Parasitic diseases such as malaria, leishmaniasis, trypanosomiasis and schistosomiasis are responsible for millions of deaths each year worldwide. The genomes of many pathogenic parasites have recently been sequenced, opening the way for rational design of targeted therapies. We analyzed genomes of pathogenic protozoan parasites as well as the genome of Schistosoma mansoni, and show the existence within them of genes encoding homologues of mammalian intracellular Ca2+ release channels: inositol 1,4,5-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs), two-pore Ca2+ channels (TPCs) and intracellular transient receptor potential (Trp) channels. The genomes of Trypanosoma, Leishmania and S. mansoni parasites encode IP3R/RyR and Trp channel homologues, and that of S. mansoni additionally encodes a TPC homologue. In contrast, apicomplexan parasites lack genes encoding IP3R/RyR homologues and possess only genes encoding TPC and Trp channel homologues (Toxoplasma gondii) or Trp channel homologues alone. The genomes of parasites also encode homologues of mammalian Ca2+influx channels, including voltage-gated Ca2+ channels and plasma membrane Trp channels. The genome of S. mansoni also encodes Orai Ca2+ channel and STIM Ca2+ sensor homologues, suggesting that store-operated Ca2+ entry may occur in this parasite. Many anti-parasitic agents alter parasite Ca2+ homeostasis and some are known modulators of mammalian Ca2+ channels, suggesting that parasite Ca2+ channel homologues might be the targets of some current anti-parasitic drugs. Differences between human and parasite Ca2+ channels suggest that pathogen-specific targeting of these channels may be an attractive therapeutic prospect.Ca Plasmodium, Toxoplasma, Cryptosporidium and Babesia, as well as several species of the kinetoplastid Trypanosoma and Leishmania parasites. Other parasitic protozoa that are major contributors to worldwide disease include Trichomonas vaginalis, Entamoeba histolytica and Giardia intestinalis. Another major cause of human disease and mortality, second only to malaria amongst parasitic diseases, is the Schistosoma family of parasitic blood flukes. Many of these parasites maintain stringent control over their intracellular Ca2+ concentration 2+ signals in response to physiological stimuli. These Ca2+ dynamics are critical for parasite function, egress, invasion, virulence and survival 2+ responses is largely unknown.Parasitic diseases collectively affect billions of people and cause millions of deaths worldwide each year Table 1.2+ release via intracellular Ca2+ channels controls numerous cellular processes, ranging from receptor signalling to growth and apoptosis 3Rs), ryanodine receptors (RyRs), two-pore channels (TPCs), and some transient receptor potential (Trp) channels. Mammalian IP3Rs and RyRs are responsible for release of Ca2+ from endoplasmic reticulum (ER) in response to the intracellular messengers IP3 and Ca2+ (IP3Rs), or cyclic ADP ribose (cADPR) and Ca2+ (RyRs), produced via a wide variety of cellular signalling pathways 2+ release from acidic organelles, including lysosomes 2+ from intracellular stores 2+ stores within their ER, mitochondria, glycosomes and acidocalcisomes 2+ channels are present in these parasite organelles and whether they mediate Ca2+ release are unknown.Ca2+ entry in mammalian cells is mediated by plasma membrane Ca2+ channels such as voltage-gated Ca2+ (Cav) channels 2+ stores following intracellular Ca2+ release. Mechanisms of Ca2+ influx and demand for it are likely to differ between life cycle stages of many parasites, due to the differential ionic conditions of their respective environments. The intracellular location of many protozoan parasites during part of their life cycles 2+ concentrations in the mammalian host cell are maintained at low levels, suggests that novel Ca2+ entry pathways may be essential for parasite survival. In the extracellular trematode parasite S. mansoni, Ca2+ influx channels are important for muscle contraction and viability S. mansoni Cav channels 2+ influx channels in pathogenic parasites remain largely unknown.Ca2+ channel activity in the plasma membrane or intracellular organelles has profound effects on cell function and survival in many organisms. This suggests that the channels underlying Ca2+ signals in parasites might represent novel drug targets. Recent advances in genomics have led to whole-genome sequencing of several parasites that are pathogenic to humans  and 31% identity with human RyR1 (hRyR1) in the full-length proteins; and one more similar to mammalian RyRs (XP_002578860), which showed 45% identity with hRyR2 and 32% identity with hIP3R1 (3R/RyR homologues (of either pore or full-length sequences) were found in the genomes of any of the apicomplexan parasites, or in the protozoan parasites E. histolytica, G. intestinalis and T. vaginalis.Mammalian IPion  and Table 2Leishmania, Trypanosoma and S. mansoni parasite proteins show a high degree of sequence similarity to IP3Rs/RyRs in the pore region, it was necessary to examine similarity in other regions before establishing them as potential functional correlates. Defining characteristics of vertebrate IP3Rs and RyRs include their large size (∼5000 residues for RyRs and ∼2700 residues for IP3Rs) and the presence of: multiple transmembrane domains near their C-terminal ends, conserved N-terminal MIR  domains, conserved RIH  sequences with homology to the IP3-binding core of IP3Rs 3Rs Although the 3R/RyR pore homologues are large proteins (2216-4998 residues)  resulted in alignment with the same Leishmania, Trypanosoma and S. mansoni proteins found in the pore homology search. However, alignments suggested that like RyRs, the parasite homologues lack many of the ten basic residues known to be important for high-affinity binding of IP3 to mouse IP3R1 data not shown). The S. mansoni protein (XP_002576843/42), which has the greatest sequence similarity to full-length human IP3Rs, also has the highest conservation of these basic residues (5 out of 10) (data not shown). All parasite homologues are predicted to contain multiple putative transmembrane domains near their C-termini, and to contain N-terminal domains with similarity to the suppressor domain of mammalian IP3Rs and the analogous A-domain of RyRs S. mansoni homologues have N-terminal MIR domains, while the S. mansoni RyR homologue, like mammalian RyRs, has four copies of a RyR motif and multiple copies of an SPRY domain  Figure 13Rs/RyRs, including the conserved GGGXGD selectivity filter motif  differ from human RyRs in the pore-lining transmembrane domain, at the position analogous to Q4934 of hRyR1, which is a determinant of ryanodine binding 3Rs and RyRs in which key functional domains are well enough conserved to suggest that these proteins might function as intracellular Ca2+ channels. However, the sensitivity of these parasite channels to drugs may differ sufficiently from those of their host to perhaps allow their selective targeting by drugs.The close similarity of the pore regions of parasite channels to those of mammalian IPer motif Figure 12+ from acidic organelles in mammalian cells. A defining characteristic of both mammalian and plant TPCs is the presence of two independent pore-forming domains in series, each consisting of multiple transmembrane domains S. mansoni and T. gondii  as well as the S. mansoni and T. gondii TPC homologues themselves were then used to search for additional parasite homologues, but none were identified. The parasite TPC homologues identified here lack the EF-hand repeat found in plant AtTPC1 channels  in mouse TrpML1 2+ release from vacuolar stores of Saccharomyces cerevisiaeIn addition to IPchannels Table 2.2+-permeable Trp channels located in the plasma membrane of mammalian cells include TrpC, TrpA, TrpV and TrpP subtypes 2+ entry in many cells T. vaginalis had a homologue of TrpV channels. In contrast, S. mansoni was found to contain TrpA and TrpC channel homologues (S. mansoni (XP_002576118) and T. vaginalis (XP_001296819) in the current study (Camologues Table 2.2+ entry (SOCE) is an almost ubiquitous feature of mammalian cells, where plasma membrane channels composed of Orai subunits mediate Ca2+ influx in response to emptying of intracellular Ca2+ stores 2+ depletion and are necessary for activation of Orai channels Plasmodium falciparumStore-operated CaS. mansoni genome contains a gene encoding an Orai1 homologue (XP_002578837), with an alternatively spliced variant (XP_002578838)  , which shows pronounced (but not perfect) similarity with Cav2B, but insufficient sequence was available to conclusively categorize this protein. Cav channel homologues were also found in all other parasites examined except Plasmodium spp., Cryptosporidium spp., T. vaginalis, G. intestinalis and E. histolytica  that are responsible for Ca2+ entry into mammalian spermatozoa v homologues identified above. Some of the Cav channel homologues identified in parasites are predicted to possess 18–24 transmembrane domains  channels are thought to have evolved from Cav channels and share a high degree of sequence similarity with them v homologues identified here may be selective for Na+ rather than Ca2+ (or they may be non-selective), although experimental testing will be required to determine this. However, all parasite homologues identified lacked the characteristic DEKA selectivity filter motif of mammalian Nav channels, which is necessary for Na+ selectivity v channel homologues possess several regularly spaced basic residues in the TMD4 region of each domain  cation channels can mediate Ca2+ influx in mammalian cells T. vaginalis which all contain the pore motif GYGD reminiscent of potassium channels +-selective channels rather than Ca2+ channels, although this requires experimental testing.Given the relative sparsity of Ca2+-permeable mammalian NMDA receptors, kainate receptors, AMPA receptors, pannexins, P2X4 purinergic receptors and nicotinic acetylcholine receptors. No convincing homologues  of these channels were found in any of the protozoan parasite genomes examined. In contrast to protozoan parasites, S. mansoni is known to have homologues of some of these receptors, such as nicotinic acetylcholine receptors We also searched protozoan genomes for genes encoding homologues of potentially Ca2+ channels are critical for many of the most fundamental cellular processes and pharmacological modulation of these channels can lead to marked changes in cell growth and viability. These channels therefore represent attractive drug targets for treatment of infectious