being perceived as unique, individual perceptions. This is most apparent in the case of mixtures, where identification of individual components of a mixture is difficult, especially as the number of components is increased While evidence exists for interactions at the receptor level The majority of these interactions were incidences of mixture suppression, in which the response to the mixture was less than the response to the strongest component. The mechanism for this, while partially peripheral, most likely involves suppression of bulbar responses via lateral inhibition. Within the bulb are two separate populations of inhibitory interneurons that could be responsible. Granule cells located deep within the bulb form reciprocal synapses with mitral/tufted (M/T) cell dendrites and mediate lateral and feedback inhibition of mitral/tufted cell output To date, it is not clear if glomerular level mitral/tufted cell mixture representations of OSN input are analytic or serve as the initial site for synthetic processing with the olfactory system. To address this question, OB glomerular mixture responses were examined using a transgenic mouse line that expresses the GFP-based calcium indicator GCaMP2 in OB M/T cells Odor-evoked GCaMP2 activity patterns were observed across the dorsal surface of the OB . To compAlthough the number of glomeruli did not change, the intensity of individual glomeruli did. Based on a classification system described previously, glomerular responses to binary mixtures were placed into one of three groups: suppression, hypoadditivity, or synergy Experimentally derived mixture response maps were then compared to the calculated sum of the activity maps for the individual components . In all The mean odor-evoked relative change in fluorescence (ΔF/F) for each glomerulus was then measured in response to the binary mixture and to each component. Similar to the odor maps, the predicted sum was calculated by summing the responses to both components. The ratio of the mixture response to the summed response was then calculated for each glomerulus. In this way, a value of 1 would indicate that the measured mixture response was identical to the calculated sum. Across all glomeruli (n = 343), the mean mixture-to-calculated sum ratio was found to be significantly less than 1 . For further analysis, all glomeruli were divided into two groups: those that only responded to one component of the mixture (non-overlapping glomeruli) and those that responded to both components (overlapping glomeruli). For overlapping glomeruli (n = 260), the mean mixture-to-sum ratio was also found to be significantly less than 1 . For non-overlapping glomeruli n = 87), mean mixture-to-sum ratio was not significantly different than 1  . These dAccording to the previous anatomical and functional studies Similar results were obtained in MV/BA mixtures . As a grAs the glomerular layer is the site of information transfer from receptor neuron to output neuron within the bulb, it provides an ideal place to investigate if the complex mixture interactions observed in mitral cells can arise from lateral processing in the early stages of the OB. With the odorants and concentrations used here, postsynaptic mixture-evoked maps appeared to reflect a simple combination of the maps evoked by each individual component with very little mixture interaction observed.Recent studies using intrinsic optical imaging in the rodent to investigate mixture interaction at the level of OSN input into the bulb have reported that glomerular responses to binary mixtures can be predicted by the linear sum of the individual component responses in vivo, in which a majority of neuron activity displayed little interaction to binary mixtures The high incidence of hypoadditivity observed in this study also suggests that there is very little mixture interaction in the early postsynaptic stages of OB processing. These results are similar to reports of mixture responses in vertebrate olfactory sensory neurons Overall, the rarity of mixture suppression observed is surprising given the extensive presynaptic and postsynaptic inhibitory networks that exist in the glomerular layer However, some evidence of mixture interaction was observed, as a small number of incidences of mixture suppression did occur. These were most frequently seen in the border glomeruli of MV/BA mixtures. It is not clear if these instances of suppression reflect postsynaptic processing by glomerular inhibitory circuits or interactions at the peripheral OSN level. A recent imaging study using the OMP-synapto-pHluorine mouse, that expresses an indicator of presynaptic OSN transmitter release, also found little mixture suppression using similar odor combinations Overall, little mixture interaction was observed in olfactory postsynaptic