Stromal interaction molecule (STIM) proteins regulate store-operated calcium entry (SOCE) by sensing Ca2+ concentration in the ER and forming oligomers to trigger Ca2+ entry through plasma membrane-localized Orai1 channels. Here we characterize a STIM2 splice variant, STIM2.1, which retains an additional exon within the region encoding the channel-activating domain. Expression of STIM2.1 is ubiquitous but its abundance relative to the more common STIM2.2 variant is dependent upon cell type and highest in naive T cells. STIM2.1 knockdown increases SOCE in naive CD4+ T cells, whereas knockdown of STIM2.2 decreases SOCE. Conversely, overexpression of STIM2.1, but not STIM2.2, decreases SOCE, indicating its inhibitory role. STIM2.1 interaction with Orai1 is impaired and prevents Orai1 activation, but STIM2.1 shows increased affinity towards calmodulin. Our results imply STIM2.1 as an additional player tuning Orai1 activation in vivo.Cellular homeostasis relies upon precise regulation of Ca STIM proteins sense calcium depletion in the endoplasmic reticulum and in response activate calcium influx through Orai1 channels located at the plasma membrane. Here, Miederer et al. identify a novel splice variant of STIM2 that fails to interact with and activate Orai1 and may act to fine-tune cellular calcium homeostasis by negatively regulating calcium influx. These diverse functions require tight control and regulation of Ca2+ influx. In immune cells, the major influx pathway for Ca2+ is encoded by components of the store-operated calcium entry (SOCE) machinery. Here, CRAC (Ca2+ release-activated Ca2+) channels encoded by the Orai genes (Orai1-3) are activated by endoplasmatic reticulum (ER)-localized stromal interaction molecule (STIM) (STIM1 and STIM2) proteins when the Ca2+ concentration of the ER  and the plasma membrane Ca2+ ATPase (PMCA) as critical components with downregulation of STIM2 decreasing basal Ca2+ and downregulation of CaM or plasma membrane Ca2+ ATPase increasing basal [Ca2+] , immediately upstream of the sequences essential for binding of Orai1  leading to an upstream end of translation and a transcript shortened by 444 bp (∼17 kDa)  flanking exon 9. + T cells as well as in Jurkat T cells and primary monocytes. Exon 9-specific quantitative reverse transcription (RT)–PCR primers were derived  and of STIM2.2  were tested using template complementary DNA (cDNA) of naive and stimulated CD4+ T cells from at least three different primary human blood donors. As also indicated by conventional PCR  is detected in naive CD4+ and CD8+ T cells. cDNA from glioblastoma samples (12 patients) showed the highest expression of STIM2.2 with little STIM2.1. In summary, we could detect expression of STIM2.1 in all tested human cell lines and primary cells, although expression was always lower than STIM2.2. Alignment of the exon 9 amino acids (VASSYLIQ) shows a high degree of conservation between different species. We also tested for STIM2.1 (VAASYLLQ) expression in lymphocytes from Mus musculus and were able to detect two bands by conventional PCR  . All rep derived , PCR proinset in , STIM2.1 contact . A reduced cells . We procpression . Highestonal PCR .+ and CD8+ T cells showed the highest absolute expression of STIM2.1 (2−ΔCq: 0.68±0.35), with an average ratio of STIM2.2/STIM2.1 expression of 1.6±0.4 (5 donors) for naive CD4+ cells and a ratio of 1.5±0.14 (3 donors) for CD8+ cells  off-target or indirect effects on STIM1, STIM2.1 , retaining 121 C-terminal aa residues . 2+], a reduced Tg-releasable store content but also an increased rate, peak and plateau of SOCE ethane-N,N,N′,N′-tetraacetic acid). As expected, STIM2.2 displayed inward currents that showed an initial rapid phase (store operated) and a second slower phase of increasing current density indicative of loss of a diffusive factor from the cytosol2+ and Orai2-mediated SOCE can be observed with STIM2.2 but not with STIM2.1  microscopy to assess the ability of STIM2.1 to oligomerize, translocate towards the ER–PM junctional regions, to recruit Orai1 to STIM2 punctae and to interact with Orai1. HEK239 cells were co-transfected with Orai1-mEGFP and STIM2.1 or STIM2.2 tagged with mcherry replacing aa 648–711 see  and trea calcium  with oveSTIM2.1 together with STIM2.2, STIM1 or an equimolar amount of YFP containing a control vector. Co-overexpression of STIM2.2 and the control vector in HEKO1 cells results in an extremely high basal Ca2+, indicating indeed a high activity of coupled STIM2.2 and Orai1. Nonetheless, STIM2.2 still elicits additional activation of Orai1 after store depletion in 0 mM Ca2+  (2+-imaging condition with only very slightly buffered intracellular Ca2+ (Fura2), we clamped the intracellular free Ca2+ concentration to 150 nM with addition of IP3 and repeated the patch-clamp analysis (P<0.05) reduced current densities (n=22 cells for each condition) with co-expression of STIM2.1 and STIM2.2. Expression of STIM2.1 did not elicit currents in either condition. Analyses of maximal current densities and exemplary IV relations are shown in + T cells as well as Jurkat T cells used in STIM1 and STIM2 (2+]i (2+ entry (green trace) was not much affected by co-expression of STIM2.2 analysed within 24 h after transfection  co-expression of both STIM2.2 and STIM2.1 increased STIM1-mediated Orai1 current densities  . To moreanalysis . Now co-analysis : betweennd STIM2 ; therefoM2 . Details on the applied algorithms and methods as well as some additional validation are given in the 14C-labelled glutathione S-transferase (GST-CaM) or with a biotinylated Orai1 C-terminal peptide. Each peptide spot contains a 15-amino-acid sequence starting with Y452 (translated from first ATG (NT 268) of NM001169118.1). The subsequent spot started with a three amino-acid shift generating spots with an overlap of 12 identical amino acids (aa) to the next neighbour (KRSTVFGTLHVAHSS) present in both splice variants  present in STIM2.1 spot 9 (LIQAEKIKKKRSTVF). STIM2.2 sequences lack this motif but also bind well to CaM with highest binding to spot 36 (MQLAIAKDEAEKIKK) .To obtain further biochemical evidence of splice variant differences and, as the insertion altered the scores of predicted calmodulin (CaM)-binding siteseighbour . Figure eighbour  or chemieighbour . AnalysiEAEKIKK) . Spots 4xclusive . To furtt manner . With sut manner . We perft manner . No bindSTIM2.1, where the sequence VAASYLIQ is spliced into the CAD/SOAR (Sα1) domain. We show that STIM2.1 is expressed in many cell types, where its relative expression ratio to the known variant STIM2.2 can differ up to eightfold in the cell types we tested. SiRNA-mediated downregulation of STIM2.1 in primary naive human CD4+ cells, which express a high amount of both STIM2 splice variants  or that STIM2.1 precludes binding of STIM2.2 or STIM1 to Orai1 subunits by steric hindrance only if cytosolic Ca2+ ions are present, the latter we believe to be more likely. A differential Ca2+ requirement for steric hindrance may explain why STIM2.1's inhibitory effect on basal Ca2+ is not always seen upon overexpression , it is highly unlikely that this constitutive entry is mediated by non-ER-localized STIM2 as postulated by Graham et al.2+ with no ability to recruit Orai1 to these ER–PM sitesBecause basal i=K*(R−Rmin)/(Rmax−R) where the values of K, Rmin and Rmax were determined from an in situ calibration of Fura 2-AM in Jurkat T cells as described in ref. 2+], the maximal Tg-induced peak in [Ca2+]i, maximal and plateau [Ca2+]i upon readdition of 0.5 mM [Ca2+]o and influx rate of cells. The minimal [Ca2+]i before addition of Tg or 0.5 mM [Ca2+]o was subtracted from Tg-peak or maximal [Ca2+]i, respectively.Human CD4STIM2.1-mcherry or STIM2.2-mcherry-pIRES and 2 μg Orai1-GFP-pMAX and seeded on 25-mm glass coverslips 24 h before measurement. Stores were depleted by incubation with 1 μg Tg in 0 Ca2+ Ringer solution for 15 min. A Leica AM TIRF MC system was used as in ref. EA) was calculated from background-subtracted images using α, β, γ and δ) were determined individually for every experimental day using single transfected cells. To ensure that FRET was compared for identical acceptor to donor ratios, only FRET values of regions of interest enclosing STIM2 clusters, which were within average acceptor to donor ratios±one s.d. of the respective experimental day, were used for final analysis.HEK293 were co-transfected with 6 μg STIM2.2 or STIM2.1 were amplified using the above mentioned pEX plasmids with primers including BamHI and XmaI recognition sites and subcloned into a self-modified pET19b vector creating an N-terminal 6 His tag. Plasmids were expressed in E. coli BL21 Rosetta strain. Overnight cultures were inoculated in 2 × YT medium containing 0.4% glucose and on the next day diluted 1:20, grown to an OD600 of 0.5 before protein production was induced with 1 mM isopropyl-b-D-thiogalactoside for 6 h at 30 °C. Lysis and protein purification were done as in ref. The cDNA coding for CAD domain of human Full details of STIM2 homology modelling and protein–protien docking analyses is given in 2 and 0.005% P20) at a flow rate of 20 μl min−1. GST-CaM was bound to the immobilized antibodies in the measuring cell. Similarly, immobilized GST in the reference cell served as a negative control. Subsequently, solutions containing increasing concentrations of purified STIM2.1 and STIM2.2 CAD domains (7.5–750 nM) were passed over the chip in the presence of 2 mM Ca2+. Each STIM application was followed by application of running buffer. The analysis was carried out employing the BIA evaluation software version 3.1 (BIACORE).SPR spectroscopy was carried out in a BIAlite X system. Briefly, monoclonal goat anti-GST serum  was immobilized on a CM5 research grade sensor chip (BIACORE) by amine coupling according to the manufacturer's instructions. The chip was equilibrated with application buffer  for 2 h at 4 °C. 14C-labelled GST-CaM was added and incubated at 4 °C overnight. The membrane was washed with binding buffer three times for 10 min each, dried at room temperature and subjected to phosphorimaging using a Typhoon-Trio imaging device . An equivalent peptide filter was incubated overnight with 10 μM biotinylated peptide in TBS with 5% biotin-free Albumin fraction V and 2.5% saccarose after blocking in buffer only for 3 h at room temperature. Blots were washed and developed after incubation with Avidin-HRP and subsequent chemiluminescence detection.Peptides encoding for STIM2 CAD domains were synthesized on acid-hardened cellulose membranes, derivatized with a polyethylene glycol spacer as described2 or 1 mM EGTA. Unbound fraction was removed by washing three times with the corresponding buffer and bound fraction was eluted by boiling at 65 °C for 15 min with 2 × Laemmli buffer and finally analysed by electrophoresis. The western blot was probed with anti-His antibody  at 1:1,000 dilution. Uncropped versions of al blot and gel images are shown in The purified STIM2.1 and STIM2.2 CAD domains were incubated at a final concentration of 2.5 μM in total volume of 250 μl with 25 μl prewashed CaM-sepharose beads  for 1 h at 4 °C with overhead rotation. The binding buffer contained 25 mM Tris, pH 7.5, 150 mM NaCl and 1 mM CaClA.-M.M., D.A. and B.A.N. conceived the project, designed the experiments and wrote the manuscript. A.-M.M. and D.A. performed the experiments and analysed the data. G.S. contributed the qRT–PCR analysis and gave technical support. P.-H.L. and V.H. undertook homology modelling and protein–protein docking analyses. M.J. designed peptide blots and performed radioactive experiments.How to cite this article: Miederer, A.-M. et al. A STIM2 splice variant negatively regulates store-operated calcium entry. Nat. Commun. 6:6899 doi: 10.1038/ncomms7899 (2015).Supplementary Figures 1-10, Supplementary Tables 1-4 and Supplementary Methods and Supplementary References