disease. Complex Ca2+ signalling processes exist in parasites and Ca2+-handling machinery including Ca2+-ATPases is present in these organisms 2+ channels and Ca2+ influx channels are present in many of the most clinically relevant pathogenic parasites (http://eupathdb.org/eupathdb) 2+ channel homologues, along with the occurrence of physiological Ca2+ signals in parasites, suggests that they participate in Ca2+ signalling within these cells. Our analysis has attempted to distinguish intracellular from plasma membrane Ca2+ channels because the distribution of Ca2+ channels profoundly affects their regulation, the amount of Ca2+ to which they have access, and the subcellular organization of the Ca2+ signals they evoke 2+ channels can function effectively in both intracellular organelles and the plasma membrane 2+ concentrations or to the 10,000-fold lower Ca2+ concentration of their host cell's cytosol, may perhaps exploit this plasticity in targeting of Ca2+ channels even more extensively than mammalian cells. Despite the difficulty of unambiguously assigning Ca2+ channel homologues to specific membrane compartments on the basis of their primary sequence alone, subsequent sections consider the parasite homologues by reference to the distribution of their mammalian counterparts.Caarasites Table 2.2+ stores including ER, mitochondria, glycosomes and acidocalcisomes, and biochemical signalling pathways analogous to those of mammalian cells have been shown to exist in parasites. Evidence exists for the presence of phospholipase C signalling pathways in parasites 3-based effects on IP3R homologues. The intracellular messenger cADPR that activates mammalian RyRs has also been shown to be involved in parasite signalling pathways 3R/RyR homologues shown in this study to exist in Leishmania, Trypanosoma and S. mansoni parasites remains to be tested. However, previous observations indicate that IP3 lacks Ca2+-releasing activity in Trypanosoma parasites 3R/RyR homologues, like mammalian RyRs, lack many of the basic residues required for high-affinity binding of IP3 in mouse IP3R1 3R/RyR homologues in these organisms might respond to a different intracellular messenger (such as cADPR), or that they might require specific conditions for activity in response to IP3.Pathogenic parasites contain a variety of intracellular Ca3R/RyR homologues, as reported by others 3R/RyR homologues in these parasites, IP3 has been reported to elicit Ca2+ release from intracellular stores of Plasmodium chabaudiE. histolytica2+ release has been attributed to IP3R/RyR-like channels in T. gondiiPlasmodium berghei3-sensitive intracellular Ca2+ channel exists in apicomplexan parasites. A similar situation is seen in S. cerevisiae, which lacks IP3R/RyR homologues, but has been reported to show IP3-induced Ca2+ release from vacuolar vesicles 2+ release from vacuolar stores of S. cerevisiae23 is not yet known. Our searches found no homologues of Yvc1p among the genomes of pathogenic apicomplexa. However, we did identify apicomplexan homologues of the PIP2-sensitive mammalian endolysosomal TrpML1 channel 3 has not been tested, so whether these homologues might mediate the IP3-sensitive Ca2+ release in apicomplexan parasites remains uncertain. The molecular determinants of IP3-evoked Ca2+ release in apicomplexan parasites therefore remain to be determined. The Trp, TPC, Cch1 and Cav channel homologues identified in this study  contain Nudix hydrolase domains, suggesting that they might be involved in signalling pathways utilizing nucleoside diphosphate derivatives such as ADPR or cADPR, as is the case with mammalian TrpM channels 2 to mammalian TrpML channels and subsequent channel activation 2+ channels identified in this study release Ca2+ from acidocalcisomes or other organelles in response to NAADP, ADPR, cADPR, PIP2, IP3, Ca2+, or other intracellular messengers therefore remains to be determined.The parasite acidocalcisome is similar in several ways to mammalian lysosomes from which TPCs 2+ influx mediates physiological signalling pathways in parasites 2+ stores following intracellular Ca2+ release. The importance of Ca2+ influx pathways for cell survival in general has been demonstrated in S. cerevisiae, where lack of the plasma membrane Cch1 Ca2+ channel impairs high-affinity Ca2+ uptake, and leads to cell death in conditions of low Ca2+ concentration or when Ca2+ influx is required Leishmania parasites, Ca2+ influx is necessary for thermotolerance 2+ levels are maintained at low levels . These parasites therefore seem likely to require unique strategies in order to enhance Ca2+ uptake. Residence of some parasites within parasitophorous vacuoles or other membraneous compartments 2+, but parasite plasma membrane Ca2+ channels or transporters with novel properties may also be essential, especially for those parasites directly exposed to the cytosol, such as T. cruzi2+ channel homologues described in this study may differentially contribute to Ca2+ influx during different phases of parasite life cycles and may have novel properties that allow their function in ionic conditions that differ substantially from those experienced by their mammalian counterparts.Cav channel homologues were found in many of the pathogenic parasites examined, suggesting that these proteins have a widespread function in parasites. The presence in these homologues of charged regions analogous to the voltage sensors of Cav channels suggests that they might be gated by transmembrane voltage, although this remains to be tested experimentally. Homologues of mammalian Ca2+-permeable TrpA, TrpC and TrpV subtypes capable of mediating plasma membrane Ca2+ influx were absent from most protozoan parasites. In contrast, TrpA and TrpC channel homologues were found in S. mansoni, although their exact subtype categorization was unclear. Since in addition to mediating intracellular Ca2+ release, TrpP channels are also capable of mediating Ca2+ influx in mammalian cells when located in the plasma membrane 2+influx in parasites. Mammalian Trp channels are modulated by diverse stimuli S. mansoni.Ca2+-sensing STIM proteins responsible for activation of Orai proteins 2+-sensing subunits of SOCE channels in these organisms. In mammalian cells, TrpC channels have also been shown to contribute to SOCE in a variety of cells S. mansoni has an alternatively spliced Orai1 homologue and a STIM1 homologue, suggesting that Orai/STIM-mediated SOCE exists in this parasite.Homologues of the Orai channels which mediate SOCE in mammalian cells 2+ channel homologues identified in this study will in future need to be measured experimentally in order to confirm their status as Ca2+ channels. Homologues of other mammalian non-selective cation channels may also be found in parasites and may contribute to Ca2+ signalling pathways in these organisms. In addition, some of the Ca2+ channel homologues identified in this study may play roles in flux of other ions such as Na+ or K+, perhaps in a lifecycle-dependent manner, given the substantial changes in ionic composition of the extracellular environment during different stages of the lifecycle in many parasites.The expression and function of the putative CaPlasmodium spp., T. gondii and Cryptosporidium spp.) with their free-living ciliate relative Paramecium reveals differences in the complement of genes encoding Ca2+ channels. IP3R/RyR homologues are absent in the Apicomplexa examined, whereas they are present in Parameciumv channels with a four-domain architecture similar to mammalian Cav channels are absent in the Apicomplexa , whereas they are present in Paramecium (data not shown). Whether the apparent absence of IP3R/RyR and Cav channels in Apicomplexa occurred as a result of the acquisition of a parasitic existence is unclear. In contrast, both the Apicomplexa examined here and Paramecium (data not shown) appear to have Trp channel homologues. ParameciumP. falciparumParamecium (data not shown), suggesting that lack of Orai homologues in Apicomplexa may not be a result of transition to parasitism. The channel-forming proteins underlying SOCE in these organisms remain to be identified.Comparison of the genomes of the parasitic Apicomplexa examined here (Trypanosoma spp. and Leishmania spp.) with their free-living choanoflagellate relative Monosiga brevicollis also reveals differences. Both M. brevicollis3Rs, Cav and Trp channels, although M. brevicollis has a greater diversity of these channels M. brevicollisA comparison of the flagellate parasites examined here  2+ signalling. We speculate that in addition to affecting their currently known targets, such as the plasmodial surface anion channel in the case of dantrolene vβ subunits in the case of praziquantel 2+ channel homologues described in this study and hence perturb Ca2+ signalling pathways involved in parasite survival. In particular, we suggest that suramin, a known RyR agonist 3R homologues shown in this study to exist in Trypanosoma, Leishmania and S. mansoni parasites. This represents a potentially novel mechanism of action for these clinically useful drugs.A role for Ca2+ channels in parasites relative to the wide array of isoforms in mammalian cells suggests that Ca2+ signalling in parasites may rely on a less redundant repertoire of Ca2+ channels. This characteristic may increase the susceptibility of parasites to drugs that target these channels.The apparent scarcity of Ca2+ channel homologues show significant similarity to their mammalian counterparts, sequence differences in functionally and pharmacologically important domains (eg. the ion-conducting pore region) exist, such as those shown to exist between human and parasite IP3R/RyR homologues , Plasmodium vivax SaI-1 (Jan 2008), Plasmodium knowlesi strain H (Apr 2008), Toxoplasma gondii ME49 (May 2008), Babesia bovis T2Bo (Mar 2008), Cryptosporidium hominis TU502 (Mar 2008), Cryptosporidium muris RN66 (Oct 2008), Cryptosporidium parvum Iowa II (Nov 2006), Leishmania braziliensis MHOM/BR/75/M2904 (Oct 2007), Leishmania infantum JPCM5 (May 2007), Leishmania major Friedlin (Mar 2008), Trypanosoma brucei TREU927 (Dec 2006), Trypanosoma cruzi CL Brener (Dec 2007), Entamoeba histolytica HM-1:IMSS (Jun 2010), Giardia intestinalis (Mar 2008), Trichomonas vaginalis G3 (Mar 2008), and Schistosoma mansoni (Jul 2011). No homologues of any Ca2+ channels examined were identified in the genomes of E. histolytica or G. intestinalis. Other genomes analyzed include those of: Homo sapiens (Dec 2010), Caenorhabditis elegans (Apr 2011), Drosophila melanogaster (May 2011), Paramecium tetraurelia (Mar 2008) and Ciona