In mammals, calsequestrin is encoded by two genes, CASQ1 and CASQ2, which are expressed almost exclusively in skeletal and cardiac muscles, respectively. Phylogenetic analysis indicates that calsequestrin is an ancient gene in metazoans, and that the duplication of the ancestral calsequestrin gene took place after the emergence of the lancelet. CASQ2 gene variants associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) in humans are positively correlated with a high degree of evolutionary conservation across all calsequestrin homologues. The mutations are distributed in diverse locations of the calsequestrin protein and impart functional diversity but remarkably manifest in a similar phenotype in humans.Calsequestrin is among the most abundant proteins in muscle sarcoplasmic reticulum and displays a high capacity but a low affinity for Ca Consequently, the SR controls excitation–contraction coupling3 without compromising Ca2+ requiring cellular processes that are normally associated with the endoplasmic reticulum4. There are two well defined structural and functional regions of the SR in cardiac muscle: the longitudinal SR that runs parallel to the myofibrils and the junctional SR that forms multiple membrane contacts with T-tubule membrane contact sites7. SR-T-tubules membrane contact sites play an important role in initiation of the excitation–contraction coupling7. The longitudinal SR is enriched with Ca2+-ATPase (SERCA) which is responsible for the rapid removal of Ca2+ from the cytoplasm to initiate muscle relaxation6. The junctional SR is the primary site of Ca2+ release via the Ryanodine Receptor (RyR)/Ca+2 channel which triggers muscle contraction. The SR luminal Ca2+ binding protein calsequestrin is a high capacity, low affinity Ca2+ binding protein that forms oligomeric structures that regulate RyR activity via interactions with RyR, triadin and junctin14. There are two isoforms of calsequestrin, which are encoded by two different genes: cardiac calsequestrin (CASQ2) and skeletal muscle calsequestrin (CASQ1)15. The crystal structure of calsequestrin indicates that the protein contains three thioredoxin-like domains reminiscent of ER luminal oxidoreductases17.The sarcoplasmic reticulum (SR) is a high specialized membrane network that supports mechanical muscle functions requiring large fluxes of Ca19. CPVT results from defects in intracellular Ca2+ handling by cardiomyocytes. Two major variants have been associated with the CPVT disorder. The autosomal dominant form is associated with mutations in the cardiac ryanodine receptor (RyR2) gene and accounts for ~ 50% cases, while a recessive form with mutations in the cardiac isoform of calsequestrin (CASQ2) accounts for 2–5% cases. Other mutations also found in the CALM1, CALM2, CALM3 , and TRDN (encodes triadin) gene account for < 2% of CPVT cases24. Thirteen mutations in the CASQ2 gene have been identified in CPVT patients, in sudden death syndrome26, and three of them are non-synonymous polymorphisms (cSNP)27. Several biochemical and cell biological studies of R33Q, L167H, and D307H calsequestrin mutants indicate that these mutations lead to impaired Ca2+ storage and Ca2+ release from the SR41. Recently new calsequestrin mutations have been identified including K180R, D351G, G332R, and P329S46.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by ventricular arrhythmias leading to sudden deathCasq1, Casq2 and pre-duplicate calsequestrin in the phylogenetic analysis. We showed that calsequestrin is an ancient protein in the metazoan, and that the duplication of the calsequestrin gene took place after the divergence of the lancelet but before divergence of Chondrichthyes. We noted that calsequestrin mutations, associated with CPVT, positively correlated with an increase in the degree of evolutionary conservation of the mutated sites. Furthermore, we carried out biochemical and biophysical analysis of seven CPVT related mutants , and whole exome sequencing variants  selected based on their linked to the human CPVT phenotype and located within highly conserved thioredoxin-like domain. The mutations are distributed in diverse locations of the calsequestrin protein but remarkably manifest in a similar phenotype in humans.In this study, we examined the evolutionary constraints of the CPVT related calsequestrin mutations, and included casq1 and casq2) was carried out with the aim of clarifying the distribution and conservation of each