glomerular responses to the odorants used in this study. In most cases, postsynaptic glomerular binary mixture representations appeared to be a combination of the component representations, regardless of the degree of overlap between the components. This suggests that at the glomerular level, M/T cell mixture representations are largely analytical and retain nearly all of the component information. These findings support recent behavioral studies showing that individual component odorant input patterns cannot be used to predict mixture quality Experiments were performed on adult transgenic mice expressing the fluorescent calcium indicator GCaMP2 under the Kv3.1 voltage-gated potassium channel promoter Imaging was performed using an Olympus BX50WI microscope equipped with 4x (0.28 NA) and 10x (0.3 NA) objectives. The OB was illuminated with a Polychrome II monochromator with an excitation wavelength centered at 480 nm. The G-CaMP2 fluorescence signal was band-passed filtered with a Chroma emission filter (HQ535/50) and recorded with a cooled, back-illuminated CCD camera (Redshirt Imaging) at 25 Hz with a 256×256 resolution. Image acquisition and analysis was performed using NeuroPlex software (Redshirt Imaging) and Matlab.et al. 2009). Odorant were delivered for 2 seconds with an inter-stimulus interval of at least 60 seconds. Each odorant was presented separately and then together as a mixture. The final concentration of the mixture was the sum of the concentrations of its components. Odorant concentrations used were between 0.06%–1% of saturated vapor concentration. Odorants used were: propanal, butanal, pentanal, amyl acetate, butyl acetate (BA), ethyl butyrate (EB), 2-heptanone (2H), and methyl valerate (MV). Mixtures consisted of 15 different combinations of odorants and concentrations.Odorants were delivered using a flow-dilution olfactometer as described previously . The odor-evoked change in fluorescence was calculated by subtracting the average of 5 frames preceding stimulus onset from the average of 5 frames centered on the peak of the response generated by the first full respiration following odor onset. The relative change in fluorescence (ΔF/F) was calculated by dividing the odor-evoked change in fluorescence by the resting fluorescence and expressed in pseudocolor. All maps are the average of several presentations of each odorant. The predicted mixture map was created by summing the corresponding pixel values from the experimentally derived component maps. The predicted map was then subtracted from the experimentally measured response to produce a difference map.For quantitative analysis of individual glomeruli, the odor-evoked response was calculated from the spatial average of 4 to 9 pixels located at the center of each glomerulus. Responses were averaged over several trials (2–8) to obtain a final mean ΔF/F value for each glomerulus for each odorant. Only glomeruli that displayed clear responses to least one component or the mixture were used for analysis. A glomerulus was considered to respond to an individual odorant if its mean ΔF/F response was greater than the background ΔF/F signal. Background signals were defined as the mean±2SD ΔF/F value obtained from regions containing no odor-evoked activity.For each glomerulus, the component that elicited the smaller response was designated as the weak component (WC) and the component the elicited the larger response was designated as the strong component (SC). The predicted mixture response was calculated by summing the responses of the two components. For each glomerulus, the experimental mixture response was then divided by the predicted response. It is possible that in some cases the predicted mixture response for a given glomerulus could be beyond the maximum response amplitude for that glomerulus. To avoid any potential confounds due to saturation, all odorants were given at low concentrations (0.06–1% s.v). In anesthetized GCaMP2 mice, these concentrations elicit ΔF/F responses that are well below the saturation level of the GCaMP2 indicator. With the concentrations used in this study, the average sum of the component responses for all glomeruli was 4.6±0.1% ΔF/F. When the same odorants are delivered at high concentrations (>10%), individual glomerular responses can often be as high as 20% ΔF/F. Based on this, it is unlikely that the sum of component responses for a given glomerulus in this study would be greater than its saturation point.To confirm this, in a subset of glomeruli in which the predicted response was larger than the mixture response, we compared the predicted (summed) mixture response to the response elicited by the strong component presented at higher concentrations than in the mixture. In 90% of the cases, higher concentrations of the strong component