intestinalis (Sept 2008).The genomes of the following pathogenic parasites were examined (annotation release date in parentheses): 3R1  or RyR1 , and full-length human sequences of TrpA1 , TrpV1 , TrpC1 , CNGA1 , CNGB1 (NP_001288), NMDA receptor NR1 (Q05586), NMDA receptor N2 (Q12879), AMPA receptor GRIA1 (P42261.2), kainate receptor GRIK1 (P39086), nAChR-alpha1 (ABR09427), purinergic receptor P2X4 (NP_002551.2), pannexin-1 (AAH16931), Orai1 (NP_116179.2), STIM1 (AAH21300), TPC1 (NP_001137291.1), TPC2 (NP_620714.2), TrpP1 (NP_001009944), TrpP2 (NP_000288), TrpM1 (NP_002411), TrpML1 (NP_065394), CatSper1 (Q8NEC5.3), mitochondrial uniporter (NP_612366.1) and Cav1.2 (NP_955630.2). Sequences of the S. cerevisiae Ca2+ channel subunit Cch1 (CAA97244) and Arabidopsis thaliana TPC1 (AAK39554) were also used to search for parasite homologues.BLASTP analysis was carried out in all cases, using the following human sequences (GenBank accession number in parentheses): full-length or pore sequences of IPH. sapiens, C. elegans, D. melanogaster and C. intestinalis were carried out against the National Center for Biotechnology (NCBI) genomic protein databases. Searches of the P. tetraurelia genome were carried out using the BLASTP facility of ParameciumDB (v.1.59) (http://paramecium.cgm.cnrs-gif.fr) S. mansoni genome was searched using the BLASTP search facility of GeneDB (http://www.genedb.org/blast/submitblast/GeneDB_Smansoni) and S. mansoni BLASTP server twinscan2 peptide prediction facility (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/s_mansoni) hosted by the Wellcome Trust Sanger Institute, UK 3R/RyR homologues) using default parameters. After using GBLOCKS at low stringency to remove regions of low confidence, and removing gaps, Maximum Likelihood analysis was carried out using PhyML v3.0 . Phylogenetic trees were depicted using TreeDyn (v198.3). MUSCLE, GBLOCKS, PhyML and TreeDyn were all functions of Phylogeny.fr (http://www.phylogeny.fr/) BLASTP searches of the genomes of protozoan parasites,


TD in animals has been used to model aging-associated neurodegeneration in the brain. The mechanisms of TD-induced neuron death are complex, and it is likely multiple mechanisms interplay and contribute to the action of TD. In this study, we demonstrated that TD significantly increased intracellular calcium concentrations i and protected cortical neurons against TD-induced cytotoxicity, whereas down-regulation of ADAR2 increased AMPA-elicited Ca2+ influx and exacerbated TD-induced death of cortical neurons.TD drastically potentiated AMPA-triggered calcium influx and inhibited pre-mRNA editing of GluR2, a CaOur findings suggest that TD-induced neuronal damage may be mediated by the modulation of ADAR2-dependent RNA Editing of GluR2. Thiamine vitamin B1) deficiency (TD) induces chronic mild impairment of oxidative metabolism and causes neuroinflammation, leading to neuronal loss in specific brain regions  deficien,3. Since2+) can enter neurons through several mechanisms. One important mechanism is through the activation of glutamate receptors i was measured using the fluorescent Ca2+ chelator Fura-2. As shown in Figure 2+]i; [Ca2+]i was approximately 200 nM and 900 nM in control and TD cultures, respectively, suggesting that TD increased the influx of Ca2+. Since AMPARs are important mediators of Ca2+ influx, we sought to determine whether TD affected AMPA-elicited Ca2+ influx. Cells were incubated with a low-affinity fluorescent Ca2+ probe Fluo-3 and challenged with a brief pulse (5 sec) of AMPA (30 μM). More than 95% of the monitored cells responded to AMPA stimulation and TD4 increased Fluo-3 fluorescence intensity . Thus, AMPA-mediated calcium elevations in cultured cells are primarily through Ca2+-permeable AMPARs. More importantly, TD4 drastically potentiated AMPA-elicited Ca2+ influx  of ADAR2. As shown in Figure 2+]i in cortical neurons; contrarily, down-regulation of ADAR2 enhanced AMPA-mediated Ca2+ influx, indicating the manipulation of ADAR2 expression was sufficient to alter Ca2+ permeability of AMPARs channels  does not significantly alter [Ca2+]i whereas TD4 raises [Ca2+]i from approximately 200 nM to 900 nM. Consistent with the profile of alterations in [Ca2+]i, TD4, but not TD1, induces the death of cortical neurons (data not shown). The close correlation suggests an association between Ca2+ influx and TD neurotoxicity. Further studies are necessary to confirm that TD-induced neurotoxicity is caused by the rise of [Ca2+]i. Ca2+ ions are necessary for proper function and survival of neurons. Ca2+ is a key regulator of synaptic plasticity. However in many pathological states, [Ca2+]i reaches critical levels leading to cell injury or death i.We have previously demonstrated that TD induces ER stress in animals and in cultured neurons . ER stre2+ levels are increased in aging neurons and the dysregulation of [Ca2+]i is implicated in neurodegeneration that occurs in AD [2+ influx and raises [Ca2+]i in neurons, resulting in neuronal death i [2+]i may be caused by the dysregulation of Ca2+ -permeable AMPARs. GluR2 is a critical subunit in determining many of the major biophysical properties of AMPARs, including, but not limited to, Ca2+ permeability, receptor kinetics, single-channel conductance and blockage by endogenous polyamines  [Ca [Ca2+]i . A TD-inlyamines . The gres (ALS)] .2+-permeable AMPARs and enhanced toxicity following neurological insults i. Thus, ADAR2-dependent GluR2 Q/R site editing may determine vulnerability of neurons to TD.The contribution of ADAR2 in AMPA-mediated Ca, (2006)  show tha, (2006) . Taken tFura-2 was obtained from Invitrogen . Fluo-3 was obtained from Calbiochem . Anti-GluR2 antibody was obtained from Chemicon . Anti-ADAR2 and ADAR1 antibodies were obtained from Santa Cruz Biotech . The secondary Texas Red conjugated donkey anti-rabbit IgG for GluR2 and FITC-conjugated donkey anti-Goat IgG for ADAR2 were obtained from Jackson ImmunoResearch Laboratories . All other chemicals were obtained from Sigma Chemical Co. .2 for 7 days in vitro (DIV7).Primary cultures of cortical neurons were generated using a previously described method ,58. Briein vitro, cortical neurons of DIV7 were treated with amprolium (1 mmol/L) for indicated times i were measured using Fura-2/acetoxymethyl ester (Fura-2/AM) as described previously with some modifications [2+]i by using the equation of [Ca2+]i = Kd*(F-Fmin)/(Fmax-F) [Kd = 0.224 mM. The Rmin and Rmax were determined by equilibrating [Ca2+]i with "calcium-free" (0 mM Ca2+/10 mM EGTA) or high Ca2+(10 mM Ca2+ in Hepes), respectively.The concentrations of intracellular free calcium [Caications . The mea(Fmax-F)  with app2+]i levels were also quantified by single cell Fluo-3 fluorescence image as described previously [2+]i. The quantification of Fluo-3 fluorescence intensity was performed using the software Rel.3.2 .[Caeviously  with somNeuronal cultures on cover slips were treated with amprolium for indicated times and fixed with 4% paraformaldehyde. The cultures were incubated with primary antibodies at 4°C overnight at the following concentrations: Anti-GluR2 antibody, 0.5 μg/ml; anti-ADAR2 antibody, 1:500. This was followed by secondary antibody treatment (Texas Red conjugated donkey anti-rabbit IgG or FITC-conjugated donkey anti-Goat IgG at a dilution of 1:100). Fluorescent images were visualized with a Zeiss LSM 510 confocal system  using a 40× oil-immersion objective lens. Differential interference contrast (DIC) images were also taken for comparison.The immunoblotting procedure has been previously described . BrieflyTotal RNA was isolated from cultured neurons with TRIzol reagent  followed by digestion with RNase-free DNase I  to eliminate possible DNA contamination. First-strand cDNA was synthesized by moloney murine leukemia virus reverse transcriptase and Oligo dT primers  .cDNA (100 ng) was subjected to PCR in a 20 μL reaction solution containing 2× Taq Premixure buffer  and 20 μM primer. The PCR amplification protocol was 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 15 s and finished with a final extension of 72°C for 10 min. PCR primers used in this study are as follows: ADAR2 forward, 5'-GCCAGTCAAGAAGCCCTCA-3'; ADAR2 reverse, 5'-TGTCCAGATTGCGGTTTT-3'; GluR2 forward, 5'-TTGAAGGGAATGAGCGTTAT-3'; GluR2 reverse, 5'-GCCGTGTAGGAGGAGATG-3'. The PCR products of ADAR2 were run on a 1% agarose gel and visualized by the Dolphin-Scan image system. The unedited PCR products of ADAR2 were 102 bp, and the edited PCR products of ADAR2 were 149 bp. The editing efficiency was quantified using Dolphin-1 D software , and the percentage of ADAR2 self-editing was calculated. The PCR products of GluR2 were sequenced and analyzed for its Q/R site editing [Real-time quantitative PCR was conducted with ABI Prism 7500 Sequence Detection System according to the instruction of the manufacturer . RT-PCR primers used in this study are as follows: GluR2 forward, 5'-CCATGAAAGTGGGAGGTAACTTG-3'; GluR2 reverse, 5'-AAGCCCCTGCTCGTTCAGT-3'; ADAR2 forward, 5'-TGTAAGCACGCGCTGTACTGT-3'; ADAR2 reverse, 5'-GACTCGTGGTATGTGGTAGGCTTAG-3'; β-actin forward, 5'-GATCATTGCTCCTCCTGAGC-3'; β-actin reverse, 5'-ACTCCTGCTTGCTGATCCAC-3'.Ct value of real-time PCR data with the ΔΔCt method normalized by an endogenous control (β-actin) [For relative comparison of each gene, we analyzed the β-actin) .6 cortical neurons were centrifuged and resuspended in 100 μL transfection buffer. One microgram of plasmids  together with one microgram of YFP was added to the neurons; the transfection was performed using an Amaxa Nucleofection apparatus. After the transfection, cells were transferred to cell culture dishes and grown in Neurobasal/B-27 media. The transfection efficiency was approximately 70% which was determined by YFP-positive cells.pcDNA6, pcDNA6-myc-ADAR2, Lentivirus-shADAR2 RNA (5'-GCAGCTGAACGAGATCAAACC-3'), Lentivirus-scramble RNA (5'-GATGTTGTCAACGACTAGTTT-3') and yellow fluorescence protein (YFP) were provided by Dr. Yong Liu . Cell transfection was performed using a Mouse Neuron Nucleofection kit  . Brieflyp was less than 0.05 were considered statistically significant.Data were presented as means ± SEM. Differences among treatment groups were assessed by ANOVA followed by Student-Newman-Keuls analyses. Differences in which The authors declare that they have no competing interests.SL, GY, YY, YL, LZ, JX, XZ and ZF carried out all of the experiments. CF carried out image analysis. ZK, JL, YL, YW and SL participated in the design of the study and the writing of the manuscript. All authors read and approved the final manuscript.