paralogue across animals, and thus deduce the timing of the gene duplication event and relate this information to calsequestrin mutants responsible for CPVT. Homology searching was undertaken in 23 metazoan genomes and three outgroup lineages to identify calsequestrin homologues. We identified unambiguous calsequestrin homologues in most of the vertebrate and invertebrate lineages . We were unable to identify any calsequestrin sequences in the insect lineage. This likely represents a bona fide loss in this line given the positive identification of a homologue in the spider Parasteatoda tepidariorum.Each of these lineages, as well the invertebrates and hemichordates, possess only a single calsequestrin gene, leaving the timing of when casq1 independently. We identified casq1 paralogues in the Chondrichthyes despite robustly classifying casq2 being present and the duplication having taken place prior to this point  to investigate Ca2+ binding, and this was monitored by circular dichroism (CD) analysis52. Upon adding Ca2+, wild-type Casq2 lost 18.3% α-helix and gained 16.79% β-sheet conformation  cross-linker  revealed that calsequestrin is an ancient protein within the metazoan, and duplication of the calsequestrin gene took place after the divergence of the lancelet but before divergence of Chondrichthyes. Duplication of the calsequestrin gene allowed for the differentiation of a muscle-specific form of the protein, namely cardiac calsequestrin (casq2) expressed in cardiomyocytes. In the heart Ca2+ release from the calsequestrin (Casq2) rich junctional SR is initiated by the Ca2+-induced Ca2+ release mechanism. In skeletal muscle, where Casq1 is expressed, Ca2+ release from the junctional SR is initiated by the depolarization-induced Ca2+ release mechanism. Notably in mammalian species Casq1 is almost exclusively expressed in skeletal muscle  even including pre-duplication Casq. Because of a specific disease phenotype of Casq2 mutants, one a priori prediction is that the mutations associated with CPVT would be in sites conserved in Casq2 but divergent in Casq1 and preduplicates, providing gain-of function amino acid changes in Casq2 which were then disrupted by the disease phenotype-associated mutations. However, this was not what we observed. Instead we found strong conservation at these positions between the paralogues or indeed across all calsequestrin homologues. This suggests that the residues at these positions are critical for calsequestrin function. Specificity of cardiac disease seen with Casq2 mutants is likely due to the tissue-specific expression patterns of the paralogues. In humans there is little or no Casq1 paralogue expressed in cardiac tissue to compensate for Casq2 malfunction in CPVT.Phylogenetic analysis of CASQ2 variants from whole-exome sequencing clinical testing, P329S, G332R, and D351G63. In agreement with previous reports64, our studies showed that Casq2R33Q, Casq2L167H, and Casq2D307H differed in their biochemical properties. Casq2D307H was reported to have significantly reduced Ca2+ binding capacity17. In this study, in label-free MST Ca2+ binding assay the D307H mutant showed no measurable Ca2+ binding and altered protein tertiary structure. R33Q and L167H mutants retained Ca2+ binding capacity but had increased sensitivity to tryptic cleavage and lost Ca2+-dependent polymerization.For our biochemical studies, we have selected mutants linked to the human CPVT phenotype and located within highly conserved positions, namely, R33Q, L167H, K180R, D307H and three R33Q and Casq2L167H mutants form large oligomers insensitive to Ca2+, indicating that they lost Ca2+ depend polymerization, and are not able to depolymerize in response to Ca2+ depletion, a critical function that affects the RyR2 channel gating response to depletion of Ca2+ during muscle contraction54. Amazingly, Casq2D307H substitution from aspartic acid to histidine, in the highly conserved hydrophobic core of third thioredoxin-like domain of Casq2, results in the loss of low affinity Ca2+ binding to Casq2. This is likely due to disruption of the third thioredoxin-like domain, a highly conserved region in Casq2. Surprisingly, Casq2D307H polymerized in a Ca2+-dependent manner, and, just like the wild-type Casq2, it exhibited Ca2+-dependent confirmation changes31. Nevertheless, the loss of low affinity and high capacity Ca2+ binding sites, due to severe Casq2D307H misfolding, results in reduced Ca2+ storage at the