resulted in glomerular responses that were larger in amplitude than that of the summed responses. This suggests that at the low concentrations used here, the lack of additivity is not due to saturation of the glomerular response. However, in some cases, it is possible that saturation of the glomerular signal at lower concentrations would to an overestimation of number of glomeruli displaying mixture responses that were less than the calculated sum.To identify mixture suppression, a suppression ratio (SR) was calculated for each glomerulus by dividing the mixture response by the strong component response. The response to a mixture was then compared to the response elicited by the strongest component. Glomeruli were then placed into one of three separate categories: suppression (mixture response<SC response), hypoadditivity (mixture response = SC response), or synergy (mixture response >SC response)


The aim of this study was to determine how InsP3 receptors can translate Ca2+ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca2+ release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element) and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP3 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca2+ transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca2+]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1  or phenylephrine , respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca2+ released from InsP3Rs is more effective than Ca2+ released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca2+ release efficiently into a depolarization of the membrane potential.The functional role of inositol 1,4,5-trisphosphate (InsP In contrast InsP3R-mediated signaling has been linked to excitation-transcription coupling. Activation of nuclear InsP3Rs was sufficient for the activation and translocation of the transcription factor HDAC that remained unresponsive to beat-to-beat changes in [Ca2+]i3Rs play a role in the induction of cardiac arrhythmia. Stimulation of InsP3R-mediated Ca2+ release results in increased spark frequency, positive inotropy, and an increase in arrhythmic spontaneous activity in atrial and ventricular myocytes 3-mediated Ca2+ release appears low and may be more relevant as a facilitator of Ca2+ release from RyRs thus contributing indirectly to excitation-contraction coupling.In contrast to the abundance of InsP3-mediated Ca2+ release could critically influence its function. Whereas sub-sarcolemmal Ca2+ release can depolarize the membrane by activation of sodium calcium exchange (NCX), Ca2+ released at the nuclear envelope might have a higher likelihood to be removed by SERCA 2+ signaling events are very pronounced in ESdCs. Localized Ca2+ release events through RyRs (sparks) can be frequently monitored throughout the ESdC, whereas localized release events through InsP3Rs (puffs) are seldom identified 3 mediated Ca2+ release can sustain spontaneous activity even after depletion of the RyR operated Ca2+ stores or in RyR2 deficient ESdCs The sub-cellular location of InsP3Rs close to the plasma membrane form functional signaling domains with NCX and that, in contrast to cytoplasmic or nuclear InsP3Rs, their Ca2+ release can be efficiently translated into INCX and a depolarization of the membrane potential (Vm). For this purpose we determined the effect i. of InsP3R-mediated release on INCX and ii. of spatial inhomogeneities in InsP3 concentration on spontaneous activity We used ESdCs as a model to test the hypothesis that InsPThe culture of mouse embryonic stem cells (mES) of the cell line CMV , their differentiation into cardiomyocytes and use for laser scanning confocal microscopy are described in detail elsewhere 3 biosensor was assembled using the InsP3R ligand-binding domain terminally fused with enhanced CFP and YFP at the amino and carboxyl termini, respectively. In FIRE-1 transfected COS-1 cells, rat neonatal, adult cat ventricular myocytes and ESdCs (As previously described nd ESdCs  FIRE-1 eGGCTCGAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTATCTCGAAGG and antisense: CCCTCGAGATACCTTTCTCTTCTTTTTTGGATCTACCTTTCTCTTCTTTTTTGGATCTACCTTTCTCTTCTTTTTTGGATCTCGAGCC). The oligonucleotides were mixed and annealed by incubation at 90°C for 5 minutes and then cooling to room temperature, followed by digestion with Xho I and ligation into similarly digested FIRE-1 plasmid 3R1 specific amino-terminal antibody  and direct visualization with fluorescence microscopy. The insert harboring the FIRE-1nuc coding region was excised with Bgl II and ligated into Bgl II digested pShuttle-CMV vector  for adenoviral production.The FIRE-1 indicator was targeted to the nucleus by insertion of a triplet tandem of the SV40 large T-antigen nls using the following oligonucleotides: (sense: CGGGTCGACCCACCATGGACTACAAGGACGAC