Anabaena under conditions replete in both combined-nitrogen and inorganic carbon. Opposite transcriptome responses to calcium treatments occur for nitrogen- and carbon-related processes.Calcium affects the primary cellular metabolism of  2+ in fine-tuning cellular responses of wild-type cyanobacteria under favourable growth conditions has not been examined. In this study, extracellular Ca2+ has been altered, and changes in the whole transcriptome of Anabaena sp. PCC 7120 have been evaluated under conditions replete of carbon and combined nitrogen. Ca2+ induced differential expression of many genes driving primary cellular metabolism, with transcriptional regulation of carbon- and nitrogen-related processes responding with opposing trends. However, physiological effects of these transcriptional responses on biomass accumulation, biomass composition, and photosynthetic activity over the 24h period following Ca2+ adjustment were found to be minor. It is well known that intracellular carbon:nitrogen balance is integral to optimal cell growth and that Ca2+ plays an important role in the response of heterocystous cyanobacteria to combined-nitrogen deprivation. This work adds to the current knowledge by demonstrating a signalling role of Ca2+ for making sensitive transcriptional adjustments required for optimal growth under non-limiting conditions.Calcium is integral to the perception, communication and adjustment of cellular responses to environmental changes. However, the role of Ca Synechocystis sp. PCC 6803 (Anabaena sp. PCC 7120 (hereafter designated as Anabaena) is one of the common models  is well known to play an essential role as second messenger and signalling molecule in all living organisms  is tightly regulated by Ca2+-binding proteins, Ca2+ pumps and other transporters to maintain low [Ca2+]i levels of about 100nM in order to avoid toxic calcium phosphate precipitation within the cell.Calcium  has been characterized  up to 1–2mM, while concentrations above that have inhibitory and toxic effects on cell growth and health  oxygen evolving complex  or in BG11 medium plus additional CaCl2 . Both media were supplemented with 10mM TES-KOH (pH 8.0). The cultures were cultivated under continuous low light (25–35 μmol photons m−2 s−1) at 30 °C and in 3% (vol/vol) CO2-containing air with mild shaking.Anabaena cells were grown under normal growth conditions in low and high Ca2+ media. After reaching OD750=1.0, the cells were centrifuged and the cell pellets resuspended to OD750=0.8 in either low or high Ca2+; for the Ca2+ treatments, pellets from high Ca2+ were resuspended in low Ca2+ medium (shifted from high to low) and pellets from low Ca2+ were resuspended in high Ca2+ medium (shifted from low to high). For the controls, pellets were resuspended in fresh medium with the same [Ca2+] (shifted from low to low or high to high). Afterwards, the cultures were again incubated under normal growth conditions. Over a time range of 24h , samples were taken for the determination of total proteins, carbohydrates, and biomass. Data were collected from three independent Ca2+ shift experiments (n=3).A 20ml culture volume was passed through a pre-washed, pre-dried and pre-weighed glass-fibre filter in a glass vacuum filtration apparatus and then washed with distilled water. Filters were dried at 60 °C for 24h, stored in a desiccator and then weighed.750 was measured against a bovine serum albumin (BSA) standard curve with a Lambda 25 UV/VIS spectrometer (Perkin Elmer).A 0.2ml volume of culture was taken in triplicate, centrifuged and washed with distilled water, and the pellets were frozen. The total protein content was determined according to a modified Lowry procedure  was represented by three independent biological replicates (n=3). One hour after the shift, the cultures were centrifuged  and the pellets immediately resuspended in 0.2ml RNA resuspension buffer  and 60 μl 0.25M ethylenediaminetetraacetic acid (EDTA) was added. A 0.3ml volume of lysis buffer  was added, followed by 0.5ml of phenol:chloroform:iso-amyl alcohol (P:C:IAA=25:24:1). Then the samples were vortexed thoroughly, incubated for 5min at 95 °C and centrifuged for 15min at 15000×g, 4 °C. The upper phase was collected, twice re-extracted with an equal volume of P:C:IAA and then extracted with C:IAA (24:1). The aqueous phase was supplemented with 10M lithium chloride to a concentration of 2.5M, and incubated overnight at –20 °C. The next day, the samples were centrifuged as before and the pellets washed with 1ml 70% ethanol by gentle mixing. After centrifugation, the RNA pellets were air-dried and resuspended in small volumes of milliQ water at 65 °C for 15min. RNA isolates were treated with DNase. RNA samples were submitted to the Turku Centre for Biotechnology  for RNA sequencing using a HiSeq 2000 (Illumina).Nostoc sp. PCC 7120, downloaded from Ensembl (EBI). Alignment was using the Tophat algorithm, and ORF calling and read-depth quantification were carried out using the open source analysis software Chipster . Significantly differentially expressed genes were identified using a false discovery rate (FDR: Benjamini–Hochberg) cutoff of 0.05. Gene descriptions were collected from Cyanobase (genome.microbedb.jp/cyanobase/) and KEGG (www.genome.jp/kegg/).RNAseq reads were aligned using the reference genome and annotations of −2 s−1), and values were normalized to the chlorophyll content (chl a estimated in 90% methanol at OD665). For the determination of Y(II), samples were diluted to a chlorophyll concentration of 5 μg chl a ml–1 and the fluorescence parameters Fv and Fm determined according to Oxygen evolution rate was measured using a Clark-type oxygen electrode (Hansatech Oxytherm) and quantum yield of PSII (Y(II)) was measured using a Dual-PAM-100 . For both, samples were dark-adapted for 5min. For the gross oxygen evolution rate in light, the rates of oxygen consumption obtained in darkness were added to oxygen evolution rates obtained in saturating light  and a Wetzlar light microscope (Leitz) on ×40 magnification. From active Nitrate concentration of filtered media was determined spectrophotometrically using a Spectroquant Nitrate Test kit (Merck).Anabaena sp. PCC 7120 to changes in external Ca2+ conditions was studied by pre-growing cells to OD750=1.0 (over approximately 3 days) in either low or high Ca2+ medium , and then replacing the medium with one of either the same Ca2+ concentration (acting as controls), or with an alternative Ca2+ concentration to create an up-shift (henceforth referred to as ‘high Ca2+’) or a down-shift (‘low Ca2+’) in [Ca2+]. The transcriptomic reaction of Anabaena cells to the change in [Ca2+] was analysed 1h post-shift by transcriptome sequencing. Gene expression in high Ca2+ and low Ca2+ were compared with the corresponding controls to identify genes that had a fold change (FC) ≥2 (log2 FC≥1) between treatment and control (FDR<0.05). Genes that were significantly differentially expressed after 1h in altered [Ca2+] were designated ‘short-term Ca2+-responsive genes’ , but showed strong differential regulation in response to the Ca2+ conditions of the 3-day pre-culture. These genes were designated ‘long-term Ca2+-responsive genes’ (see The response of nes’ see . Genes wnes’ see . The absnes’ see .3–) uptake were among the most strongly affected short-term Ca2+-responsive genes. All eight components of the three known HCO3– transporters in Anabaena, which are encoded on three separate operons, underwent 4–6.5 FC up-regulation (log2 FC of 2–2.6) in high Ca2+ shift. Conversely, the same genes were strongly down-regulated in low Ca2+ shift . Additionally, several Calvin cycle and metabolic enzymes, including fructose bisphosphate aldolase, transketolase and the petF ferredoxin , shared the Cluster 1 expression profile.Genes related to bicarbonate , and a desiccation-responsive dehydrogenase  was also strongly up-regulated in high Ca2+.Cluster 2 genes were strongly up-regulated in the short-term response to high Caalr5182; . The cya2+ and up-regulated in low Ca2+  genes and several members of the nif operon that encode the heterocyst-specific nitrogen fixing machinery in Anabaena  and ECF sigma factor sigG . Heterocyst frequency data are shown in Supplementary Table S1 at JXB online. To investigate this further, the combined nitrogen levels were determined in the filtered media taken from all time points, and shown to be approximately 200mg L−1 NO3−-N for both high and low Ca2+ media.Heterocyst frequency was evaluated at 10h, 20h and 2 d after the change in Ca2+ ions in signal transduction is widely recognized in eukaryotic organisms , Bic and Sbt, as well as the Mrp operon that is involved in supplying Na+ to the latter two HCO3− symporters. These transporters are components of the carbon-concentrating mechanism (CCM) that also includes CO2 transport machinery in the plasma and thylakoid membranes . Notably, translocation of extracellular HCO3− by the cmpA subunit requires co-binding of Ca2+ and HCO3− ions to cmpA . Carbohydrate measurements in this study showed no difference between high and low Ca2+ that would reflect the up- and down-regulation of HCO3− transporters. Rapid induction of several photosynthetic genes under high Ca2+ may be connected with the increased uptake of HCO3−, which is converted to CO2 for photo-assimilation  and by 2-OG, a metabolite which represents cellular C:N levels. These indicators respectively activate the ntcB and ntcA transcriptional activators of nirA  was observed under high Ca2+ conditions in this study  genes are up-regulated by high Ca2+ in media replete with nitrate , decreasing ccbP expression and increasing free [Ca2+]i. Free [Ca2+]i is also increased in heterocysts through the degradation of CcbP by HetR, the master regulator of heterocyst development. Collectively, the [Ca2+]i of heterocyst cells reaches concentrations severalfold higher than that of vegetative cells  gene cluster .This study has found that changes in Ca2+]i in Anabaena (2+]e may most likely be a metabolic adjustment in response to the up-regulation of carbon metabolism in the short term. In this case, the observed increase in nif expression may be induced through increased 2-OG levels as a consequence of increase of Cmp-transported HCO3−. Nonetheless, the possibility that even trace quantities of [Ca2+]i can influence gene expression through Ca2+-binding transcription regulators, despite strict [Ca2+]i maintenance, should not be dismissed.Considering the tight regulation of  in the expression data of the current study.Table S1. Heterocyst counts in Ca2+ shift experiments.


MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψm) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψm in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in  This transfer of electrons induces the pumping of protons out of the mitochondrial matrix by complexes I, III and IV to generate a mitochondrial membrane potential (Δψm), which is subsequently used by complex V (FoF1-ATP synthetase) to generate ATP  was calculated using Chelator software c in control (CON) and ND5 mutant cybrids was the same, whereas resting [Ca2+]c was significantly higher in ρ0 cells (p<0.05)  and the mitochondria into the cytosol. Ionomycin treatment revealed significantly less stored Ca2+ in ND5 mutant cybrids (45.3 ± 11.0% of control (CON) cybrids, p<0.05)  (We used the ratiometric dye fura-2 to perform a quantitative comparison of cytosolic free Ca(p<0.05) . This isell type , 13. The p<0.05) . Stored  p<0.05) .2+ in the absence of extracellular Ca2+ (Ca2+ free RS) (2+]c in control (CON) cybrids, ND5 mutant cybrids or ρ0 cells  and ρ0 cells  compared to the controls  . Under tρ0 cells . Howevercontrols . The toted in 2+ .2+ specifically from the ER, followed by ionomycin, which will then release calcium sequestered in the mitochondria. Thapsigargin inhibits the Ca2+-ATPase, causing depletion of ER Ca2+ and an increase in [Ca2+]c. Subsequent addition of ionomycin, in the absence of extracellular free Ca2+, will then release Ca2+ from any remaining intracellular pool, which will be dominated by the mitochondria.We next used thapsigargin to measure the release of Ca2+ free buffer, followed by the addition of thapsigargin. This resulted in the release of Ca2+ from ER stores in both control (CON) cybrids and ND5 mutant cybrids  and ND5 mutant cybrids, however, stored Ca2+ in ND5 mutant mitochondria was significantly lower   (Subsequent addition of ionomycin released Ca p<0.05) . Releasa p<0.05) .2+, cybrids were loaded with fluo-4 and then permeabilized with digitonin in a cytosol-like buffer  containing 62 nM free Ca2+. In addition, thapsigargin was added to deplete ER Ca2+ stores. Under these conditions, fluo-4 is lost from both the cytosol and ER, with the only dye remaining trapped in the mitochondria  were measured simultaneously by confocal microscopy.In order to measure the ability of the mitochondria to buffer increases in Cachondria . Aliquot2+] to 327 nM caused no change in [Ca2+]m in control cybrids  [2+] of 62 nM  to maintain Δψm in ND5 mutant mitochondria m only 24% of control values when [Ca2+] was elevated to 2.48μM  and Δψm  were made at various [Ca2+] in control (CON) cybrids, ND5 mutant cybrids and ρ0 cells are shown  cybrids up to 35 min of treatment (2+]c increased significantly in ρ0 cells after only 5 min 2DG treatment (p<0.05), followed by a slight reduction for the remainder of the experiment (In control (CON) cybrids,  reached 2.48 μM in control cybrids, a large depolarization of Δψm was observed, indicating the initiation of mitochondrial membrane permeability transition. Interestingly, a large membrane depolarization was not observed in either the ND5 mutant cybrids or ρ0 cells. This may be due to the fact that resting Δψm in both these cell types is lower than Δψm in control cybrids following membrane permeability transition. Nevertheless, it is possible that mitochondrial permeability transition, or low conductance pore opening, is occurring in both ND5 mutant cybrids and ρ0 cells at high [Ca2+].When [CaCalcium is particularly important in neuronal cells, where calcium signaling is critical for signal processing. In these cells, defects in mitochondrial metabolism will have severe functional consequences, as the reduction in ATP generation will lead to elevated cytoplasmic calcium levels that disturb cell signaling pathways, confound normal signal processing, and may even promote cell death via calcium overload and/or excitotoxicity . In neurIt has long been postulated that disruption of cell death signaling pathways may be a contributing factor to mtDNA disease pathogenesis . FurtherMT-ND5 mutation studied here was originally isolated from a MELAS patient who also carries a mutation in POLG, which encodes the catalytic subunit of the mitochondrial DNA polymerase [MT-ND5 mutation disrupts both mitochondrial respiratory function and calcium handling, and suggests that the MT-ND5 mutation may play a role in modulating the disease phenotype in conjunction with the POLG mutation.The ND5 mutant cybrids showed a reduced ability to generate ATP and exhibit defects in mitochondrial calcium accumulation, two features which serve to compound mitochondrial disease pathology. Of note, the m.13565C>T lymerase . Neverthm. This will have a profound impact on cell metabolism, with energetic failure or collapse of Δψm leading to cell death [Changes in cytosolic calcium concentration signal the mitochondria to match energy supply with demand, in particular by regulating mitochondrial dehydrogenase activity –28. Howell death . As suchS1 File(XLS)Click here for additional data file.


No significant difference in the coagulation function was observed between the control and drug-treatment groups. These results indicate that naringin improved platelet aggregation and inhibited the excessive release of P-selectin and PF4 in hyperlipidemic rabbits. This study suggests that the antiplatelet effect of naringin may be due to its ability to regulate the levels of blood cholesterol and [Ca2+]i in platelets. Naringin also did not cause bleeding in the hyperlipidemic rabbits.This study investigated the effects of naringin on platelet aggregation and release in hyperlipidemic rabbits, and the underlying mechanisms. The safety of naringin was also investigated. The rabbits were orally administered 60, 30 or 15 mg/kg of naringin once a day for 14 days after being fed a high fat/cholesterol diet for four weeks. Following the two weeks of drug administration, the degree of platelet aggregation induced by arachidonic acid, adenosine diphosphate and collagen was significantly reduced by naringin at certain doses compared with those in the rabbits of the model group (P<0.01). The levels of P-selectin and platelet factor 4 (PF4) also decreased following treatment with naringin compared with those of the model group. Certain doses of naringin significantly reduced the total cholesterol (TC) levels and elevated the ratio of high-density lipoprotein cholesterol to TC compared with those in the model group, and significantly decreased the cytosolic free calcium concentration ([Ca Platelets are central mediators of primary homeostasis and mediate pathological thrombosis. Activated platelets stimulate thrombus formation in response to the rupture of an atherosclerotic plaque or endothelial cell erosion, thereby promoting atherothrombotic disease . AntiplaThe present study explored the effect of naringin on the aggregation and release of activated platelets in hyperlipidemic rabbits.Male New Zealand white rabbits (weight = 2.0–2.5 kg) were provided by Beijing Animal Breeding Center . The animals were acclimated for at least one week under standard conditions with free access to a standard diet and water. All procedures were approved by the Animal Care and Use Committee of Tianjin University of Traditional Chinese Medicine  and conformed to the Guide for the Care and Use of Laboratory Animals published by the U. S. National Institutes of Health .2+ testing kit was obtained from Genmed Scientifics Inc. .Naringin was obtained from Shanghai Meilian Biotechnology Co., Ltd. . Adenosine diphosphate (ADP), arachidonic acid (AA) and collagen (COLL) were purchased from Chrono-Log Corp. . ELISA kits for P-selectin and platelet factor 4 (PF4) were obtained from R&D Systems Inc. . Fibrinogen (FIB), activated partial thromboplastin time (APTT) and prothrombin time (PT) kits were purchased from Stago Diagnosis Technology Co., Ltd. . An intracytoplasmic Caad libitum. After four weeks, blood was collected from the ear edge vein of the rabbits. The 24 hyperlipidemic rabbits in group M were selected based on their significantly higher TC values compared with those of the control group and then were divided into four groups, namely, model group (model), high-dose naringin treatment group , medium-dose naringin treatment group , and low-dose naringin treatment group .A total of 30 male rabbits were divided into two groups (control group and group M) according to the total cholesterol (TC) in their plasma. The rabbits in the control group  were fed with a basic diet during the experimental period. The rabbits in group M  were fed a high fat/cholesterol diet  for four weeks. The amount of daily diet for each animal was restricted to 50 g, and water was supplied The rabbits in the treatment groups were orally administered naringin once a day for 14 consecutive days. The rabbits in the four groups, with the exception of the control group, continued to be fed a high fat/cholesterol diet five times a week to maintain the model.9 platelets/ml using the PPP.The rabbits were locally anesthetized with 2% lidocaine (1 ml), and then blood was collected from the common carotid artery (CCA) 2 h after the final drug administration and anticoagulated with citrate . PRP was obtained by centrifugation at 800 rpm for 15 min, and the remaining blood was further centrifuged at 3,500 rpm for 10 min to prepare the PPP. The platelet concentration of the PRP was adjusted to 3–5×10Platelet aggregation was measured using an aggregometer  according to the methods of Born and Cross . In a tyBlood was collected from the CCA and anticoagulated with citrate . The plasma was separated by centrifugation at 3,500 rpm for 10 min. The levels of FIB in the plasma, and the PT and APTT were determined with an automatic blood coagulation analyzer Blood was collected from the CCA. Following placement in a water bath for 30 min at 37°C, the serum was separated by centrifugation at 3,500 rpm for 15 min. The levels of serum TC, TG, HDL and LDL were determined with an automatic analyzer .Blood was collected from the CCA. Following placement in a water bath for 30 min at 37°C, the serum was separated by centrifugation at 3,500 rpm for 10 min. The levels of serum P-selectin and PF4 were determined with the ELISA kits according to manufacturer’s instructions.2+-free Tyrode’s buffer , the platelets were suspended in Ca2+ Tyrode’s buffer . The platelet concentration was adjusted to 2×108 platelets/ml, and then [Ca2+]i was determined using the intracytoplasmic Ca2+ testing kit according to the manufacturer’s instructions.Following washing twice with CaAll data are expressed as the mean ± standard error of the mean. Statistical analysis was performed using analysis of variance. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS software, version 11.5 .The maximum gathered rates induced by AA, ADP and COLL in the model group were significantly increased compared with those of the control group (P<0.01), as shown in 2+]i significantly increased in the model group compared with that in the control group (P<0.01), and the low dose of naringin had no significant effect on the platelet [Ca2+]i compared with that in the model group. However, the medium and high doses of naringin significantly reduced the platelet [Ca2+]i compared with that of the model group . Naringin reduced the levels of FIB in the plasma and prolonged the PT and APTT to improve the blood hypercoagulable state of the hyperlipidemic rabbit; however, no significant difference was identified in the results for the naringin groups compared with those of the control group . This inAfter four weeks of high-fat feeding, the levels of TC, HDL and LDL significantly increased compared with those in the control group . Howeveret al (Hyperlipidemia reportedly triggers platelet activation, lipid peroxidation, platelet granulocyte aggregation and platelet aggregation. Sener et al  demonstret al . Naringiet al . The preThe present study demonstrated that naringin exerted positive modulatory effects on the levels of blood lipids in hyperlipemic rabbits. The medium and high doses of naringin significantly decreased the TC levels and increased the proportion of HDL in the TC. Naringin also reduces the sensitivity of platelets by adjusting the levels of blood lipids, thereby achieving the desired effect of antiplatelet aggregation.2+]i activates myosin light chain kinase, which leads to platelet deformation and shrinkage. Multiple Ca2+-dependent proteases play an important role in platelet activation. Ca2+ activates phospholipase C and phospholipase A2, leading to the release of thromboxane A2 and platelets. The activation of protein kinase C, which is involved in the release and aggregation of platelets and AA metabolism, requires the participation of Ca2+. The activation state of platelet [Ca2+]i in the hyperlipemic animal model in the present study was significantly higher than that in the control group. The results of the present study confirm that naringin reduces the [Ca2+]i, indicating that naringin inhibits platelet contraction and release by decreasing the platelet [Ca2+]i concentration and thereby inhibiting myosin light chain phosphorylation.As a second messenger, calcium is involved in platelet deformation, aggregation and the release reaction to stimuli. Increased platelet [CaNaringin inhibited platelet aggregation induced by AA, ADP and COLL in the present study to appreciable degrees and had a marked dose-dependent effect, indicating that naringin inhibits platelet activation via multiple pathways. The present study also confirmed that naringin had no marked influence on the normal coagulation function in the experimental animals. Further studies are required to elucidate the specific mechanisms of naringin. In conclusion, the results of the present study suggest that naringin inhibits platelet overactivity by regulating the levels of blood lipids and the concentration of platelet cytoplasmic calcium.


PKD2L1 is a Ca2+-permeable cation channel that is activated by extracellular Ca2+ (on-response) in Xenopus oocytes. PKD2L1 co-expressed with PKD protein 1 Like 3 (PKD1L3) exhibits extracellular acid-induced activation  but whether PKD1L3 participates in acid sensing remains unclear. Here we used the two-microelectrode voltage-clamp, site directed mutagenesis, Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence, and showed that PKD2L1 expressed in oocytes exhibits sustained off-response currents in the absence of PKD1L3. PKD1L3 co-expression augmented the PKD2L1 plasma membrane localization but did not alter the observed properties of the off-response. PKD2L1 off-response was inhibited by an increase in intracellular Ca2+. We also identified two intra-membrane residues aspartic acid 349 (D349) and glutamic acid 356 (E356) in the third transmembrane domain that are critical for PKD2L1 channel function. Our study suggests that PKD2L1 may itself sense acids and defines off-response properties in the absence of PKD1L3.Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), also called transient receptor potential polycystin-3 (TRPP3), regulates Ca Lack of PKD2L1 in mice was associated with defective hedgehog signalling and development of inverted colon13Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1) belongs to the transient receptor potential (TRP) superfamily of which members are implicated in a number of sensory functions, such as detection of light, force, osmolarity, temperature, odor, taste and pain1Xenopus oocytes by immunofluorescence, surface biotinylation and/or electrophysiology5121316151623In mammalian cells and oocytes, PKD2L1, in complex with PKD protein 1 Like 3 , was activated by acids in an off-response manner, i.e., the PKD2L1/PKD1L3 channel complex mediated a large current only after the removal of the extracellular acid1517151720182+ permeation and cation selectivity of the PKD2L1/PKD1L3 channel complex, and may thus be involved in channel gating and selectivity.PKD2L1/PKD1L3 channel complex responded to both weak and strong acids. It was reported that weak acids, such as citric acid, induce larger off-response currents in HEK cells expressing the PKD2L1/PKD1L3 complex than strong acids, such as HCl, at the same pH. All these acids possessed similar activation threshold values of around pH 3.020Xenopus oocyte expression model to examine how acids and PKD1L3 modulate PKD2L1 channel activity. In addition, we examined roles of Ca2+ and intra-membrane acidic residues in this modulation. For this we utilized two-microelectrode voltage clamp in combination with mutagenesis, Western blotting, RT-PCR and immunofluorescence.Despite recent significant progresses towards understanding the biological and physiological roles of PKD2L1 and PKD1L3, how PKD2L1 and/or PKD1L3 sense acid is not fully understood. In the current study, we employed Xenopus oocytes responds to acidic stimuli. In the absence of extracellular Ca2+ (to avoid the on-response effect of Ca2+ on PKD2L1), we found that increasing extracellular acidity (from pH 7.5 to pH 2.5–4.5) to PKD2L1-expressing or control oocytes does not induce appreciable currents. However, in PKD2L1-expressing, but not water-injected control, oocytes voltage clamped at −50 mV a large inward current was elicited following substitution of an acidic extracellular solution with a control solution at pH 7.5  of PKD2L1 mRNA the off-response was observed only in the presence of PKD1L3 co-expression (injected at 25 ng mRNA per oocyte) . Our immOur voltage clamp data showed that the PKD2L1-associated off-response is dose-dependent with half maximal activation value of pH 3.5 ± 0.5 (N = 5–9) in PKD2L1-expressing oocytes voltage clamped at −50 mV . Previou151819In order to determine whether the off-response is sensitive to the duration of acid application, we applied a solution of pH 3.0 for different periods of time. We found that oocytes over-expressing PKD2L1 exhibit similar off-response amplitudes for different application durations, ranging from 10 sec to 1 min . Our data values of both acids, the resulting solutions should contain 49.1 and 12.9 mM of the undissociated membrane-permeable form of acetic and citric acids, respectively. Additionally, we found that the off-response current induced by acetic acid, but not citric acid, is significantly larger than that induced by HCl   . This pa2+ induces PKD2L1 channel activation followed by an ensuing channel inactivation2+-free conditions (2+-induced on-response with inactivation and acid-induced off-response (in the absence of Ca2+) without inactivation.It was previously reported that application of extracellular Canditions . Thus, t2+-induced PKD2L1 activation2+-induced activation is completely abolished when extracellular pH drops to pH 3.0 , PKD2L1-expressing oocytes responded with a robust off-response current followed by channel inactivation, forming a spike of current . For this, we pre-injected Xenopus oocytes over-expressing PKD2L1 with 50 nL of 50 mM ethylene glycol tetra-acetic acid (EGTA) tetra -sodium salt solution 1 hr before the same electrophysiological measurements as those for 2+. We found that while EGTA pre-injection does not prevent PKD2L1 from exhibiting the off-response, it substantially reduces the ability of extracellular Ca2+ to inhibit the off-response   . These r2+. We added 1 mM EGTA to our extracellular solutions to chelate any trace amounts of Ca2+ ion and, consistently, found a 36.7 ± 3.8%  increase in the off-response current  for the Ca2+ inhibition by applying different [Ca2+]o values, as shown in 2 is sufficient to completely inhibit the off-response current and that Ki for [Ca2+]o is equal to 167 ± 33 μM (N = 11) for the off-response current generated from pH 3.0 to 7.5  may account, at least in part, for the difference in the off-response current with and without extracellular EGTA  and 77.2 ± 2.8% , respectively  can be repeatedly induced immediately one after another. Further, Ca2+-induced channel activation was followed by channel inactivation while acid-induced channel activation was sustained without inactivation in the absence of Ca2+. These data also suggest that the Ca2+-dependent on-response and acid-dependent off-response are not completely governed by the same mechanism.PKD2L1 was first reported in 1999 to be a non-selective cation channel activated by and highly permeable to Ca2+ and reached a plateau, similar to reported data using oocytes2+, the off-response was still present but was followed by an ensuing inactivation, producing a current spike that is similar to the reported observations in HEK cells for PKD2L1/PKD1L3 under a comparable condition2+ was added during an off-response activation, then an immediate inhibition of the off-response current was observed, with a Ki value (50% inhibition) for [Ca2+]o of only 0.17 mM. These data together indicated that extracellular Ca2+ regulates (somehow inhibits) the off-response currents. In contrast, other previous reports using HEK cells expressing PKD2L1 and PKD1L3 stated that the acid-induced off-response requires the presence of extracellular Ca2+ and results in an increase in [Ca2+]i2+ will have to be clarified in further studies.In fact, acid-induced off-response activation in PKD2L1-expressing oocytes occurred in the absence of extracellular Ca2+ chelator did not significantly alter the off-response, it abolished the ability of extracellular Ca2+ to inhibit the off-response. This