junctional SR, and impaired Casq2/Ca2+-dependent regulation of RyR2 activity65. Animal studies revealed that in Casq2 D307H /D307H knock-in mice there is a reduction in the abundance Casq2 D307H protein66. Others reported no effect of D307H mutation of protein stability in either animal model or in cultured cardiomyocytes65. Expression of Casq2 L167H mutant did not affect the level of the endogenous wild-type Casq234. However, in R33Q knock-in mice there was a significant reduction in the abundance of Casq2R33Q/R33Q protein30. This likely contributes to the CPVT phenotype.The Casq227. Knollman’s group recently reported a CPVT-like phenotype in a K180R heterozygous knock-in mouse model46. Here we discovered that the Casq2K180R protein has indistinguishable biochemical properties from the wild-type Casq2, including Ca2+ binding affinity, secondary structure and conformation change in response to increased Ca2+ concentration, protein flexibility and conformation stability upon trypsin proteolysis, protein folding, and Ca2+ dependent polymerization. Recently, crystal structure studies of the Casq2K180R maps the mutation to the filament-forming interface67, and it was proposed that disrupted Casq2 polymer formation may be responsible for Casq2 mutant-associated CPVT. Casq2 binds directly to the luminal domain of ER stress sensor IRE1α at the junctional SR to prevent the activation of IRE1α55. Interestingly, of all mutants tested in this study, only Casq2K180R showed altered binding to the luminal domain of IRE1α. Whether this is associated with CPVT remains to be established.K180R, P329S, G332R, and D351G mutants have not been previously studied with respect to their biochemical properties. K180R is a newly identified Casq2 mutant, and the first autosomal dominant mutant found of Casq2D351G42, Casq2P329S39 and Casq2G332R39 are three novel Casq2 variants42 located in a highly conserved third thioredoxin-like domain. Casq2P329S and Casq2G332R have been identified as heterozygous carrier39. They are localized in the hydrophobic core of the highly conserved beta-sheet of the third thioredoxin-like domain and are highly conserved throughout the metazoan including Casq1 and pre-duplicate Casq. To our knowledge, there have been no reports on the biochemical and biophysical analysis of these mutants. Casq2D351G showed protein folding and function similar to wild-type protein, whereas Casq2P329S and Casq2G332R showed similar properties to Casq2D307H, including severe disruption in protein folding and impaired Ca2+ binding, indicative of an important structural and functional role for the highly conserved beta-sheet in the third thioredoxin-like domain of Casq2.Casq22+ binding, mutations in this region including D307H, P329S, and G332R, and all result in severely misfolded protein with reduced or lost Ca2+ binding. R33Q, L167H and K180R are located at the Casq2 front-to-front and back-to-back polymerization interface, causing dysfunction in the protein’s Ca2+ depend polymerization/depolymerization that would affect filament formation, as proposed by Titus et al.67. This may be the unifying feature of Casq2 mutants association with CPVT67.Overall, the three CPVT disease causing Casq2 mutants  and two heterozygous variants (P329S and G332R) may lead to CPVT via different mechanisms. The third thioredoxin-like fold domain contains one highly conserved beta-sheet consist of four beta-strands, which are essential for correct folding and CaHomo sapiens, Canis lupus familiaris, Bos taurus, Oryctolagus cuniculus, Sus scrofa, Rattus norvegicus, Mus musculus, Xenopus tropicalis, Gallus gallus, Taeniopygia guttata, Crocodylus porosus, Latimeria chalumnae, Danio rerio, Callorhinchus milii, Leucoraja erinacea, Branchiostoma floridae, Ciona intestinalis, Helobdella robusta, Drosophila melanogaster, Apis mellifera, Manduca sexta, Daphnia pulex, Parhyale hawaiensis, Parasteatoda tepidariorum, Caenorhabditis elegans, Nematostella vectensis, Mnemiopsis leidyi, Trichoplax adhaerans, Amphimedon queenslandica, Monosiga brevicollis, Salpingoeca rosetta, Capsaspora owczarzaki. Ensembl: Helobdella robusta. Skatebase.org: Leucoraja erinacea. hymenopteragenome.org: Apis mellifera. wfleabase.org: Daphnia pulex.The genomes used in the comparative genomics and phylogenetics analyses are publicly available and include the following from NCBI: H. sapiens CASQ1 and CASQ2 sequences as queries, BLASTp  searches were performed on the genomes