and antisense: GCCGTCGACTCACTGCACGACACAACA). The PCR product was digested with Sal I, ligated into similarly digested pCMV-5 vector, and expression was verified in COS-1 cells by transient transfection and Western blotting with anti-FLAG antibody (Affinity BioReagents) (The coding region of the mouse 43 kDa inositol polyphosphate 5′-phosphatase (m43) with N-terminal FLAG-tag from pcDNA3  was amplified by PCR using the following primers (sense: eagents) . The FLAin vivo homologous recombination with either pShuttle-CMV-m43 or pShuttle-CMV-FIRE-1nuc. The pAdEasy-1-m43 or pAdEasy-1-FIRE-1nuc insert containing plasmids were separately transformed into DH5α and produced in bulk. Purified pAdEasy-1-m43 or pAdEasy-1-FIRE-1nuc was used to transfect AD-293 cells for virus amplification. Both viruses were plaque-purified, amplified, CsCl gradient-purified, and stored at −80°C.The adenoviruses were created using the commercially available AdEasyTM XL adenoviral vector system kit . Briefly, the bacterial cell line BJ5183-AD-1, pre-transformed with the plasmid pAdEasy-1 was used for 3 biosensor FIRE-1 F488) and >560 nm (F560), respectively. Changes in InsP3 activity are defined as the relative change in the background corrected ratio of F560/F488. To obtain a reliable reproducible readout for the changes induced by the pharmacological agents, the fluorescence was determined after 3 min of superfusion. The change was then quantified as the average fluorescence over the time period of 5 min. The experiments were conducted at room temperature. FRET between CFP and YFP was confirmed by photobleaching of the accepter molecule , U73122 and U73343 were dissolved in dimethylsulphoxide (DMSO) and further diluted >1,000 fold for experiments. All chemicals were purchased from Sigma. Results are presented as mean ± SEM and n represents the number of experiments. Statistical differences between two groups were analyzed by student's t-test and considered significant at P<0.05. Multiple comparisons were performed by analysis of variance (ANOVA) and significant differences between the groups were identified with the Tukey HSD Test indicating significance at P<0.05. A detailed description of the confocal imaging, electrophysiological recordings, and immunocytochemistry can be found in Endothelin-1 (ET-1), phenylephrine (PE), and caffeine were diluted in H3-mediated Ca2+ release plays a critical role in the generation of spontaneous activity in ESdCs 2+]i correlate with changes in membrane voltage (Vm) we recorded action potentials (APs) in Fluo-4/AM loaded ESdCs with the perforated patch technique. As shown in 2+]i closely correlated with changes in Vm showing a clear increase in basal [Ca2+]i during the diastolic depolarization. This increase in [Ca2+]i was spatially homogeneous and did not correlate with a specific location inside the cell e.g. the nuclear envelope 3Rs in ESdCs, cells were stained with antibodies against InsP3Rs type-1 and type-2. As shown in 3R isoforms. Pronounced peri-nuclear staining was identified, together with extensive endoplasmic reticulum staining throughout the cell that extended to the plasma membrane. This localization pattern suggests that InsP3R-mediated Ca2+ release is not restricted to the nuclear envelope.In our previous study we demonstrated that InsP2+ transients have been recorded already when the cells first develop spontaneous activity 3Rs control different functional pools of Ca2+ stored in the SR we superfused ESdCs with ET-1 (100 nmol/L) after the caffeine sensitive stores were depleted . FIRE-1 exhibits an increase in the fluorescence ratio (F560/F480) upon binding of InsP33 production in ESdCs was stimulated by ET-1 (100 nmol/L) or PE (10 µmol/L) a positive chronotropic effect was determined in the frequency of the spontaneous Ca2+ transients (560/F488) was determined that reached a steady state after about 2.5 min of superfusion. For ET-1, a 3.7±0.6% change (560/F488 (3 production. U73343 (1 µmol/L) the inactive analog of U73122 remained without effect  and 18.6±0.36% (n = 2), and 27.05±1.5% (n = 3), respectively 2+-free, n = 3; 2-APB: 2 µmol/L, n = 3; or BAPTA/AM: 1 µmol/L, n = 2) was a change in the FRET ratio (F560/F488) measured indicating that changes in FIRE-1 did not depend on changes in [Ca2+]i or spontaneous activity.To determine the spatial organization of InsP% change ; n = 4 i% change ; n = 2. % change . When FI560/F488 ; n = 5 wt effect ; n = 2. er 2-APB , Ca2+-frsolution  or BAPTAsolution  the flor2+ transients was attenuated  compared to non-transduced cells  of the same age (day 16). The results indicate that the InsP3 buffer capacity of FIRE-1 in ESdCs reduces the beating frequency. To determine if frequency regulation through InsP3-mediated Ca2+-release depends on defined InsP3 signaling domains we