suggests that an increase in the intracellular free Ca2+ is required for Ca2+ to inhibit the off-response activation. On the other hand, we previously reported that an increase in intracellular Ca2+ is required for both the Ca2+-induced on-response activation and the ensuing inactivation+ concentrations. Putting all data together, although Ca2+ and acid distinctly induce PKD2L1 channel activations they seem to share the same or similar way of inactivation, i.e., by an increase in [Ca2+]i.Interestingly, while preloading oocytes with Ca2+ and proton ions trigger channel activations although Ca2+ plays roles in both phenomena. It is worthy to note that the PKD2L1 C-terminus possesses an EF hand, a coiled-coil domain, a cytoplasmic regulatory domain  and phosphorylation sites302+, or through activating downstream signalling molecules. More recently, CRD binding to Ca2+ was reported to change the α-helical property of the C-terminus2+-induced activation. However, whether the same domain is involved in the Ca2+ inhibition associated with acid-dependent off-response needs further studies. On the other hand, we found two negatively charged residues D349 and E356 to be critical for both on- and off-response functions. Because aspartate and glutamate residues have their pKa values of pH 3.7 and pH 4.0, respectively, which are well within the pH range in which PKD2L1 exhibits the off-response, our work suggests that protons may interact with these residues as part of the off-response activation process. Further, because of their importance for the Ca2+-induced on-response activation, these acidic residues may also interact with Ca2+ during the on-response process.It has remained unclear as to how CaXenopus oocytes exhibits extracellular acid-induced off-responses with characteristics resembling those associated with PKD2L1/PKD1L3 and that this off-response is inhibited by an increase in the intracellular Ca2+. Further studies will help better understanding how PKD1L3 and Ca2+ modulate the PKD2L1 channel function.In summary, in the present study we discovered that PKD2L1 expressed alone in Human PKD2L1 gene was cloned as previously describedPKD2L1 cDNA were linearized with Mlu I, followed by phenol/chloroform purification and ethanol precipitation.Plasmids pCHGF harbouring WT or a mutant in vitro synthesize capped mRNAs using the T7 mMessage mMachineTM kit . Stage V-VI oocytes were isolated from Xenopus laevis under an approved institutional protocol. Defolliculation of oocytes was performed through incubation in Barth’s solution2O containing 25–50 ng mRNAs or as stated in the experiment. An equal volume of H2O was injected into control oocytes. Injected oocytes were incubated at 16–18 °C in Barth’s solution, for 2–4 days prior to experiments. For intracellular Ca2+ chelation studies, oocytes were injected with 50 nL of 50 mM EGTA  to allow a final intracellular concentration of ~5 mM 1 hr prior to recordings.Linearized plasmids were used to Western blotting was performed as previously describedXenopus oocytes were washed in PBS, fixed in 3% paraformaldehyde for 15 min, and washed 3 times in PBS plus 50 mM NH4Cl, and then permeabilized with 0.1% Triton X-100 for 4 min. Oocytes were then washed 3 times in PBS for 5 min each time, blocked in 3% skim milk in PBS for 30 min, and then incubated overnight with the PKD2L1 polyclonal antibody. This was followed by 3 times 10 min washes in PBS. The oocytes were then incubated with a secondary AlexaFluor 488-conjugated donkey anti-rabbit antibody  for 30 min, followed by 3 times washes in PBS and mounting in Vectashield . The slides were examined on an AIVI spinning disc confocal microscopy .2, 10 HEPES adjusted to pH 7.5 by TRIS base. Low pH (2.5–4.5) standard solutions were prepared from the standard solution adjusted by HCl. Solutions containing extracellular Ca2+ were prepared from the standard solution with the addition of CaCl2.The standard extracellular solution contained (in mM): 100 NaCl, 2 KCl, 1 MgClCurrent and voltage signals were measured with the conventional two-microelectrode voltage clamp technique with a commercial amplifier . Electrodes were fabricated from borosilicate glass  by a micropipette puller  and filled with 3 M KCl with typical tip resistance of 0.5–3 MΩ. Digidata 1320A converter and pClamp 9.2  were used for data acquisition and analysis. In experiments using a ramp or gapfree protocolXenopus laevis oocytes, tongue, kidney, and brain tissues. Oocytes were collected and defolliculated as described above. Tissues were washed in PBS and kept on ice. RNAs were then extracted at room temperature with TRIzol reagent . According to the manufacturer’s manual, 1 mL TRIzol was used to homegenize 50–100 mg of tissue. Chloroform was used to isolate the RNA followed by precipitation, wash and resuspension steps. One-step RT-PCR was carried out using SuperScript®III One-step RT-PCR kit (Invitrogen Life Technologies) by following the instruction manual. The oligonucleotide primers for Xenopus laevis β-actin and PKD1L3 for the first PCR were as follows: β-actin, sense GAGATGAAGCTCAAAGCAAAAG and antisense CAGGATTCCATACCAAGGAAAG; PKD1L3 sense GCAGATTGTGAGGAAGAAAGG and antisense TGCTGAGAGCTGGTAGGGTAGT. 10X diluted first PKD1L3 PCR products were used as templates to run the second (nested) PCR using inner primers to increase PCR specificity and efficiency. Sequences of the PKD1L3 inner primers were: sense AGATTGTGAGGAAGAAAGGGGG and antisense TTTGCTAAAGTCTGGTGGGTTG.Total RNAs were prepared from female Data were presented as mean ± (SEM). All data were collected from at least three different experiments. Student’s t-test was used to compare two groups of data for statistical significance indicated by a p value. P values of less than 0.05 and 0.01 were considered significant and very significant, respectively.How to cite this article: Hussein, S. et al. Acid-induced off-response of PKD2L1 channel in Xenopus oocytes and its regulation by Ca2+. Sci. Rep.5, 15752; doi: 10.1038/srep15752 (2015).


This route for Ca2+ influx is regulated by the filling state of the intracellular Ca2+ stores communicated to the plasma membrane channels by the proteins of the Stromal Interaction Molecule (STIM) family, STIM1, and STIM2. Store-dependent, STIM1-modulated, channels include the Ca2+ release-activated Ca2+ channels, comprised of subunits of Orai proteins, as well as the store-operated Ca2+ (SOC) channels, involving Orai1, and members of the canonical transient receptor potential family of proteins. Recent studies have revealed the expression of splice variants of STIM1, STIM2, and Orai1 in different cell types. While certain variants are ubiquitously expressed, others, such as STIM1L, show a more restricted expression. The splice variants for STIM and Orai1 proteins exhibit significant functional differences and reveal that alternative splicing enhance the functional diversity of STIM1, STIM2, and Orai1 genes to modulate the dynamics of Ca2+ signals.Store-operated Ca Evolution has provided the cells with a highly complex machinery, which finely tunes, orchestrates and coordinates intracellular Ca2+ homeostasis. Physiological agonists modulates [Ca2+]c by the activation of more or less selective Ca2+ channels and transporters, both in the intracellular Ca2+ compartments  and in the plasma membrane (PM). Once the stimulus ends, [Ca2+]c returns to basal level and the cell is ready for a new stimulation , evokes the opening of channels in the PM leading to Ca2+ entry from the extracellular medium , a protein discovered in 2+ sensor that stimulate Ca2+ entry, triggering the activation of store-operated channels located in the PM  channels (2+ selective store-operated Ca2+ (SOC) channels, which require the interaction of Orai1 with the canonical transient receptor potential (TRP) family member TRPC1 , a major mechanism for Car medium . After in the PM . Concernchannels . In addier TRPC1 . STIM1 aer TRPC1  also ideer TRPC1 ; the Oraer TRPC1  and the er TRPC1 . SOAR dier TRPC1 . The acter TRPC1 , and theer TRPC1 .2+ entry activated by arachidonate  and STIM2 , have highly conserved structure and present slightly divergences giving them different functions. Upon store depletion, STIM1 oligomerizes and redistributes into discrete puncture nearby the PM  proteins that are located mainly in the ER , but als2+ entry . Next, S2+ entry . SAM is n occurs . Locatedn occurs . Furthernt STIM1 , 2013. Tnd CC1α3 . Furthernd CC1α3 . Beside nd CC1α3 .Figure 3). Both, N- and C- termini are required for STIM1 interaction and regulation  channels, a store-independent channel regulated by the STIM1 population located in the PM  within the N-terminus ; the hydrophobic cavity  and a basic region . It is surrounded by three rings, subsequently comprised by TM2, TM3, and TM4  inserted in the cytosolic region , an actin-binding domain, that allows STIM1L to interact with Orai1 Ca2+ channels to form permanent clusters  in comparison with other cells where full SOCE activation requires several seconds  domain that contains three CC domains (CC1-3) including the including the SOAR/CAD, which has been reported to bind to Orai and TRPC channels . STIM2 s2+ concentrations and modulates prolonged Ca2+ entry and response to low concentrations of physiological agonists , the conventional isoform of STIM2, which will be named STIM2.2 from now on.Two recent studies have identified three STIM2 splice variants: STIM2.1, STIM2.2, and STIM2.3 . The strFigure 2). The expression of the STIM2.1 variant has been reported to be ubiquitous and its abundance relative to STIM2.2 depends upon the cell type but is significantly high in naive T cells, where the expression of both variants is similar  in its SOAR/CAD region, encoded by an additional exon 9, that disrupts binding to Orai . The function of the STIM2.3 variant is unknown at present and its expression seems to be quite limited and has not been detected in lymphocytes, where the other two variants are significantly expressed  as well as store-independent (ARC) channels. The analysis of the expression ratios of the different variants in a particular cellular model might be of great interest to understand the fine modulation of Ca2+ entry.Summarizing, different STIM1, STIM2, and Orai1 variants have been reported to be expressed as a result of alternative splicing in a number of cell types. The expression of certain variants, such as STIM1L, is quite restricted while that of other variants is ubiquitous. As it has been hypothesized , alternaJR designed the manuscript, contribute to write it and performed the final edition. RD, TS, and IJ contributed to write the manuscript and discussion.The 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.


HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca2 + concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca2 + transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca2 + removal , L-type Ca2 + current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca2 + content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca2 + current (ICa-L) in control cells reproduced both the decrease in Ca2 + transient amplitude and increase of SR Ca2 + content observed in voltage-clamped HF cells. During β-AR stimulation Ca2 + transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca2 + content was highest in HF cells during β-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca2 + transient amplitude and increased SR Ca2 + content observed in voltage-clamped cells.Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during β-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca •2 + is reduced in atrial myocytes.In an ovine model of heart failure systolic Ca•2 +]i.Action potential duration shortening is not responsible for changes in [Ca•2 + current underpin the smaller systolic Ca2 + transient.Reductions in L-type Ca•2 + extrusion increased.NCX current is reduced in heart failure but sarcolemmal Ca•2 + buffering power accelerates sarcolemmal Ca2 + extrusion via NCX.Decreased Ca Using a brief period of rapid ventricular pacing in the dog, Yeh et al. 2 + transients that were attributed to increased SR Ca2 + loading  and a prolonged action potential duration in ‘HF’ myocytes; however, a role for changes in ICa-L, or other Ca2 + homeostatic mechanisms such as SERCA activity and Ca2 + buffering ICa-L in cardiac disease states 2 +, SR Ca2 + content and cellular Ca2 + homeostasis in the atria during HF is unknown.Despite the extensive data showing that 2 + homeostasis, systolic Ca2 + and SR Ca2 + content and how these are modified by β-AR stimulation. We used an ovine model of dilated cardiomyopathy produced by rapid ventricular pacing and found that HF leads to a shortening of the atrial action potential duration (APD) and reduction in Ca2 + transient amplitude but SR Ca2 + content, determined from the amplitude of the caffeine evoked rise of [Ca2 +]i, was unaltered. Under voltage clamp conditions the Ca2 + transient remained smaller in HF and this occurred due to a reduction in ICa-L rather than a decrease in SR Ca2 + content, which was paradoxically increased. During β-AR stimulation the Ca2 + transient was increased more in HF than control cells. However, whilst SR dependent Ca2 + removal and ICa-L were both augmented by β-AR stimulation in HF cells, they remained reduced compared to control cells. Interestingly, SR Ca2 + content was highest in voltage-clamped HF atrial cells during β-AR stimulation. In action potential clamped myocytes the shortened HF APD does not explain the smaller systolic Ca2 + transient. We therefore conclude that the decreased ICa-L in HF atrial myocytes is the major factor causing the smaller systolic Ca2 + transient.Thus, the objectives of the present study were to assess the impact of HF on atrial myocyte intracellular Ca2All procedures were in accordance with The United Kingdom Animals (Scientific Procedures) Act, 1986 and European Union Directive 2010/63. Institutional approval was received from The University of Manchester Animal Welfare and Ethical Review Board.A detailed description of the experimental methods used in this study is available in the online supplement.2.1HF was induced in 25 female Welsh sheep  by transvenous right ventricular tachypacing as described in detail previously 2.22 + concentration ([Ca2 +]i) were monitored using the acetoxymethyl ester of either Fluo-5F  were performed at 37 °C. Changes in intracellular Ca5F 5 μM;  or Fura-5F 5 μM; . Where F2 + content was calculated by the rapid application of 10 mM caffeine and integration of the resulting Na+–Ca2 + exchange (NCX) current 2 + buffering capacity of cells was also obtained from the caffeine evoked Ca2 + transients SR Ca2.32 until use. Samples were prepared in RIPA buffer and subject to denaturing SDS-PAGE as described previously Left atrial tissue was snap frozen and stored in liquid N2.4n cells and N animals . Continuous scale data was log10 transformed n) were obtained from each experimental subject (N) (IBM SPSS Statistics v20). Where treatments/effects were within the same animal, cellular differences were assessed using a paired Students t-test and considered significant when P < 0.05.Initial estimates of sample sizes were determined by power analysis using Sigmaplot 11  assuming α = 0.05 and β = 0.8 and were based on the level of effects and standard deviations observed previously in this model 33.1P < 0.001). Atrial cellular hypertrophy was noted in isolated left atrial myocytes  with an increase in planar width from 15.0 ± 0.2 to 18.1 ± 0.4 μm and length from 127.5 ± 1.7 to 168.5 ± 3.5 μm . Membrane capacitance also increased from 59.6 ± 2.1 to 125.9 ± 9.3 pF . From simultaneously derived confocal 3-dimensional rendered cell volumes and membrane capacitance measurements P < 0.001. 19/5 control; 15/5 HF). However, the calculated surface area to volume ratio was unaltered . The respective surface area to volume ratio was used to express all integrated cellular Ca2 + fluxes and SR Ca2 + contents to total cellular volume Clinical signs of HF (lethargy and dyspnoea) were present after a median 33.5 (29.8–37) days of tachypacing. Due to the anatomic orientation of the cardiac apex over the sternum in sheep we were unable to obtain trans-thoracic four-chamber views and thus measures of atrial dimensions or E/A ratios. However ventricular dilatation was present as we have found previously 3.22 + transient. Under current clamp conditions, action potential evoked systolic Ca2 + transients were elicited at 0.5 Hz and representative traces shown in 2 +]i was reduced in current-clamped HF myocytes control, 60.7 ± 4.5; HF, 37.3 ± 4.8 nmol/l, P = 0.001. However, the amplitude of the action potential evoked rise of systolic Ca2 + was reduced in HF atrial myocytes by 46 ± 17% . Conversely, despite the smaller systolic rise of [Ca2 +]i, the amplitude of the caffeine evoked rise of [Ca2 +]i, used as a qualitative measure of SR Ca2 + content, was unaltered following current clamp stimulation  with the time to 90% repolarization decreasing from 357 ± 26 ms in control to 280 ± 15 ms in HF.We first sought to determine if the cellular hypertrophy and chamber dilatation were associated with alterations to the systolic Camulation B.ii. The2 + transient amplitude was reduced in HF atrial myocytes. In the first series of experiments the action potential clamp technique was used to apply the averaged control and HF action potential waveforms to myocytes isolated from control atria . Following stimulation with the HF atrial action potential there was no detectable change in the amplitude of the systolic Ca2 + transient or caffeine-evoked rise of [Ca2 +]i or SR Ca2 + content measured from the integral of the NCX current following caffeine application . Thus, changes in action potential duration do not explain the smaller atrial systolic Ca2 + transients observed in this model of HF.We then sought to determine the potential mechanisms by which the systolic CaICa-L, cellular Ca2 + fluxes and SR Ca2 + content. Representative Ca2 + transients following steady-state voltage clamp stimulation are illustrated in 2 + transient was reduced by 28 ± 9% . Given the strong dependence of Ca2 + transient amplitude on SR Ca2 + content and the indication, from the slowed decay of [Ca2 +]i in HF  was applied rapidly to the cell and the resulting NCX current integrated. This data is summarized in 2 + content was increased from 83.9 ± 4.5 to 100.1 ± 6.7 μmol/l . Therefore, the reduced Ca2 + transient amplitude observed in HF atrial cells cannot be explained by a decrease of SR Ca2 + content.We then performed voltage clamp experiments to facilitate quantitative analysis of ]i in HF B.ii that2 + transients observed in HF atrial cells would be an increase in the Ca2 + buffering power of the cells. Representative Ca2 + buffering curves are illustrated in total and free Ca2 + is shallower in the HF atrial cells  was reduced by 43.7 ± 8.2% in HF . Therefore, as with SR Ca2 + content, changes in Ca2 + buffering power do not explain the smaller systolic Ca2 + transient in HF atrial cells.Another possible explanation for the smaller systolic Ca2 +]i was also assessed from the caffeine evoked rise of [Ca2 +]i. The representative traces and summary data .The dependence of NCX on i and inward NCX current are increased following nicardipine exposure (2 + content from 74.1 ± 7.6 μmol/l in control to 89.9 ± 10.5 μmol/l in nicardipine (P < 0.01). This data therefore suggests that it is the reduction of ICa-L in HF atrial cells that is responsible for both the decrease of Ca2 + transient amplitude and increase of SR Ca2 + content.The role that the decreased exposure C.i and tICa-L therefore explains the smaller Ca2 + transients and increased SR Ca2 + content observed in voltage-clamped cells we also sought to determine if changes in SERCA or NCX activity also occur in HF and thus may contribute to the observed changes in systolic Ca2 +. Firstly we determined if SERCA activity was altered in HF using the approaches outlined in 2 + transients from a control and HF atrial cell are shown in 2 + transient decays more slowly in HF. This was quantified by fitting a single exponential to the decay phase of the Ca2 + transient to obtain the rate constant of decay of systolic Ca2 + . The underlying mechanism for the reduction of ksys in HF atrial cells was then determined by also fitting the decay phase of the caffeine evoked Ca2 + transient with a single exponential (kcaff). The SR dependent rate of Ca2 + removal (kSR) was then derived by subtraction (kSR = ksys − kcaff). In HF, sarcolemmal dependent Ca2 + extrusion by NCX and PMCA (kcaff) was increased by 25.2 ± 10% . Conversely, the SR dependent rate of Ca2 + removal (kSR) was decreased . Thus the increase in surface membrane-mediated Ca2 + extrusion (kcaff) is inconsistent with the decrease in the slope of the relationship between INCX and [Ca2 +]i  an effect of reduced cellular Ca2 + buffering power  exists indicating that as Ca2 + buffering power decreases then caffk increases. We have also determined, by blocking NCX with 10 mmol/l Ni2 + and measuring the rate of decay of the caffeine evoked rise of [Ca2 +]i, that the contribution of PMCA to Ca2 + extrusion is no different between control and HF atrial myocytes . The unaltered kPMCA, despite reduced Ca2 + buffering, is further evidence that the decrease in Ca2 + buffering power in HF accounts for the observed increase in kcaff.Whilst the decrease in  + ksys, B.ii. On [Ca2 +]i C. There ng power B. The ef3.42 +-calmodulin kinase II- (CAMKII) dependent Thr-17 residue ; an effect most likely due to the 320 ± 40% increase (relative to control) in CAMKII expression in HF (P < 0.001). The unaltered PKA-dependent phosphorylation of PLN was associated with an increase in protein phosphatase 1 . No differences in RyR expression, PKA-dependent Ser-2808 RyR phosphorylation or CAMKII-dependent Thr-2814 RyR phosphorylation were observed between control and HF atrial tissues.We then examined additional molecular correlates of the reduced Ca3.52 + transient observed in HF atrial cells could be ‘rescued’, we examined the effects of the non-specific β-AR agonist isoprenaline (100 nmol/l) on Ca2 + transient amplitude  such that during β-AR stimulation, Ca2 + transient amplitude in HF was indistinguishable from that in control (not shown). Isoprenaline also accelerated the rate of decay of the systolic Ca2 + transient in both control and HF atrial cells ; an effect attributable to an increase in SERCA activity with kSR increasing from 1.80 ± 0.36 s− 1 to 5.19 ± 0.68 s− 1 in control cells (P < 0.001) and, 0.93 ± 0.17 s− 1 to 3.47 ± 0.50 s− 1 in HF atrial cells (P < 0.001). Again, during β-AR stimulation HF and control cells were statistically indistinguishable . The increase in SERCA activity during β-AR stimulation also increased SR Ca2 + content in both control  and HF  atrial cells . The isoprenaline-mediated augmentation of Ca2 + transient amplitude was also associated with an increase