of 28 metazoan organisms  was used for site-directed mutagenesis PCR with primers (Supp. Table A pET22b casq2 and casq2 mutants was cloned into pET22b expression vector. Proteins were expressed in E. coli BL21(DE3) and purified. Cells were grown in LB medium until the A600 reaches 0.6 at 37 °C, then induced with 1 mM IPTG at 37 °C for 3 h. Cells were crashed into a buffer containing 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, and 10% glycerol, then purified by using a HisTrap HP purification column  and AKTA pure chromatography system (GE lifesciences 29018224). Purification was performed using binding buffer containing 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, and protein eluted with buffer containing 50 mM Tris–HCl, pH 8.0, 300 mM NaCl, and 250 mM imidazole. The 6xHis tagged ER luminal domain of IRE1α (IRE1-NLD) was expressed in COS-1 cells and purified by Ni-NTA agarose chromatography55. Protein concentration was determined using Bio-Rad DC protein assay  as recommended by the manufacturer. Protein concentration was also determined by measuring A280 absorbance using Nanovue plus spectrophotometer .cDNA encoding wild-type canine cardiac 2PO4, pH 7.4, and 5 mM KCl, as NaCl interferes with CD analysis. CD scans were recorded using a quartz cell with a path length of 1 mm, response time of 2 s, scan speed of 10 nm/min, and band width of 1.0 nm. Ca2+-induced changes in CD spectra were monitored in the presence of 1 mM of EGTA and 6 mM of CaCl2. CD spectra analysis was carried out at 24 °C. The final spectra was an average of 5 measurements, after baseline subtraction. Analysis of the spectra was performed using K2D368. The following calculations were performed for analysis:CD spectra were recorded on a Jasco model J-810 spectropolarimeter. Far UV CD spectra were collected with 4.82 μM protein in buffer containing 10 mM NaH2dmol−1, θobs is the observed ellipticity in degrees, Mean residue ellipticity was calculated with formula:2, 250 μM EGTA, 0.05% Tween-20, and 2.5% glycerol. CaCl2 and EGTA concentrations were adjusted to obtain the desired free Ca2+ concentration: no Ca2+ , 5 mM . Free Ca2+ concentration was calculated using the Ca2+-EGTA Calculator TS v1.3 web tool69.MST analyses were carried out using a Monolith NT.115 instrument  or Monolith NT.LabelFree instrument . The ER-luminal domain of IRE1α (IRE1-NLD) was labelled using the Monolith NT Protein Labeling Kit RED-NHS  following the manufacture’s protocol. All experiments were carried out at room temperature in standard capillaries with 20% LED power (fluorescence lamp intensity) and 40% MST power (IR-laser intensity). The assay buffer contained 50 mM HEPES, pH 7.4, 150 mM KCl, 500 μM CaCl2+ binding to wild-type Casq2 or Casq2 mutants were carried out using a Monolith NT.LabelFree instrument in standard capillaries with 20% LED power and 60% MST power. The proteins were incubated for 10 min in a buffer containing 50 mM HEPES, pH 7.4, 150 mM KCl, 0.1% pluronic F-127, and 50 μM EGTA. An increasing concentration of CaCl2  was used. All MST data was analyzed by Monolith Affinity Analysis v2.2.6 software.Ca2, 250 μM EGTA, 0.05% Tween-20, and 2.5% glycerol.Thermal denaturation analysis of wild-type Casq2 or Casq2 mutants was carried out using Tycho NT.6. This label-free system is based on measurement of a protein’s intrinsic tryptophan fluorescence and records a protein’s unfolding profile in real-time as temperature is increased from 35 to 95 °C. Ten µl of 0.25 mg/ml protein was used in buffer containing 50 mM HEPES, pH 7.4, 150 mM KCl, 500 μM CaCl2+ concentration (167 µM), a discontinuous Tris–glycine polyacrylamide gel system consisting of a 5% stacking gel and a 10% separation gel was used under non-denaturing conditions. Proteins were diluted 3 × with non-denaturing loading dye . Proteins were separated in a Mini-PROTEAN electrophoresis chamber (BioRad) in a running buffer containing 25 mM Tris, pH 8.8, and 192 mM glycine, at 100 V, for 2 h at 4 °C. The proteins were stained with Stains-all solution70, Coomassie-blue, or transferred to nitrocellulose membrane for immunoblotting analysis.To determining the oligomerization state of Casq2 or Casq2 mutants in the presence of the same free Ca2, 250 μM EGTA, 0.05% Tween-20, and 2.5% glycerol. CaCl2 and EGTA concentrations were adjusted to the desired free Ca2+ concentration: no Ca2+ , 5 mM . Free Ca2+ concentration was calculated using the Ca-EGTA Calculator TS v1.3 web