G-Nitro-Arginine (LNNA) in males, but not in females. Here, we investigated whether this gender difference is based on differences in the cytosolic Ca2+ concentration and/or the Ca2+ sensitivity of the myofilaments. Vascular diameter, myogenic tone, cytosolic Ca2+, and Ca2+ sensitivity were evaluated in pressurized SMA segments isolated from male and female gerbils using laser-scanning microscopy and microfluorometry. The gender difference of the LNNA-induced tone was compared, in the same vessel segments, to tone induced by 150 mM K+ and endothelin-1, neither of which showed an apparent gender-difference. Interestingly, LNNA-induced tone in male SMAs was observed in protocols that included changes in intramural pressure, but not when the intramural pressure was held constant. LNNA in male SMAs did not increase the global Ca2+ concentration in smooth muscle cells but increased the Ca2+ sensitivity. This increase in the Ca2+ sensitivity was abolished in the presence of the guanylyl cyclase inhibitor ODQ or by extrinsic application of either the nitric oxide (NO)-donor DEA-NONOate or the cGMP analog 8-pCPT-cGMP. The rho-kinase blocker Y27632 decreased the basal Ca2+ sensitivity and abolished the LNNA-induced increase in Ca2+ sensitivity in male SMAs. Neither LNNA nor Y27632 changed the Ca2+ sensitivity in female SMAs. The data suggest that the gender difference in LNNA-induced tone is based on a gender difference in the regulation of rho-kinase mediated Ca2+ sensitivity. Rho-kinase and NO thus emerge as critical factors in the regulation of cochlear blood flow. The larger role of NO-dependent mechanisms in male SMAs predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.Cochlear blood flow regulation is important to prevent hearing loss caused by ischemia and oxidative stress. Cochlear blood supply is provided by the spiral modiolar artery (SMA). The myogenic tone of the SMA is enhanced by the nitric oxide synthase (NOS) blocker L-N Both, neuronal activity and the generation of the endocochlear potential, which are essential for hearing, are exquisitely sensitive to ischemia and oxidative stress The main blood supply to the cochlea originates from the anterior inferior cerebellar artery that feeds the spiral modiolar artery (SMA) which is located in the center (modiolus) of the cochlea. The SMA supplies the capillary beds in the modiolus, where the cochlear innervation is housed, and branches into radiating arterioles that supply the capillary beds in the cochlear lateral wall, where the endocochlear potential is generated.G-Nitro-Arginine (LNNA). Interestingly, male SMAs exhibit a significantly greater myogenic response in the presence of the NOS inhibitor than female SMAs 2+ concentration, or a greater increase in the Ca2+ sensitivity of the contractile myofilaments.Increases in the intramural pressure cause constriction of the SMA. This myogenic response is enhanced by inhibition of nitric oxide synthase (NOS) with L-N2+ influx mainly via L-type Ca2+ channels. This Ca2+ influx leads to an increase in the cytosolic Ca2+ concentration 2+ sensitivity of the myofilaments 2+ sensitivity is determined by the relative activity of myosin light chain kinase and myosin light chain phosphatase. Myosin light chain phosphatase activity itself is heavily regulated 2+ sensitivity. Rho-kinase phosphorylates myosin phosphatase target subunit 1 and thereby inhibits myosin light chain phosphatase In general, myogenic responses depend on a pressure-induced Ca2+ channels that control Ca2+ influx and vasoconstriction 2+ sensitivity of the myofilaments 2+ concentration or a difference in the Ca2+ sensitivity.Prior studies have demonstrated that the SMA is endowed with L-type voltage-dependent Ca2+ concentration were monitored simultaneously by laser scanning microscopy and microfluorometry using the Ca2+ indicator fluo4. The results reveal that male and female SMAs do not differ in their maximal contractile strength and that the greater LNNA-induced tone in male SMAs is due to a greater Ca2+ sensitivity that depends on rho-kinase and cGMP. From this study, NO and rho-kinase emerge as critical factors in the regulation of cochlear blood flow. The larger role of NOS-dependent mechanisms in the male SMA predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.In the present study, we isolated, pressurized and superfused SMAs from male and female gerbils. Vascular diameter and changes in the global cytosolic CaAll procedures involving animals were approved by the Institutional Animal Care and Use Committee at Kansas State University (IACUC#: 2613 and 2961). Male and female gerbils, 5–11 weeks of age , were deeply anesthetized with tri-bromo-ethanol (560 mg/kg i.p) and sacrificed by decapitation. Temporal bones were removed and spiral modiolar arteries (SMAs) were isolated by microdissection at 4°C.−1. The temperature control entailed a triple system that maintained the superfusate, the bath chamber and the 40x oil-immersion microscope objective at 37°C .Methods for the pressurization and superfusion have been described previously 2, 1 CaCl2 and 5 glucose, pH 7.4. Ca2+-free solution contained (in mM) 150 NaCl, 5 HEPES, 3.6 KCl, 1 MgCl2, 1 EGTA and 5 glucose, pH 7.4 and 150 mM K+ solution contained (in mM) 150 KCl, 5 HEPES, 1 MgCl2, 1 CaCl2 and 5 glucose, pH 7.4. Compounds added to these solutions included 10 µM L-NG-nitro-arginine , 10 nM endothelin-1 , 100 µM papaverine (Sigma-Aldrich), 8 µM diethylammonium (Z)-1-diazen-1-ium-1,2-diolate , 10 µM trans-4-[(1R)-1-aminoethyl]-N-4-pyridinyl-cyclo-hexane-carboxamide dihydrochloride , 10 µM 1H-2+ concentration was increased to 3 and 10 mM by addition of CaCl2. Stock solutions of 20 mM LNNA, 10 mM Y27632, 50 mM ODQ and 250 mM papaverine were prepared in DMSO. The final concentration of DMSO did not exceed 0.1%. A stock solution of 2 µM endothelin-1 was prepared in PSS, stock solutions of 1.5 mM 8-Br-cGMP and 1.5 mM 8-pCPT-cGMP were prepared in deionized H2O, and a stock solution of 8 mM DEA-NONOate was prepared in 0.01 N NaOH. Addition of the DEA-NONOate stock to the final solution did not change the pH by >0.01 pH-units.PSS solution contained (in mM) 150 NaCl, 5 HEPES, 3.6 KCl, 1 MgClpassive – IDactive) ×100/ IDpassive, where IDpassive is the inner diameter in Ca2+-free solution and IDactive is the inner diameter in PSS or 150 mM K+ solution with or without compounds.Brightfield images of pressurized vessel segments were obtained by laser scanning microscopy at a rate of 1 image/s . Images were analyzed using a custom program written in IDL 6.3  by Withrow Gil Wier  and analyzed with a custom program written in Origin 6.0  by P. Wangemann . Myogenic tone (%) was calculated as  at 37°C. Fluo4 was excited by a 488 nm argon laser and emissions were filtered by a 488 notch, a 490 nm dichroic and a 505 nm long pass filter. Global Ca2+ concentration changes were obtained by averaging fluo4 intensity measurements from 5 smooth muscle cells per vessel segment. Data presented are averages from multiple vessel segments.Changes in the global Ca2+ were simultaneously measured in vessel segments pressurized at 60 cmH2O. Following equilibration for 15 min in PSS, arteries were superfused with PSS solutions containing 0, 1, 3 and 10 mM Ca2+ each for 2 min. Data points were obtained by averaging diameter and fluorescence intensity measurements over the last 30 s of each Ca2+ step and normalizing these values against the average value obtained in PSS containing 1 mM Ca2+. Data points from individual vessels were fitted to a modified Hill equation:Dia is the normalized diameter, Base is the minimal diameter independently obtained in the presence of 10 nM ET-1 to be 53% in female and 55% in male SMA, Max is the diameter in PSS containing 1 mM Ca2+, 2+][Ca is the normalized cytosolic Ca2+ concentration, h is the slope coefficient, and 50FC is the fold-change in the global cytosolic Ca2+ concentration that is necessary for a half-maximal constriction. The slope coefficient h was arbitrarily set to −5.2 and Max was clamped to 100%. Two 50FC values describing a control and an experimental period were obtained from each vessel segment. For presentation, data points were averaged and curves representing the averaged 50FC values were drawn.Vascular diameter and changes in global Ca2+ sensitivity in the presence of 10 µM LNNA, a stock solution (8 mM) of the NO donor DEA-NONOate was prepared in 0.01 N NaOH where the NO donor is stable. Prior to the beginning of every 2 min step in the protocol, 10 µl of the stock was added to 10 ml of the superfusing solution  and quickly mixed (within 15 seconds) to obtain a concentration of 8 µM DEA-NONOate. With a reported decomposition rate of 1.5 mol NO per mol of DEA-NONOate and the half-life of 2 min at pH = 7.4 and 37°C, the concentration of the released NO at the end of the 2 min step in the protocol is expected to drop to ∼6 µM, a saturating concentration that is approximately 30-fold higher than the 50EC reported for rat aorta For evaluating the effect of the NO donor on Ca50FC values was obtained by paired Student's