employed two different strategies. First we limited peri-nuclear and nuclear InsP3 signaling by expression of FIRE-1 with a nuclear localization sequence (FIRE-1nuc), second we limited sub-sarcolemmal InsP3 signaling by over-expression of the membrane associated inositol polyphosphate 5-phosphatase m43 3 by removing the 5′ phosphate FIRE-1 transduction changed the spontaneous activity in ESdCs. The overall number of spontaneously active cells was reduced and in beating cells the frequency of spontaneous CaThe spatially defined localization of both FIRE-1nuc and m43 was confirmed by adenoviral transduction of the two constructs in atrial and ventricular myocytes as well as ESdCs. FIRE-1nuc was readily identified by its YFP fluorescence and the m43-phosphatase was visualized with an antibody against the incorporated FLAG-tag. 3 signaling effects the spontaneous activity of ESdCs we measured [Ca2+]i in cells expressing m43 or FIRE-1nuc 24 hours post adenoviral transduction. 2+ transients in an ESdC expressing FIRE-1nuc. Non-transduced 14 days old cells obtained from the same isolation served as control. Control ESdCs and ESdCs transduced with FIRE-1nuc exhibited no significant difference in their Ca2+ transient frequency  in control and 24±1%  in FIRE-1nuc transduced ESdCs. The data indicate that the ET-1 induced positive chronotropic effect persists when InsP3R is buffered in the nucleus of ESdCs. In contrast, cells transduced with m43 exhibited a significantly reduced beating frequency in comparison to control and FIRE-1nuc cells Click here for additional data file.Figure S1A. Fluorescent images of an ESdC taken at >560 nm (top) and 488 nm (bottom) before (left) and after bleaching (right). B. Bar graphs display the change in CFP (right) and decrease of YFP (right) fluorescence after photobleaching . The results are comparable to bleaching experiments in FIRE-1 expressing COS-1 cells (TIF)Click here for additional data file.Figure S2Western blot of the cytoplasmic (soluble) and membrane fraction (pellet) of M43 transfected COS cells. Blots probed with the anti-tag antibody show positive M43 immunostaining only in the membrane fraction.(TIF)Click here for additional data file.


We investigated a panel of toxins -salane complexes) in four different mammalian cell lines covering three different species . All tested compounds induced an increase in free cytosolic Ca2+ within the first 5 s after toxin application. Cytosolic Ca2+ shifts occurred independently of the chemical structure in all tested cell systems and were persistent up to 3 h. The linear increase of free cytosolic Ca2+ within the first 5 s of drug treatment correlates with the EC25 and EC75 values obtained in Alamar Blue assays one day after toxin exposure. Moreover, a rise of cytosolic Ca2+ was detectable independent of induced cell death mode as assessed by caspase and poly(ADP-ribose) polymerase (PARP) activity in HeLa versus MCF-7 cells at very low concentrations. In conclusion, a cytotoxicity assay based on Ca2+ shifts has a low limit of detection (LOD), is less time consuming (at least 24 times faster) compared to the cell viability assay Alamar Blue and is suitable for high-troughput-screening (HTS).The determination of the cytotoxic potential of new and so far unknown compounds as well as their metabolites is fundamental in risk assessment. A variety of strategic endpoints have been defined to describe toxin-cell interactions, leading to prediction of cell fate. They involve measurement of metabolic endpoints, bio-energetic parameters or morphological cell modifications. Here, we evaluated alterations of the free cytosolic Ca The development of assays estimating the cytotoxic potential of drugs and chemicals is of fundamental interest in early risk assessment to prioritize them for further testing. Moreover, a few years ago, the European Union (EU) initiated a regulation for the Registration, Evaluation and Authorisation of Chemicals (REACH). Around 30 000 chemical substances, which are manufactured, imported or, used in the EU require validation ,2. The iIn the past, a variety of different biological endpoints have been defined for cytotoxicity testing. These include the assessment of energy status , cell membrane integrity  leakage), DNA-strand breaks (COMET) as well as metabolic parameters -2,5-diphenyltetrazolium bromide (MTT), Alamar Blue) [2O3), gossypol, hydrogen peroxide (H2O2) and staurosporine in Alamar Blue assays with toxin-induced elevations of cytosolic Ca2+  oxidizing macromolecules, and a disturbed endoplasmic reticulum function [1. Asfunction -13. Howe2. The racematic organic compound gossypol isolated from cotton seed and its metabolites display a wide pattern of cytotoxic cell alterations because of the complexity of gossypol chemistry and its potential