of L-type Ca2 + current  in both cell types (P < 0.001) although peak ICa-L was smaller in HF cells than in control cells (P < 0.05). In ISO the integrated Ca2 + entry on ICa-L was indistinguishable between control and HF mycoytes. Finally, by Western blotting we were unable to detect differences in the expression of the G-proteins Gαs and Gαi(1/2/3) .To determine if the reduced Camplitude A.i & ii.al cells C. Howeve3.62 + homeostasis in atrial cells occurring in HF. How these impact on the total Ca2 + economy of the cell are summarized in 2 + transient decreases by 28 ± 9% in HF. However, as a consequence of the decreased Ca2 + buffering power (β) of the cell in HF the total Ca2 + requirement to generate the systolic Ca2 + transient is decreased by 59 ± 7% (P < 0.001). This total Ca2 + requirement is met from two sources; i) Ca2 + entry via ICa-L and, ii) the release of Ca2 + from the SR. Given that the integrated Ca2 + entry via ICa-L is unaltered and SR Ca2 + content is increased in HF, it is not surprising therefore that the fractional release of Ca2 + from the SR is reduced in HF by 64 ± 7% (P < 0.001). Finally, the gain of excitation contraction coupling can be calculated from the integrated Ca2 + entry and release of Ca2 + from the SR during the systolic Ca2 + transient is reduced by 67 ± 7% (P < 0.001) in HF.The previous sections highlight multiple changes in cellular Ca42 + transients that decay more slowly, iii) have a reduction in ICa,L, iv) have a reduced Ca2 + buffering power and, iv) have a greater increase in Ca2 + transient amplitude in response to β-AR stimulation. The reduction of ICa-L is sufficient to explain the smaller systolic Ca2 + transient and the accompanying increased SR Ca2 + content observed in voltage clamped cells. The reduced Ca2 + buffering capacity of HF atrial myocytes is insufficient to maintain systolic Ca2 +, but it does facilitate the enhanced sarcolemmal-mediated rate of Ca2 + extrusion observed in HF atrial myocytes. The restoration of systolic Ca2 + during β-AR stimulation likely occurs as a result of increases in ICa-L and SR Ca2 + content. The above changes in cellular Ca2 + homeostasis provide a mechanism for the reduction in Ca2 + transient amplitude observed in atrial myocytes isolated from failing hearts.The main findings from the present work are that in failing hearts atrial myocytes, i) are hypertrophied, ii) have smaller systolic Ca4.12 + homeostasis. As such it is important to note  that the high ventricular pacing rate used to initiate HF does not increase atrial rate. The changes in Ca2 + homeostasis and cellular remodelling are therefore not due to atrial tachypacing in contrast to those observed in various models of atrial fibrillation e.g. 2 + cycling, a measure of the surface area to volume ratio is required to relate sarcolemmal Ca2 + fluxes and SR Ca2 + content to the volume of the cell. In this model of HF, atrial myocytes are markedly hypertrophied showing a ~ 20% increase in cell diameter, ~ 30% increase in cell length and ~ 70% increase in cell depth. Both the increase of cell dimensions and loss of t-tubules that occurs in atrial cells in this HF model Two major considerations in the present manuscript are how HF induced by right ventricular tachypacing mediates changes in cellular geometry and atrial cellular Ca4.2ICa-L accompanied the observed decrease of the systolic Ca2 + transient. Such a decrease of atrial ICa-L has been observed previously in various models of cardiac disease e.g. ICa-L is causal of the decreased Ca2 + transient is demonstrated by the observation that pharmacologically inhibiting ICa-L in control cells with nicardipine reproduces the effect of HF on ICa-L, Ca2 + transient amplitude and SR Ca2 + content. Whilst we do not have data for changes in dihydropyridine binding or L-type Ca2 + channel α-subunit expression in the atria in HF, the loss of atrial t-tubules that occurs in this model of HF ICa-LWe find that a decrease of ICa-L may also explain the observed increase of SR Ca2 + content in voltage-clamped myocytes. The L-type Ca2 + current has two effects on SR Ca2 +; i) it triggers release and therefore an increase of ICa-L would be expected to decrease SR Ca2 + and, ii) on the other hand it loads the cell and therefore the SR with Ca2 +. It is therefore difficult to predict the net effect of change in ICa-L on SR Ca2 + content. We find that in the atria, as in the ventricle ICa-L leads to an increase of SR Ca2 + content. However, the increase in SR Ca2 + content is insufficient to restore the systolic Ca2 + transient which remains smaller in HF atrial myocytes despite the increase in SR Ca2 + content.The decrease of 4.32 + transient observed in HF atrial cells can be mimicked in control cells by inhibition of ICa-L with nicardipine, additional factors regulating the systolic Ca2 + transient also change in HF. These include, i) reductions in the Ca2 + buffering capacity and, ii) a decrease in the gain of excitation contraction coupling. The net effect of these changes however remains a smaller rise of Ca2 + in HF atrial cells. A key factor likely contributing to the reduction in excitation contraction coupling gain is the reduction in peak (trigger) ICa-L in HF. Again, the loss of t-tubules in HF atrial myoyctes ICa-L2 + transient amplitude we observe in HF atrial cells in the sheep.Whilst the smaller systolic Ca2 +]i is reduced in HF is consistent with either a reduction of NCX expression at the cell surface in HF or, if this is unaltered, an increased density of NCX on the t-tubules which have subsequently been lost in HF. Given the importance of NCX in arrhythmogenesis via Ca2 + dependent mechanisms we would suggest that, on balance, the decrease in NCX current we see in this model of end-stage heart failure might mean that other mechanisms e.g. as a result of action potential shortening or K+ current remodelling, may be more important in increasing the susceptibility of the atria to arrhythmias in HF. However, it is important to note that the data presented here is from a model of end-stage heart failure where contractile dysfunction rather than arrhythmias may be more pathologically relevant.The finding in the present study that, despite the reported loss of t-tubules in HF atrial myocytes 4.42 + transient in HF atrial cells is indistinguishable from that of control cells. However, neither the rate of decay of the systolic Ca2 + transient (ksys) nor SR dependent rate of Ca2 + removal (kSR) is fully restored by β-AR stimulation. Nevertheless, SR Ca2 + content is increased by β-AR stimulation in both control and HF cells and importantly, in HF cells, SR Ca2 + content remains greater than in control cells. It is therefore likely that the combined effect of increasing SR Ca2 + content and ICa-L during β-AR stimulation is responsible for normalizing systolic Ca2 + in HF atrial myocytes. Thus, during β-AR stimulation the increase in excitation contraction coupling gain and fractional release of Ca2 + from the SR 2 + transient to the cell centre and restore systolic Ca2 + despite the loss of atrial t-tubules in HF In the present study we find that, during β-AR stimulation, the amplitude of the systolic Ca2 + content from the amplitude of the caffeine evoked Ca2 + transient, should SR Ca2 + content preferentially increase during β-AR stimulation in HF atrial cells following current clamp stimulation (the in vivo situation) this would also provide a conducive pathway to DAD formation and act as an important precursor for new onset AF 2 + content following β-AR stimulation during current clamp stimulation, the shorter action potential duration noted in HF atrial cells, via reductions in effective refractory period would also serve to promote arrhythmias although via re-entrant mechanisms.Although not addressed specifically in the present study, due to the difficulty in quantitatively measuring changes of SR Ca4.52 + transient and SR dependent Ca2 + uptake. Whilst changes in SERCA expression or activity have only modest effects on SR Ca2 + content 2 + content on SERCA activity 2 + content was increased in voltage-clamped HF atrial cells. Importantly, the changes in SERCA activity were noted despite the decrease in Ca2 + buffering power and therefore, as presented, likely represent an underestimate of the actual decrease in SERCA function. Whilst we were unable to detect changes in SERCA, total PLN, Ser-16 phosphorylated PLN or the ratio of PLN to SERCA, we did observe an increase in Thr-17 phosphorylated PLN, CAMKII, PP1, PP2a and GRK-2 expression. Thus the reported changes in protein expression in HF atria do not readily account for the observed reduction in SERCA activity. In the atria SERCA is also regulated by an additional protein, sarcolipin A major observation in the present study is that SERCA activity is reduced in HF atrial cells resulting in reductions to the rate of decay of the systolic CaICa-L in HF atrial cells is consistent with the increased expression of the G-protein receptor kinase GRK-2 ICa-L has on SR Ca2 + content, the increases in GRK-2, PP1 and PP2a may be important in maintaining the increased SR Ca2 + content in HF atrial cells during β-AR stimulation because they attenuate any β-AR mediated increase in ICa,L.The failure of β-AR stimulation to completely restore SERCA function and 4.62 + homeostasis and the response to catecholamine stimulation are altered in the atria in HF. Whilst the changes in cellular Ca2 + homeostasis and, in particular SR Ca2 + content, may provide a substrate for the generation of Ca2 + dependent arrhythmias we have not established if the threshold SR Ca2 + content is altered in HF, if HF atrial myocytes are more excitable due to concomitant alterations in K+ channel expression or function e.g. 2 + release and DADs were more prevalent in HF atrial myocytes ICa-L a holding potential of − 40 mV was used and we did not correct for any liquid junction potential artefacts (~ 8 mV).We have examined how intracellular CaFor the in vivo assessments of cardiac function by ECG and echocardiography animals were gently restrained. As such we cannot exclude the possibility that adrenergic outflow may be influencing our measures of cardiac function in both control and HF animals; and potentially differentially. However, the use of sedation or performing such measurements in anaesthetized animals would also be expected to influence these measurements and again may do so differentially.2 + uptake rate and impaired ICa-L that are present under basal and β-AR stimulation conditions in HF atrial myocytes. Moreover, we did not determine if changes in PMCA activity occur in this model of HF and therefore whether the correction factor non-NCX mediated Ca2 + extrusion differs between control and HF myocytes. However, even if PMCA activity were to change by 50% this would result in an under or overestimation of SR Ca2 + content by less than 10%.In the present study we have used Western blotting to measure the expression of protein phosphatases and kinases and have not investigated their activity directly. However, the expression of protein phosphatases and G-protein receptor kinases has previously been demonstrated to correlate directly with their activity 52 + homeostasis that occur as a consequence of HF induced by right ventricular tachypacing. Specifically, the decrease in ICa-L and previously reported loss of atrial t-tubules in this model of HF serve to decrease the systolic Ca2 + transient amplitude and increase SR Ca2 + content in voltage-clamped cells. However, the changes in SR Ca2 + content observed in voltage-clamped cells, are insufficient to prevent the smaller rise of systolic Ca2 +. Similarly, the observed decrease in cellular Ca2 + buffering capacity is also insufficient to maintain systolic Ca2 + although it does contribute to an increased rate of Ca2 + removal from the cell via the sarcolemmal pathways despite the reduction in NCX current in the atria in heart failure.In summary we describe a number of changes to atrial CaBritish Heart Foundation , European 6th Framework Award (Normacor), University of Manchester Biomedical Research Centre Award (George Lancashire Award) and Wellcome Trust Institutional Strategic Support Fund (ISSF) award (097820).This work was supported by grants from the None.