t-test. Significance was assumed at p<0.05.The number of experiments (n) represents the number of vessel segments. Data are presented as mean ± sem. Each mean consists of vessel segments that originated from more than one animal. Statistical significance for differences in tone between multiple treatments was obtained on raw data by 2-way repeated-measures ANOVA . Statistical significance for 2O and superfused with 10 µM LNNA, during which the pressure was either held constant at 60 cmH2O to provide constant stretch or altered between 20 and 80 cmH2O (red trace). Interestingly, tone was not enhanced, when the pressure was held constant (black trace). These results demonstrate that NOS inhibition in conjunction with changes in the intramural pressure promotes myogenic vasoconstriction in the male SMA.Nitric oxide (NO) opposes myogenic constrictions in most large and small arteries and thereby exerts a vasoprotective role in response to physiological stimuli such as pressure-induced stretch and flow-induced shear that cause vasoconstriction 80 cmH2O . When pr+ and ET-1, in male and females SMAs. Tone was measured in PSS, 10 µM LNNA, 150 mM K+ and 10 nM ET-1 , 1, 3 or 10 mM Ca2+. Changes in the diameter were plotted against changes in the global cytosolic Ca2+ concentration . NOS inhibition with 10 µM LNNA produced no significant effect on the Ca2+ sensitivity in female SMA, however, caused a marked increase in the Ca2+ sensitivity in male SMA . These observations are consistent with a mechanism whereby NO stimulates guanylyl cyclase to generate cGMP and cGMP leads to inhibition of rho-kinase. The lack of an effect of ODQ itself suggests the presence of compensatory mechanisms that regulate rho-kinase and/or the Ca2+ sensitivity in the absence of cGMP.The effects of NO in vascular smooth muscle cells are commonly mediated by the activation of soluble guanylyl cyclase leading to an increase of the second messenger cGMP male SMA . Howeverminished , suggestsitivity . Howeversitivity . A simil2+ concentration, but the result of a difference in the rho-kinase mediated increase in Ca2+ sensitivity. The results suggest that NO controls the Ca2+ sensitivity of the male SMA via the regulation of rho-kinase in a cGMP-dependent manner.The most salient findings of the present study are that changes in intramural pressure during NOS inhibition by LNNA enhances myogenic vasoconstrictions in the male but not in the female SMA, that this gender difference is neither a manifestation of a difference in the maximal achievable contractility, nor the consequence of an increase in global CaMyogenic tone in male SMAs was enhanced during NOS inhibition when the vessel was subjected to a protocol of pressure changes but not when subjected to a single constant intramural pressure . This obin vitro and large arterioles in vivoIn the coronary and cerebral vasculature, it is well-established that the NO-induced reduction in vascular tone is gender-dependent 2+ concentration of smooth muscle cells by different mechanisms. Some of these mechanisms involve activation of K+ channels, hyperpolarization of the membrane potential and closure of voltage-dependent, mainly L-type, Ca2+ channels that mediate Ca2+ influx from the extracellular medium. For instance, NO hyperpolarizes the membrane by activation of large-conductance Ca2+ activated K+ channels either directly ATP K+ channels 2+ sparks, which are spatially confined Ca2+ releases from the sarcoplasmic reticulum that occur via ryanodine-sensitive Ca2+ channels and activate closely apposed large-conductance Ca2+ activated K+ channels 2+ concentration of smooth muscle cells entails stimulation of the Ca2+ ATPase in the sarcoplasmic reticulum that increases the uptake of Ca2+ into the sarcoplasmic reticulum 2+ concentration and cause relaxation of smooth muscle cells. Common to all of these mechanisms is that NOS inhibition with LNNA would be expected to lead to a vasoconstriction via an increase in the cytosolic Ca2+ concentration. Interestingly, LNNA-mediated NOS inhibition in the male SMA increased tone without an increase in the global cytosolic Ca2+ concentration (NO can decrease the cytosolic Cantration . This ob2+ concentration in smooth muscle cells but also the Ca2+ sensitivity of the myofilaments. NO has been shown to inhibit rho-kinase activity leading to disinhibition of myosin light chain phosphatase, since rho-kinase increases the Ca2+ sensitivity by inhibiting myosin light chain phosphatase 2+ sensitivity (2+ sensitivity is a major pathway of controlling tone in the male SMA. The finding that the LNNA-mediated increase in the Ca2+ sensitivity was abolished by Y27632 (2+ sensitivity and the target of regulation by NO in the male SMA.NO not only modulates the cytosolic Casitivity  and thatsitivity  demonstry Y27632  establis2+ sensitivity in males, but not in females (2+ sensitivity (2+ sensitivity in female SMAs may be attributable to a lower basal rho-kinase activity. This hypothesis is supported by the observation that rho-kinase inhibition did not decrease basal Ca2+ sensitivity in female SMAs whereas an inhibitory effect was observed in male SMAs (2+ sensitivity 2+ sensitivity.A striking gender difference was found between male and female SMAs in the action of NO. LNNA-mediated NOS inhibition increased tone and the Ca females  vs 5B. Tsitivity . The lacale SMAs  vs 6B. A2+ as well as the lowering of Ca2+ sensitivity via the inhibition of Rho-A and rho-kinase 2+ sensitivity in the male SMA was profoundly diminished in the presence of the guanylyl cyclase inhibitor ODQ and completely abolished by the cGMP analog is consistent with such a mechanism (2+ sensitivity indicates that other mechanisms may be involved. Nevertheless, our results suggest that cGMP is essential for the rho-kinase mediated regulation of Ca2+ sensitivity by NO.The most common mechanism through which NO regulates smooth muscle function involves the activation of soluble guanylyl cyclase that leads to increased formation of the second messenger cGMP echanism . The fac2+ sensitivity and tone during NOS inhibition and reveals mechanisms by which NOS inhibition induces vasoconstriction in male SMA. These gender differences suggest that loss of NO is likely to have a greater impact on the regulation of cochlear blood flow in males than in females, which is consistent with animal experiments and clinical outcomes that suggest a gender preference in hearing loss, with males being more susceptible In summary, this study demonstrates gender differences in the regulation of rho-kinase mediated Ca


However, the mechanisms underlying the effect are not well known. This study is to investigate whether anandamide alter Na+/Ca2+ exchanger current (INCX) was recorded and analysed by using whole-cell patch-clamp technique and [Ca2+]i was measured by loading myocytes with the fluorescent Ca2+ indicator Fura-2/AM.NaNCX was enhanced significantly after perfusion with simulated ischemic external solution; [Ca2+]i was also significantly increased by simulated ischemic solution. The reversal potential of INCX was shifted to negative potentials in simulated ischemic external solution. Anandamide (1–100 nM) failed to affect INCX and [Ca2+]i in normal solution. However, anandamide (1–100 nM) suppressed the increase in INCX in simulated ischemic external solution concentration-dependently and normalized INCX reversal potential. Furthermore, anandamide (100 nM) significantly attenuated the increase in [Ca2+]i in simulated ischemic solution. Blocking CB1 receptors with the specific antagonist AM251 (500 nM) failed to affect the effects of anandamide on INCX and [Ca2+]i in simulated ischemic solution. CB2 receptor antagonist AM630 (100 nM) eliminated the effects of anandamide on INCX and [Ca2+]i in simulated ischemic solution, and CB2 receptor agonist JWH133 (100 nM) simulated the effects of anandamide that suppressed the increase in INCX and [Ca2+]i in simulated ischemic solution. In addition, pretreatment with the Gi/o-specific inhibitor pertussis toxin  eliminated the effects of anandamide and JWH133 on INCX in simulated ischemic solution.We found that INCX during perfusion with simulated ischemic solution; the effects may be mediated by CB2 receptor via PTX-sensitive Gi/o proteins. This mechanism is importantly involved in the anti-ischemia injury caused by endocannabinoids.Collectively, these findings suggest that anandamide suppresses calcium overload through inhibition of I The NCX removes a Ca2+ ion in exchange for the import of three Na+ ions and is considered one of the most important mechanisms for Ca2+ removal in cardiac muscle 2+ extrusion) produces an inward current  and an outward currents is induced  if NCX works at the reverse mode (Ca2+ influx) + and Ca2+ gradients 2+ concentration [Ca2+]i during ischemia is due to Ca2+ entry by reverse-mode NCX 2+ homeostasis in cardiomyocytes, especially during cardiac ischemia.Na2+ overload? Therefore, in this study, by using whole-cell patch clamp and Fura-2/AM fluorescence ratio image method, we explored the effects of anandamide