chemical reactions with other macromolecules. Gossypol cytotoxicity includes ROS induction, microsomal enzyme inhibition, glutathione-S-transferase inhibition, mitochondrial dysfunction, caspase dependent and independent cell death associated with DNA degradation, and was described to interfere with the anti-apoptotic bcl-2 protein -18.2O2 is used as surrogate for ROS. It oxidizes directly macromolecules including lipids, proteins and DNA. This can lead to a complex cytotoxicity response with the involvement of stress activated kinases, caspase and calpain activation, mitochondrial apoptosis induction factor (AIF) translocation, endoplasmic reticulum stress, nuclear poly(ADP-ribosylation), DNA degradation and many more [3. In this study, Hany more -22.4. The bacterial alkaloide staurosporine is intensively investigated as inducer of a classical apoptotic cell death. It was initially described as an inhibitor of protein kinases -25. On cThe Alamar Blue assay was considered as a benchmark cytotoxicity test because of its improved performance compared to other pertinent assays, e.g. detection of cell densities as low as 200 cells/well ,30. More2+ were investigated using the fluorescent Ca2+ binding dye Fluo-4 during the application of four toxins in all cell lines . Multipfunction . It is wll death .2+ assay. Sensitivity in our setting is defined as the ability to detect a significant effect of the used compounds at a specified concentration, whereas LOD is the lowest concentration level determined to be statistically different from blank. Here we show that As2O3, gossypol, H2O2 and staurosporine induce a dose-dependent increase in cytosolic Ca2+ at lethal (EC75) and sublethal (EC25) concentrations immediately after application in all tested cell lines. The cytosolic Ca2+ elevation follows linear kinetics for the first 5 s under all test conditions. Cytosolic Ca2+ shifts occur independent of the chemical structure of the toxin in all tested cell systems and are persistent up to 3 h. Moreover, the increase of free cytosolic Ca2+ is detectable independent of the mode of cell death as investigated by caspase and PARP activity. Therefore, we suggest the determination of early cytosolic Ca2+ shifts as a rapid, highly efficient, inexpensive cytotoxicity test that is at least as sensitive as the established metabolic assay Alamar Blue.In our study, we focussed on the sensitivity, the species-specificity and the limit of detection (LOD) of the Fluo-4 Ca2+ was assessed using the fluorescence dye Fluo-4  after its intracellular bio-activation by esterases [Cytosolic Ca4 Figure B,C. Thissterases . Therefo2+ homeostasis. The cytosolic Ca2+ levels remained unaffected for the whole measuring period in the absence of a toxic insult  as well as EC75 (100 μM) induced cytosolic Ca2+ shifts in HeLa cells  and 2 mM (EC75) of H2O2 were analyzed regarding cytosolic Ca2+ imbalances. A dose dependency in the cytosolic Ca2+ response was already significant within the first 5 s of measurements  and 1 μM (EC75) determined in Alamar Blue assays correlate with linear increases in cytosolic Ca2+ levels for the first 5 s of Fluo-4 measurements -salane complexes  were analyzed in Fluo-4 assays directly after application. Cytoplasmic Ca2+ was not altered in untreated control MCF-7 cells within 2 h . The cytoplasmic Ca2+ slopes of the tested toxin concentrations were dose dependent and the RFUs at 5 s  and staurosporine (0.5 μM and 4 μM) – concentration indicative of sublethal and lethal cell stress – were analysed in a similar way , H25 and EC75 values for As2O3, gossypol, H2O2 and staurosporine as assessed in Alamar Blue assays  and lethal (100 μM) concentrations of Ass Figure A. A dose2+ following drug treatment was linear for both concentrations analysed in a dose dependent manner until 5 s post application. The fluorescence units were significantly different between 75 μM and 100 μM gossypol at this time point (1.4±0.71 RFU versus 4.2±1.12 RFU). The difference in rise of cytosolic Ca2+ levels seen at 5 s was consistently maintained during the whole period of observation  and EC75 (10 mM) for H2O2 in Vero 76 cells as assessed in Alamar Blue viability assays were investigated in Fluo-4 assays  and lethal (500 nM) concentrations (1.1±0.43 RFU versus 5.0±0.83 RFU) evident still at 3 h after drug application  of the four test compounds, caspase 7 and 9 were only processed in HeLa cells into their active form as assessed by Western blot analysis 4 h after treatment  from mitochondria to the nucleus, leading finally to cell death [75) challenges with As2O3, gossypol, H2O2 and staurosporine in both HeLa  glycohydrolase (PARG) are responsible for extracellular Ca2+ gated by transmembranous transient receptor mediated Ca2+ channel (TRPM2). On the other hand, free cytosolic Ca2+ origins