Most of the research on Ca2+ signaling in plants has been focused on the transport mechanisms for Ca2+ across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca2+ signals, but how intracellular organelles such as mitochondria are involved in the process of Ca2+ signaling is just emerging. The combination of the molecular players and the elicitors of Ca2+ signaling in mitochondria together with newly generated detection systems for measuring organellar Ca2+ concentrations in plants has started to provide fruitful grounds for further discoveries. In the present review we give an updated overview of the currently identified/hypothesized pathways, such as voltage-dependent anion channels, homologs of the mammalian mitochondrial uniporter (MCU), LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore (PTP), that may contribute to the transport of Ca2+ across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca2+ homeostasis for ensuring optimal bioenergetic performance of this organelle.Plants, being sessile organisms, have evolved the ability to integrate external stimuli into metabolic and developmental signals. A wide variety of signals, including abiotic, biotic, and developmental stimuli, were observed to evoke specific spatio-temporal Ca In animals, when PTP opens only transiently, it possibly mediates Ca2+ release from mitochondria  and function have been identified  mediated by VDACs is unclear, however AtVDAC1 has been shown to interact in two-hybrid yeast system with CBL1, a Ca2+-sensor  selectivity filter sequence (http://aramemnon.uni-koeln.de/). Whether all these isoforms are indeed targeted to mitochondria, whether they form ion channels able to provide a permeation pathway for Ca2+ and whether the various isoforms operate in different tissues and/or at different developmental stages still awaits clarification. Recently obtained experimental evidence indicates that at least one of the isoforms is indeed targeted to mitochondria in Arabidopsis  in vitro -binding domains are located in the cytosol, possibly sensing the cytosolic glutamate/aminoacid concentration  that show high aminoacid sequence similarity to SPG7  and to AFG3L2 . Among those with the highest score, AtFtsH3 and AtFtsH10 are present in mitochondria , the above-described uptake and efflux pathways for calcium might cooperate. For example, Caoduction .In summary, a combination of genetics, fluorescent probe imaging, electrophysiology, bioenergetics and physiology will hopefully provide answers to the numerous open questions related to mitochondrial calcium homeostasis in plants.All authors contributed to the works published by our groups which are described in the minireview. Furthermore, all authors actively participated in writing the manuscript.The 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.


In addition, alteration of intracellular Ca2+ signaling underlies the pathophysiology of schizophrenia, while it remains unclear how typical or atypical antipsychotics affect intracellular Ca2+ mobilization in microglial cells. This mini-review article summarizes recent findings on cellular mechanisms underlying the characteristic differences in the actions of antipsychotics on microglial intracellular Ca2+ signaling and reinforces the importance of the ER of microglial cells as a target of antipsychotics for the treatment of schizophrenia.Microglia are resident innate immune cells which release many factors including proinflammatory cytokines, nitric oxide (NO) and neurotrophic factors when they are activated in response to immunological stimuli. Recent reports show that pathophysiology of schizophrenia is related to the inflammatory responses mediated by microglia. Intracellular Ca Both CaMKII and NCS-1 have also been reported to be involved in the pathophysiology of schizophrenia , which leads to the change of gamma rhythms and the impairment of cognitive functions observed in patients suffered from schizophrenia. In addition, dysregulation of the redox signaling pathway might provide an explanation for the developmental origins of schizophrenia because there appears to be a link between maternal viral infections during gestation and the incidence of schizophrenia. During viral infections, the increase of the IL-6 release and the resultant activation of redox signaling pathway promote the hypofunction of NMDARs in the GABAergic interneurons  depends on a number of different types of voltage- or ligand-gated ion channels that are permeable to inorganic ions such as sodium, potassium, chloride and calcium. While the former three ions predominantly support the electrogenic role, Caa Lidow, . A possin et al. , based o2+ currents mediated by L-type voltage-gated calcium channels recorded in olfactory neuroepithelial cells obtained from patients with schizophrenia were 50% smaller than those from healthy subjects. Because these patients with schizophrenia were taking antipsychotics, the finding does not simply support the genetic studies suggesting that dysfunction of L-type voltage-gated calcium channels occurs in patients with schizophrenia.Recently, there are many reports that have shown that possible involvement of single-nucleotide polymorphisms (SNPs) within two L-type voltage-gated calcium channel subunits, CACNA1C and CACNB2, and neuropsychiatric disorders including schizophrenia, suggesting that dysfunction of L-type voltage-gated calcium channels occurs in patients with schizophrenia  receptors on the ER membrane. Stimulation of G protein-coupled metabotropic receptors results in the activation of the phospholipase C (PLC), production of two second messengers including the diacylglycerol (DAG) and the InsP3 and the release of Ca2+ from the ER. Importantly, the depletion of ER activates the store-operated Ca2+ entry (SOCE), known as a capacitative Ca2+ influx, mediated by plasmalemmal channels such as calcium release-activated Ca2+ (CRAC) channels and/or transient receptor potential (TRP) channels plasmic reticulum Ca2+-ATPases (SERCA). The concentration of Ca2+ in the ER is precisely controlled by SERCA. The influx of Ca2+ through the TRP channels plays an important role in many inflammatory processes including the activation of microglia  or atypical (risperidone) antipsychotics affects TG-induced intracellular Ca2+ mobilization, which represents a cellular stress response. In rodent microglial cells, we observed that opposite effects of haloperidol and risperidone on the TG-induced intracellular Ca2+ mobilization . There are two other reports showing opposite effects of haloperidol and risperidone on intracellular Ca2+ mobilization. In cultured astrocytes derived from rat cortex and striatum, intracellular Ca2+ imaging showed that pretreatment with risperidone but not haloperidol suppressed the dopamine-induced increase in [Ca2+]i (Reuss and Unsicker, 2+]i, while risperidone completely suppressed it, suggesting that opposite effects of haloperidol and risperidone on rotenone-induced neurotoxicity could be mediated by their differential effects on intracellular Ca2+ mobilization (Tan et al., 2+ signaling triggers the activation of cell death programs (Orrenius et al., 2+ mobilization, which might lead to the differences in the actions of typical and atypical antipsychotics on the induction of the ER stress response, promotion of the inflammatory responses and/or initiation of apoptosis in microglia (Figure In some electrically non-excitable cells such as macrophages, adipocytes, β-cells and oligodendrocytes, perturbation of the calcium homeostasis in the ER results in the accumulation of unfolded proteins, the induction of the ER stress response, the promotion of the inflammatory processes and the initiation of apoptosis (Zhang and Kaufman, a Figure .2+]i elevation, which was mediated by an initial PLC/InsP3-driven Ca2+ release from the ER that followed by a long-lasting activation of the SOCE via the up-regulation of cell-surface TRPC3 channels in rodent microglial cells (Mizoguchi et al., 2+ mobilization in microglia.Brain-derived neurotrophic factor is also well known for its involvement in the pathophysiology of neuropsychiatric disorders including schizophrenia (Autry and Monteggia, 2+]i elevation in microglial cells (Kato et al., 2+ is the ER. suggesting the importance of the ER as a therapeutic target of antipsychotics for the treatment of schizophrenia.There is increasing evidence suggesting that pathophysiology of schizophrenia is related to the inflammatory responses mediated by microglia (Müller and Schwarz, 2+]i, suggesting the importance of microglial intracellular Ca2+ signaling as a target of antipsychotics for the treatment of schizophrenia. Although the electrical activity of neurons mainly depends on voltage-gated calcium channels and NMDARs, the generation of intracellular Ca2+ signaling in non-excitable cells such as microglia is mainly regulated by the ER. These suggest the importance of the ER as a therapeutic target of antipsychotics for the treatment of schizophrenia.Microglia can release many factors including proinflammatory cytokines, NO and BDNF when they are activated in response to immunological stimuli. There is increasing evidence suggesting that pathophysiology of schizophrenia is related to the inflammatory responses mediated by microglia. In addition, we have previously reported that pretreatment with antipsychotics significantly inhibits the release of proinflammatory cytokines and/or NO from activated microglial cells, possibly through the suppression of the elevation of [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.