on INCX and [Ca2+]i in ventricular myocytes.Anandamide can protect heart against ischemia-reperfusion injury. However, it is not clear if NCX is involved an anandamide-induced protective effect during ischemia-reperfusion injury to alleviate CaWe carried out this study in adult male Sprague-Dawley rats (weighting 230–280 g) obtained from the Experimental Animal Center of Hebei Province. This study was performed conforming to the Guide for the Care and Use of Laboratory Animals described by Directive 2010/63/EU of the European Parliament. Animal work was approved by the Ethics Committee for Animal Experiments of the Hebei Medical University, in compliance with NIH Guidelines, and carried out in compliance with China Government Guidelines.2+-free Tyrode's solution via the aorta at a perfusion rate of 4 ml/min for 5 min. The Ca2+-free Tyrode's solution contained (in mM) NaCl 135, KCl 5.4, MgCl2 1.0, NaH2PO4 0.33, glucose 5, and HEPES 10 (pH was adjusted to 7.4 with NaOH). Then, the heart was perfused with Tyrode's solution containing CaCl2 (34 µM) and collagenase II (300 mg/L) at 37 °C for 12 min. Finally, the left ventricle was removed and teased into smaller pieces in Kreb's solution containing (in mM) KOH 80, KCl 40, NaH2PO4 25, MgSO4 3, glutamic acid 50, taurine 20, EGTA 1, HEPES 10, and glucose 10 (pH was adjusted to 7.4 with KOH). Single myocytes were harvested after filtration through a nylon mesh (pore size 200 µm) and stored in Kreb's solution at room temperature for at least 1 h, then the concentration of Ca2+ in Kreb's solution was gradually increased to 1.0 mM before the experiment.Single ventricular myocytes were isolated from the heart of adult rat by using enzymatic dissociation as described previously 2, 20 NaCl, 5 MgATP, 6 CaCl2, 4 MgCl2, 10 HEPES, 20 tetraethyl ammonium chloride (TEA), and 21 EGTA (pH 7.4 adjusted with CsOH) NCX. Ni2+ (5 mM) was added to block the INCX. The protocol was repeated in the presence of 5 mM Ni2+ to obtain Ni-insensitive currents. INCX was measured as the Ni-sensitive currents which were calculated as a subtraction of residual currents in the presence of Ni2+ from the total currents . After 30 min settling to the bottom of chamber, the myocytes were superfused with the external solution for 10 min at a rate of 2–3 ml/min at room temperature. Whole-cell recordings were performed on these cells and membrane current was assessed by using Axopatch 200B patch-clamp amplifier and Digidata 1200B acquisition board . Patch-pipettes were pulled by a micropipette puller  with a resistance of 2 to 5 MΩ when it was filled with electrode internal solution containing (in mM) 65 CsClcurrents . In the NCX was recorded when myocytes were superfused by normal external solution and simulated ischemic external solution to mimic ischemic environment NCX recording contained (in mM) NaCl 140, CaCl2 2.0, MgCl2 2.0, HEPES 5.0, glucose 10, Oubain 0.02, BaCl2 1.0, CsCl2 2.0, nifedipine 0.001 . Simulated ischemic external solution contained (in mM): NaCl 137.8, MgCl2 1.0, CaCl2 1.8, NaHCO3 3.8, NaH2PO4 0.9, Oubain 0.02, BaCl2 1.0, CsCl2 2.0, nifedipine 0.001, Sodium lactate 20 and was gassed with 95% N2 and 5% CO2 (pH 6.8) for 30 min. Glibenclamide 20 µM was added to external solutions to block IKATP. Cariporide 10 µM, a selective Na+/H+ exchanger (NHE) inhibitor, was also added to external solutions to block Na+/H+ exchanger current (INHE) I2+]i was measured with a video-imaging-system . Cells were illuminated alternately at 340- and 380-nm excitation wavelengths, excitation light was provided by a monochromator (Till Photonics). Then, 500-nm emission light images were captured by an image-intensifying, charge-coupled device (CCD) camera  and digitized by an image processing system . The monochromator and CCD camera were controlled by TillVision software, which was also for image analysis. Ratios were converted to Ca2+ concentrations as previously described Myocytes were incubated with 2 µM Fura-2/AM for 40 min at 37°C to load Fura-2/AM into the cell. Briefly, the coverslip was placed in a small superfusion chamber on the stage of an inverted microscope . [Ca2+]i was recorded when myocytes were superfused with normal Tyrode's solution and simulated ischemic solution to mimic ischemic environment 2 1.05, CaCl2 1.80, NaHCO3 1.2, glucose 11.0, and Tris 5.0 . The composition of simulated ischemic solution (in mM) was: NaCl 137.8, KCl 8.0, MgCl2 1.0, CaCl2 1.8, NaHCO3 3.8, NaH2PO4 0.9, Sodium lactate 20 . Glibenclamide 20 µM was added to solutions to block IKATP. Solutions were maintained at a temperature of 37.0 ± 0.5°C throughout the experiment.i was calculated from fluorescence emission ratios obtained from 340- and 380-nm excitation of the Ca2+-bound and Ca2+-free forms of fura- according to the following formula: [Ca2+]  =  Kd·[(R- Rmin)/(Rmax- R)]·S, where Kd (224 nM) is the dissociation constant for the association of the deesterified Fura-2 molecule with ionized calcium; R, Rmax, and Rmin are, respectively, the fluorescence ratio (340∶380) for the sample at time t, following the addition of 100 µl of 0.1% Triton X-100 and following the addition of 100 µl of 0.5 mM EGTA to the cell suspension; S is the ratio of fluorescence at 380 nm in EGTA-treated samples over Triton X-100-treated samples. Excitation of Fura-2 and electrical stimulation were restricted to brief recording periods. Calibrations in single cells were carried out at the end of each experiment. Although the calculated values of [Ca2+]i may be sensitive to calibration reference and other factors, the overall conclusions drawn from the data are not altered because each cell serves as its own control. The differences between groups were compared using one-way ANOVA or within groups before and after drug application using ANOVA with repeated measures. When significant differences were found using ANOVA, the Dunnet's post hoc test was used to determine which groups were significantly different. Statistical significance was accepted at P<0.05.Data were expressed as mean ± SEM. For electrophysiological recording data analysis were replayed and analysed by using Pclamp10.0 (Axon Instruments). i to 139.9 ± 15.8 nM, but the [Ca2+]i was still higher than that in normal Tyrode's solution  or CB2 antagonist AM630 (100 nM) for 15 min in the simulated ischemic solution. Blockade of CB1 receptors with AM251 (500 nM) or CB2 receptors with AM630 (100 nM) had no effect on basal [Ca2+]i level in cardiac myocytes. However, pretreatment of cells with AM630 (100 nM), but not AM251 (500 nM), for 15 min abolished the inhibitory effect of anandamide (100 nM) on [Ca2+]i in the simulated ischemic solution  and cyclic guanine mono-phosphate (cGMP) production in the cardiac myocardium 2+ sensitivity mediated exclusively by a cGMP-PKG pathway without significantly changing the amplitude or kinetics of [Ca2+]i2+ responsiveness without alteration of [Ca2+]i.Although anandamide and other cannabinoids have little effect on cardiovascular hemodynamics in conscious animals NCX (both reverse mode and forward mode) was enhanced obviously in simulated ischemic external solution at 30 min. Furthermore, the reversal potential was shifted to the negative potentials. The intracellular Na+ increases during ischemia through enhanced activity of Na+-H+ exchange (NHE) + channels + current and Na+/H+ exchanger activity contribute to the augmentation of the reverse NCX activity during hypoxia or acute ischemia in ventricular myocytes + concentration, coupled with the depolarized plasma membrane, results in an increase in the reversal NCX activity to bring more Ca2+ into the cardiac myocytes NCX. In the simulated ischemic external solution, anandamide reduced both outward and inward INCX, the reversal potential was also recovered to the level of normal external solution. It was reported anandamide had little effect on Na+ channel in cardiac myocytes NCX directly. However, there were some reports that INCX was inhibited during cardiac ischemia NCX after perfusion of simulated ischemic solution for 30 min. Therefore, this discrepancy is possibly due to the measurement in different time points after ischemia.We found that I2+]i of myocardium during ischemia produces irreversible tissue damage. Therefore, a manipulation that reduces the rise in [Ca2+]i can attenuate or delay the onset of irreversible cardiac injury 2+]i during ischemia. In this regard, the rise in cytosolic Ca2+ during ischemia is due to Ca2+ entry through an increased activity of NCX in reverse mode 2+]iNCX in the simulated ischemic external solution. Therefore, we reasoned that anandamide decrease [Ca2+]i through inhibition of INCX to protect cardiomyocyte against ischemia injury. Consistent with previous study 2+]i was increased during perfusion of simulated ischemic solution. Anandamide partially, but significantly, inhibited the increase in [Ca2+]i in simulated ischemic solution. These result is consistent with the finding that anandamide elicited a concentration-dependent inhibition of depolarization-evoked Ca2+ transients in oligodendroglial somata 2+ transients 3-mediated nuclear Ca2+ release in isolated cardiac nuclei NCX at a concentration of 1 µM 2+ overload during I/R injuries 2+ overload 2+]i in simulated ischemic solution. These data suggest that anandamide inhibits [Ca2+]i during ischemia through inhibition of INCX.Increasing lines of studies indicate that a rise in [CaNCX and [Ca2+]i in cardiac myocytes in simulated ischemic solution. To further clarify the effect of CB2 receptor, the effect of CB2 specific agonist JWH133 was tested. The effects of JWH133 was similar to anandamide, which also inhibited the enhancement of INCX and partially suppressed the increase in [Ca2+]i in cardiac myocytes in simulated ischemic solution. However, a CB1 receptor antagonist AM251 failed to affect the inhibitory effects of anandamide on INCX and [Ca2+]i, even at higher concentrations. These findings indicated that the inhibitory effects of anandamide on INCX and [Ca2+]i were mediated by CB2 receptor but not CB1 receptor. Many studies have emphasized the role of CB2 receptors in cardioprotection. For example, it has been shown that blockade of CB2 receptors eliminates cardiac protective effect of endocannabinoids in rat isolated hearts exposed to low-flow ischemia and reperfusion Two types of cannabinoid receptors, the CB1 and CB2 have been cloned NCX, but also the effect of CB2 receptor agonist JWH133 on INCX. Thus, at least we can deduce that anandamide inhibites INCX through CB2 receptor via PTX sensitive Gi/o proteins. But the next signaling pathway after activation of Gi/o need further exploration.Recent studies have reported that NCX function is regulated by tyrosine phosphorylation NCX during perfusion with simulated ischemic solution, the effects may be mediated by CB2 receptor via PTX-sensitive Gi/o proteins.In conclusion, these findings suggest that anandamide suppresses calcium overload through inhibition of I


Here, we examined the cytoplasmic Ca2+ levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca2+ level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 . We found that the extracellularly added Ca2+ caused a dose-dependent increase in the cytoplasmic Ca2+ level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca2+ peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca2+ increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca2+ increase is abolished by the addition of the Ca2+ chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca2+ channel complex, indicating that the synergistic increase is caused by the Ca2+ influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca2+ influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.The regulation of cytoplasmic Ca Small changes in cytoplasmic Ca2+ levels can activate various Ca2+-sensing proteins, such as calmodulin and calcineurin, which then lead to the induction of various downstream signal transduction pathways. In response to external or internal stress the cells have evolved mechanisms to regulate cytoplasmic Ca2+ homeostasis. In mammalian cells, various channels including the voltage-gated calcium channels and the transient receptor potential (TRP) channels play important roles in regulating cytoplasmic Ca2+Saccharomyces cerevisiae, vacuolar TRP channel Yvc1p mediates the release of Ca2+ from the vacuole in response to hyperosmotic shock 2+ influx at the plasma membrane Schizosaccharomyces pombe, the TRP channel Pkd2 plays important roles in cell wall synthesis and membrane trafficking Calcium signaling is an important regulator in all eukaryotic cells for a wide variety of physiological processes. The cells have evolved mechanisms to regulate cytoplasmic Ca2+ levels for 10 min using aequorin (AEQ) in living fission yeast cells transformed with pREP1-AEQ 2+ levels over a long period of time. It was reported that a GFP-AEQ fusion protein linked by 19 amino acid (GFP-19-AEQ) closely approximates the Aequorea victoria jellyfish GFP and results in an efficient bioluminescence resonance energy transfer to provide a highly sensitive Ca2+ sensor 2+ level in living fission yeast cells. By monitoring cytoplasmic Ca2+ under various conditions, we showed that two TRP homologues, Trp1322 and Pkd2, mediate the cytoplasmic Ca2+ rise caused by the extracellularly added CaCl2, and that the Cch1-Yam8 channel complex mediates the cytoplasmic Ca2+ rise caused by the extracellularly added NaCl plus FK506. Also, we showed that calcineurin play important roles in these two processes.In our previous study, we monitored the cytoplasmic CaS. pombe strains used in this study are listed in trp1322::ura4+). Gene disruptions are abbreviated by the gene preceded by Δ . Proteins are denoted by Roman letters and only the first letter is capitalized .nmt1 promoter adh1 promoter ′-GAT CTC CCG GTA CTG CCA CTC CCG CCA CTA CTC CCA CTA CTG CCC CCA CTG CCG GTA CTG GTC TGC AGG GAT CCT GC-3′5 and ′-GCG GCC GCA GGA TCC CTG CAG ACC AGT ACC GGC AGT GGG GGC AGT AGT GGG AGT AGT GGC GGG AGT GGC AGT ACC GGG A-3′5, were mixed in equimolar amounts, reannealed, and cloned into the BamHI/NotI site of the pREP1-N-GFP vector (pKB4663) to give pREP1-GFP-19 amino acid (pKB5995). Second, the apoaequorin (AEQ) gene was amplified by polymerase chain reaction (PCR) from the pEVP11/AEQ plasmid XhoI/NotI fragment containing GFP-19-AEQ was subcloned into the XhoI/NotI pKB1894 (pREP41-N-GST) to give pREP41-GFP-19-AEQ (pKB6045).For ectopic expression of proteins, the thiamine-repressible adh1 promoter was amplified by PCR using the pART1 vector containing an adh1 promoter ′-CGC GGA TCC TGC AGT GCA TGC CCT ACA ACA ACT AAG-3′5, and the antisense primer ′-CGC GGA TCC TCG AGG AAT TCT CTT GCT TAA AGA AAA G-3′5. The amplified product containing the adh1 promoter was digested with BamHI, and the resulting fragment was subcloned into BlueScriptSK (+) (Stratagene) to give pKB6881. Then the PstI/XhoI fragment of pKB6881 was ligated into the PstI/XhoI site of pKB1894 to give pKB6891. Finally, the above-mentioned XhoI/NotI fragment containing GFP-19-AEQ from pKB6011 was ligated into XhoI/NotI site of pKB6891 to give adh1-GFP-19-AEQ (pKB6892).The DDPstI/SacI fragment containing pREP1-GST-Pek1DD was subcloned into the PstI/SacI site of pKB5049 bearing the arg1+ marker StuI, and transformed into KP2101 cells The constitutively active Pmk1 MAPKK Pek12+ levels were determined using a previously described method with minor modifications 2+ level, a method previously described was used with minor modifications 2.The cytoplasmic Ca+trp1322 gene (SPCC1322.03), the sense primer used was 5′-GGA AGA TCT ATG AAG CCT CGC CCG TAC TCT G-3′, and the antisense primer was 5′-GGA AGA TCT TTA TGG ATG TGG AAT CGA CC-3′. For the +trp663 gene (SPCC663.14c), the sense primer used was 5′-GGA AGA TCT ATG AAG CTA ATA CTC TTA GC-3′, and the antisense primer was 5′-GGA AGA TCT CGA ACA GTA CGC TGC ATT TCA TTA ATC CCG-3′. For the +pkd2 gene, the sense primer used was 5′-CGC GGA TCC CAT ATG AGG CTT TGG AGA AGC CC-3′, and the antisense primer was 5′-CGC GGA TCC CGA CGA AAA GCA TTG TTA GGT AAT GG-3′. The amplified product containing the +trp1322 or +trp663 gene was digested with BglII, and the resulting fragment was subcloned into BlueScriptSK (+) (Stratagene) in which BamHI site was changed to a BglII site. The amplified product containing the +pkd2 gene was digested with BamHI, and the resulting fragment was subcloned into BlueScriptSK (+). As described previously +trp1322 was amplified by PCR and was ligated to the N-terminus of the GFP carrying the S65T mutation +pkd2 or +trp663 was amplified by PCR and was ligated to the C-terminus of the GFP carrying the S65T mutation The three TRP genes were amplified by PCR with the genomic DNA of wild-type cells as a template. For the +trp1322 gene (SPCC1322.03), a one-step gene disruption by homologous recombination was performed trp1322::+ura4 disruption was constructed as follows. The BglII fragment containing the ORF of the +trp1322 gene was subcloned into BlueScriptSK (+) in which BamHI site was changed to a BglII site. Then, a BamHI fragment containing the +ura4 gene was inserted into the BamHI site of the previous construct. The fragment containing the disrupted +trp1322 gene was transformed into diploid cells. Stable integrants were selected on medium lacking uracil. The disruption of the gene was checked by genomic Southern hybridization (data not shown).To knockout the yam8+ gene was disrupted by the insertion of KanMX6 cassette at the BamHI site of the Yam8 ORF. The yam8::6KanMX cells were constructed using a previously described method with minor modifications yam8 gene were transformed into KP456 cells. The stable integrants were selected on G418 plates (100 mg/l), and disruption of the gene was checked by genomic Southern hybridization (data not shown).The For DAPI staining, the cells grown to log-phase were fixed for 10 min in a solution of 3.7% formaldehyde and washed twice in 1×PBS, and then were resuspended in 100 µl 1×PBS. Suitable quantities of the cells were stained with DAPI for 10 min, and then were observed under the microscope.Methods in light microscopy, such as