also from intracellular sources. For instance, protein markers of endoplasmic reticulum (ER) stress were detected pointing to Ca2+ released from ER stores in parallel. Blocking the influx of Ca2+ protected the cells from oxidative insults.In recent publications, we reported a correlation between cytosolic Cae stress ,21. Usin2+ shifts are generally predictive of cytotoxicity, we investigated here a wide spectrum of toxins in cell lines from different species origin. The toxicity of arsenic trioxide, hydrogen peroxide, gossypol and staurosporine was tested in human, mouse, and monkey cells using Alamar Blue assay. These compounds have different cellular targets and induce different cell death pathways, ranging from general macromolecule damage, especially to DNA, by oxidative stressor H2O2 to the apoptotic model compound staurosporine, which has been shown to inhibit a wide spectrum of kinases without damaging DNA. The toxicity data were compared to cytosolic Ca2+ measurements at the respective sublethal (EC25) and lethal (EC75) doses. Our fluorimetric assay revealed in all settings a rapid rise in cytosolic Ca2+, regardless of species-origin and toxin applied. Moreover, it has a low LOD. Thus, our data provide evidence that Ca2+ shifts are a common denominator in cytotoxic insults, independent of the mode of cell death. Interestingly, this can be monitored with an unmatched speed and at doses that show hardly significant changes in cell viability assays. Even sublethal (EC25) toxin concentrations generated slopes of free cytosolic Ca2+ increases significantly different from solvent controls indicative for the superior sensitivity of the Fluo-4 Ca2+ assay. Moreover, this assay discriminates between structurally closely related titanium(IV)-salane complexes, i.e. toxic TC52 and non-toxic TC53. In an additional data set, we tested the toxicity of a physiological compound, i.e. ATP. High extracellular concentrations have been reported to induce cell death [2+ shifts in our assay after application of ATP in a similar setting as before (EC25 and EC75). However, low dose extracellular ATP induces Ca2+ shifts if cells express members of P2X and P2Y transporter family, as it is the case in HeLa cells [2+ dependent signaling processes. In contrast, engineered substances inducing a rise in free cytosolic Ca2+ as presented in this study are indicative of unwanted biological effects. Therefore we conclude that cytosolic Ca2+ increases within the first 5 s of exposure as measured with Fluo-4 dye are predictive of the cytotoxic potential of a xenobiotic compound.In order to see whether Call death ,39-45. ILa cells . TherefoLa cells . To avoi2+ increase in the first 5 s of exposure shows the same or even higher statistical predictivity than the standard Alamar Blue assay. Thus, this fluorimetry-based method is a rapid predictor of cytotoxicity, superior to other assays in speed and cost effectiveness.Our newly developed assay is applicable in cells from different species and with a wide variety of toxins, acting on different signaling pathways and modes of cell death. Measuring the free cytosolic Ca2) atmosphere, in complete Dulbecco’s modified Eagle’s medium  containing 1 g/L glucose and supplemented with 10% (v/v) FBS and Penicillin/Streptomycin .In this study HeLa, immortalized mouse embryonic fibroblasts, MCF-7 and Vero 76 cells were investigated Figure B. All ceN-(2-Quinolyl)valyl-aspartyl-methylketone (Q-VD-OPh) was from Calbiochem . N--N,N-dimethylacetamide, HCl (PJ-34) was obtained from ENZO Life Sciences . HOECHST 33342 was from Invitrogen. Titanium(IV)-salane complexes TC52 and TC53 were both synthesized in the Chemistry Department (Thomas Huhn Group) of University of Konstanz/Germany. All other chemicals were from Applichem , Fluka , Merck  or Sigma. All chemicals used as inhibitors were simultaneously administered with toxin treatment.2O2  diluted in OPTI-MEM I (Gibco) to the desired concentration. After 1 h, 3 parts (150 μL) complete D-MEM were added. Gossypol (Sigma) was dissolved in DMSO to a stock solution of 100 mM. Then diluted in OPTI-MEM I to the desired concentration. Staurosporine  and As2O3  were diluted in D-MEM directly to the concentration needed. TC52 and TC53 were dissolved in DMSO to a stock solution of 2.5 mM and diluted in D-MEM to the desired concentration. ATP Mg2+ salt (Sigma) was diluted in PBS supplemented with 2 mM Ca2+ to the concentration needed. After 30 min of treatment the ATP solution was replaced with complete D-MEM. All toxin treatments were maintained without any alterations until the end of the experiment.Cells were challenged with 1 part (50 μL) HCells were seeded in 96-well-plates (15 000 cells/well) and incubated overnight  supplemented with 1 part 1 M HEPES (pH 7.2) (Assay Buffer) containing CaCl2 or