differential interference contrast (DIC) and fluorescence microscopy, that were used to observe the GFP-19-AEQ were performed as described 2+ levels in fission yeast, we expressed GFP-19-AEQ adh1 promoter or the thiamine-repressible nmt1 promoter, and the fluorescence was checked by microscopy. Under the nmt1 promoter of the strong expression vector pREP1, GFP-19-AEQ aggregated as dots in the cells (data not shown). Under the nmt1 promoter of the attenuated expression vector pREP41, the fluorescence of GFP-19-AEQ localized to both the cytoplasm and nucleus . As shown in 2, the cytoplasmic Ca2+ level increased immediately and reached a peak level, and then decreased to a new steady-state within 2–3 min in a dose-dependent manner. The steady-state was sustained for at least 4 hours (data not shown). This pattern suggests a markedly improved sensitivity over our previous report using pREP1-AEQ and is consistent with the pattern of CDRE-reporter assay adh1-GFP-19-AEQ .To investigate whether the CaS. pombe databases led to the identification of three open reading frames which are as follows, SPCC1322.03 (named as +trp1322 in this study), SPCC663.14c (named as +trp663 in this study), and SPAC1F7.03 (Pkd2) 2+ levels in wild-type cells. The results showed that the overexpression of Trp1322 or Pkd2 markedly induced an approximately 10∼20 fold burst-like increase in the cytoplasmic Ca2+ levels when stimulated by the extracellularly added CaCl2 as compared with the control vector .To investigate whether the increased cytoplasmic Cativity 2 . Also, tnmt1 promoter did not show any clear localization signals in the presence of thiamine, and showed a similar localization to that of the plasmid-based GFP-Trp1322 in the absence of thiamine (data not shown). Regarding Trp633, a strain with the integrated GFP constructs under endogenous promoters was made, but no signals were observed (data not shown), suggesting that Trp633 did not show any clear localization signals when produced under physiological conditions.We then observed the intracellular localization of the three TRP homologues. GFP-Pkd2 localized to the membrane structures as described previously by other investigators (data not shown) 2+ level at 60 to 90 min after the addition of CaCl2, suggesting that Pkd2 channel may be responsible for the release of Ca2+ from the intracellular organelles. To investigate whether the Golgi or the vacuole is responsible for the biphasic increase, we monitored the effect of Pkd2 overexpression in the knockout cells of Pmr1 (Golgi membrane Ca2+ pump), Pmc1 (vacuolar Ca2+ ATPase), and Vcx1 (vacuolar H+/Ca2+ exchanger). In Δpmr1, Δpmc1 and Δvcx1 cells respectively, the burst-like first peaks were also observed (data not shown). Interestingly, in Δpmr1 cells the second peak was not observed , or NaCl plus FK506. Wild-type cells expressing GFP-19-AEQ were cultured as described in 2+ level in wild-type cells .Our previous result showed that high extracellular NaCl concentration caused an increase in the cytoplasmic Ca2+ level was caused by the inhibition of calcineurin activity, we monitored the level of cytoplasmic Ca2+ in the knockout cells of the ppb1+ gene, encoding a single catalytic subunit of fission yeast calcineurin ppb1 cells, the extracellularly added NaCl caused a persistent increase in the cytoplasmic Ca2+ level, and the addition of FK506 did not cause any further increase  ethane-N, N, N′, N′-tetraacetic acid). As shown in 2+ level is dependent on the influx across the Ca2+ channel that exists on the plasma membrane.To investigate whether the increase in the cytoplasmic Cayam8+ or cch1+ gene that encodes the putative subunit of a Ca2+ channel abolished the NaCl-induced activation of calcineurin 2+ level . Knockout of the genes encoding MAPKK pek1+ and MAPKKK mkh1+, also completely abolished the synergistic increase and suppressed the cell death (data not shown).It was also reported that the protein kinase C-MAP kinase pathway regulates the Cch1-Yam8 channel complex of salts , Δpmk1. DD is the constitutively active mutant of MAPKK, Pek1 DD overexpression on the cytoplasmic Ca2+ level, we constructed the pREP1-GST-Pek1DD integrated cells as described in DD was overexpressed, NaCl plus FK506 caused an approximately 2-fold increase in RLU . The deletion of the yam8+ gene also abolished the effect of Pek1DD overexpression (data not shown). These results suggest that the calcineurin and the Pmk1 MAPK pathways antagonistically regulate the Ca2+ influx via the Cch1-Yam8 channel complex. Notably, when Pek1DD was overexpressed, FK506 elicited a marked increase in RLU . These results suggest that a persistently increased level of cytoplasmic Ca2+, but not Cl−, causes cell death.In our previous study, we showed that the Δpmk1 cells with DAPI to visualize the nuclei in the presence of MgCl2 plus FK506. Fluorescence microscopic imaging of DAPI-stained cells revealed nuclear fragmentation in wild-type cells treated with MgCl2 plus FK506, whereas the nuclei of Δpmk1 cells remained big, round and intact for the same time course of treatment . Thus, both the overexpression and deletion of the trp1322+ gene antagonize the synergistic effect of NaCl plus FK506 on the Ca2+ influx via the Cch1-Yam8 channel complex. Deletion of the trp663+ gene had no effect on the synergistic Ca2+ increase (data not shown).Our results also showed that in Δppressed . ConsistDD, a constitutively active mutant of MAPKK trp1322 cells. Overexpression of Pek1DD recovered the synergistic effect of NaCl plus FK506 in Δtrp1322 cells . Altogether our results indicate that the overexpression of some calcium channels  mediate a rise in the cytosolic Ca2+ level whereas other channels  do not. Also, the release from the intracellular organelles  may play an important role in the cytosolic Ca2+ increase.The dose-dependent rise in the cytosolic Ca2+ level  is likelincrease . Consist2+ level , the burst-like peak was also observed in wild-type cells.The dose-dependent rise in the cytosolic Ca2+ level  showed a mutants  and Δncspkd2+ may encode a mechanosensitive ion channel based on the observation that modulators of mechanosensitive ion channels such as lysophosphatidylinositol or gadolinium affected the growth of the Pkd2-depleted strain 2 also produced a dose-dependent rise in the cytoplasmic Ca2+ level, we suggest that Trp1322 and Pkd2 are Ca2+ permeable, but not mechanosensitive, channels. Another TRP homologue, Trp663, on the other hand does not seem to be involved in this process, although Trp663 has a higher amino acid sequence identity to Pkd2, as compared with that of Trp1322.Palmer et al. speculated that 2 the effect of Trp1322 and Pkd2 overexpression is totally abolished in Δppb1 cells, and is partially abolished in Δprz1 cells (pmr1 cells (pmc1 or Δvcx1 cells (data not shown), the burst-like peaks were observed with an increased magnitude of the peak. Furthermore, the FK506 treatment 1 hour before the CaCl2 stimulation failed to suppress the burst-like peak while the FK506 treatment 24 hours before CaCl2 stimulation completely abolished the peak (data not shown). Altogether these results suggest that the effect of calcineurin gene deletion is mainly due to the long-term inhibition of Prz1-dependent transcription, but not due to the inhibition of the direct TRP dephosphorylation by calcineurin.Our results also showed that upon the addition of CaClz1 cells . In Δpmrr1 cells , Δpmc1 o2+ level in fission yeast cells? What is the molecular mechanism that generates the synergistic Ca2+ signal? How do NaCl and FK506 work together? In Δprz1 cells, the synergistic increase in the cytoplasmic Ca2+ level was clearly observed suggesting that the regulation of calcineurin on Cch1-Yam8 is not due to the transcriptional regulation by Prz1. The deletion of the pmk1+ gene markedly suppressed the Ca2+ influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the Ca2+ influx, indicating that Pmk1 MAPK pathway may have a positive regulation on the Cch1-Yam8 channels.Why does NaCl plus FK506 cause a slow and persistent increase in the cytoplasmic Capmk1 cells, the basal cytoplasmic Ca2+ level in Δtrp1322 cells was extremely low, and the synergistic increase in the cytoplasmic Ca2+ level caused by NaCl plus FK506 was markedly suppressed. Second, similar to the deletion of pmk1, the deletion of the trp1322+ gene significantly suppressed the cell death caused by salts plus FK506. Third, similar to MAPKK Pek1, the overexpression of Trp1322 suppressed the salts-sensitive phenotype of Δppb1 cells. Fourth, in Δtrp1322 cells, the overexpression the constitutively active Pek1DD also elicit the synergistic effect of NaCl plus FK506  were transformed with a control vector and pREP1-Cch1-GFP respectively. The transformants were grown to exponential phase in the absence of thiamine for 36 hours to co-overexpress GFP-19-AEQ, Yam8 and Cch1. The monitoring of the cytoplasmic Ca2+ level was performed as described in (TIF)Click here for additional data file.Methods S1Overexpression of the cch1+ and yam8+ genes.(DOCX)Click here for additional data file.