not. 100 μL Fluo-4-NW-dye-mix from Molecular Probes (Invitrogen) was added and incubated for 30 min at 37°C, followed by 30 min incubation in the dark at room temperature  in LS55 luminescence spectrometer (Perkin-Elmer) after toxin treatment. Stock solutions of toxins were diluted in Assay Buffer to the desired concentration. Free cytosolic Ca2+ was monitored for the indicated time with a measure frequency of 1 s or less.This was performed as described before . BrieflyImmunoblots were performed as described previously . The folst antibody (diluted 1:200 in Blocking Buffer). After an incubation for 1 h at 37°C, coverslips were washed three times with TBST (each 5 min), followed by a 2nd antibody incubation  for 1 h at 37°C in the dark. Afterwards, probes were washed three times with TBST (each 5 min). DAPI staining was performed for 5 min and coverslips were washed with H2O and dried afterwards. The samples were further processed with ProLong Antifade kit (Invitrogen) according to the manufacturer’s protocol and analyzed with a fluorescence microscope (Nikon) connected to a digital camera .Cells were seeded on coverslips  in 24-well-plates (Costar Corning Incorporated) and let attach overnight. The toxin treatment was performed in D-MEM for 5 min. Cells were fixed with ice-cold methanol and stored at −20°C for 7 min. Coverslips were subsequently washed twice with 1xTris buffered saline  and incubated with Blocking Buffer (1xTBS/0.2% Tween 20 (TBST), 1% BSA) for 30 min at 37°C. Monoclonal 10H anti-poly(ADP-ribose) (PAR) antibody  was usedIf not stated differently, all results are shown as mean±SD of the indicated number of independent experiments. All statistical analyses were calculated with Prism Software 5.0b .The authors declare that they have competing interests. A patent application protecting the invention has been filed (EP 12/187234).PW, CB and TP planned and performed the experiments. All authors analysed the data. PW, CB, SB and FRA wrote the manuscript. All authors read and approved the final manuscript.Control measurements of Fluo-4 free cytosolic calcium assay. (A) HeLa cells . (B) MCF-7 cells . (C) Murine fibroblasts . (D) Vero 76 cells . (E) Ca2+ shift endpoint at 5 s after 1 μM, 2 μM or 5 μM As2O3 with  compared to control in HeLa cells. Ca2+ shift endpoint at 5 s after 5 μM, 10 μM or 75 μM gossypol with  compared to control. Ca2+ shift endpoint at 5 s after 100 nM, 200 nM or 400 nM staurosporine with  compared to control. (F) Alamar Blue endpoint at 24 h after 1 μM, 2 μM or 5 μM As2O3 with  compared to control in HeLa cells. Alamar Blue endpoint at 24 h after 5 μM, 10 μM or 75 μM gossypol with  compared to control. Alamar Blue endpoint at 24 h after 100 nM, 200 nM or 400 nM staurosporine with  compared to control. (G) Chemical structures of the investigated compounds TC52 and TC53. (H) Alamar Blue endpoint at 24 h after 4 μM or 10 μM TC52 or 10 μM TC53 with  compared to control in HeLa cells (I) Ca2+ shift endpoint at 5 s after 4 μM or 10 μM TC52 or 10 μM TC53 with  compared to untreated control in HeLa cells.Click here for fileImpact of toxic compounds on cytosolic Ca2+ levels in HeLa cells. (A) Ca2+ shifts after 5 μM or 50 μM As2O3 with  at 1800 s. (B) Ca2+ shifts after 75 μM or 100 μM gossypol with  at 1800 s. (C) Ca2+ shifts after 0.5 mM or 2 mM H2O2 with  at 180 s and  at 1800 s. (D) Ca2+ shifts after 400 nM or 1000 nM staurosporine with  at 1800 s.Click here for fileAssessment of ATP-induced toxicity in HeLa cells. (A) Alamar Blue assay in presence of ATP as indicated . (B) Ca2+ shifts after 25 mM or 40 mM ATP . (C) Statistical evaluation of 25 or 40 mM ATP treated HeLa cells in Fluo-4 analyses .Click here for fileImpact of toxic compounds on cytosolic Ca2+ levels in MCF-7 cells. (A) Ca2+ shifts after 20 μM or 50 μM As2O3 with  at 10800 s. (B) Ca2+ shifts after 60 μM or 75 μM gossypol with  at 1800 s. (C) Ca2+ shifts after 5 mM or 10 mM H2O2 with  at 1800 s. (D) Ca2+ shifts after 200 nM or 400 nM staurosporine with  at 180 s and  at 1800 s and  at 10800 s.Click here for fileImpact of toxic compounds on cytosolic Ca2+ levels in murine fibroblasts. (A) Ca2+ shifts after 45 μM or 50 μM As2O3 with  at 180 s. (B) Ca2+ shifts after 75 μM or 100 μM gossypol with  at 180 s and  at 1800 s. (C) Ca2+ shifts after 0.5 mM or 5 mM H2O2 with  at 180 s and  at 1800 s. (D) Ca2+ shifts after 500 nM or 4000 nM staurosporine with  at 180 and 1800 s.Click here for fileImpact of toxic compounds on cytosolic Ca2+ levels in Vero 76 cells. (A) Ca2+ shifts after 35 μM or 100 μM As2O3 with  at 10800 s. (B) Ca2+ shifts after 75 μM or 150 μM gossypol with  at 180 s with  at 1800 s and  at 10800 s. (C) Ca2+ shifts after 8.5 mM or 10 mM H2O2 with  at 1800 s. (D) Ca2+ shifts after 200 nM or 500 nM staurosporine with  at 10800 s.Click here for file