tool69. Proteins (150 μg of protein in 200 μl) were incubated in a reaction buffer with the desired free Ca2+ concentration for 20 min at 25 °C before addition of trypsin at the trypsin/protein ration of 1:50 (w/w), and samples were taken for SDS-PAGE analysis at 1, 2, 5, 10, 20, 30, 60, and 120 min. The samples were mixed with 4 × SDS-PAGE sample buffer (Bio-Rad) containing PMSF, and boiled at 100 °C for 2 min before SDS-PAGE. All experiments were repeated 3 times with protein from 2 separate purifications. The gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad).Cardiac calsequestrin (Casq2) and mutant proteins were subjected to proteolysis in a buffer containing 50 mM HEPES, pH 7.4, 150 mM KCl, 500 μM CaCl55. Protein concentration of wild-type and Casq2 mutant proteins was determined using Bio-Rad DC protein assay and the proteins were diluted to a final concentration of 10 µM in a reaction buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 250 μM EGTA, 500 μM CaCl2, and 0.05% Tween-20. Proteins were incubated with 20-fold molar excess of DSS for 1 h at 22.5 °C. The reaction was then quenched for 15 min with 100 mM Tris pH 7.4 followed by SDS-PAGE (10% acrylamide). Proteins were transferred to nitrocellulose membrane follow by immunoblotting with mouse anti-6xHis antibodies  or anti-calsequestrin antibodies .The homobiofunctional protein cross linker disuccinimidyl suberate (DSS)  was dissolved in DMSO at a final concentration of 10 mMp-value determined to be significant if less than 0.05.Statistical analysis was performed using GraphPad Prism version 7.0. The Student’s t-test was used to compare the mean of two independent groups, and one-way Anova was used to compare the mean of three or more independent groups, with a Supplementary Information 1.Supplementary Table S1.


These data indicate that PI3K-C2α and PI3K-C2β play the redundant but essential role for vascular smooth muscle contraction and blood pressure regulation mainly through their involvement in Rho activation.Class II phosphatidylinositol 3-kinases (PI3K), PI3K-C2α and PI3K-C2β, are involved in cellular processes including endocytosis, cilia formation and autophagy. However, the role of PI3K-C2α and PI3K-C2β at the organismal level is not well understood. We found that double knockout (KO) mice with both smooth muscle-specific KO of PI3K-C2α and global PI3K-C2β KO, but not single KO mice of either PI3K-C2α or PI3K-C2β, exhibited reductions in arterial blood pressure and substantial attenuation of contractile responses of isolated aortic rings. In wild-type vascular smooth muscle cells, double knockdown of PI3K-C2α and PI3K-C2β but not single knockdown of either PI3K markedly inhibited contraction with reduced phosphorylation of 20-kDa myosin light chain and MYPT1 and Rho activation, but without inhibition of the intracellular Ca Rho-MLCP pathway as well as Ca2+-MLCK pathway coordinately and effectively increase MLC20 phosphorylation, thus playing a critical role in contraction induced by various agonists including NA, endothelin-1 and thromboxane A2. We and others previously showed that an increase in [Ca2+]i induced by membrane depolarization and a Ca2+ ionophore also resulted in the activation of Rho-MLCP pathway in smooth muscle i response and contraction of control siRNA-transfected HASM. Control siRNA-transfected, the fluorescent Ca2+ indicator fluo-8 loaded HASM cells were stimulated with ionomycin (0.3 µM) at 1 min, and contractile responses and changes in [Ca2+]i response were continuously monitored for 10 min .Additional file 2: Video S2. The time-lapse imaging of ionomycin-induced [Ca2+]i response and contraction of PI3K-C2α siRNA-transfected HASM. PI3K-C2α specific siRNA-transfected, the fluorescent Ca2+ indicator fluo-8 loaded HASM cells were stimulated with ionomycin (0.3 µM) at 1 min, and contractile responses and changes in [Ca2+]i response were continuously monitored for 10 min .Additional file 3: Video S3. The time-lapse imaging of ionomycin-induced [Ca2+]i response and contraction of PI3K-C2β siRNA-transfected HASM. PI3K-C2β specific siRNA-transfected, the fluorescent Ca2+ indicator fluo-8 loaded HASM cells were stimulated with ionomycin (0.3 µM) at 1 min, and contractile responses and changes in [Ca2+]i response were continuously monitored for 10 min .Additional file 4: Video S4. The time-lapse imaging of ionomycin-induced [Ca2+]i response and contraction of PI3K-C2α and -C2β siRNA-transfected HASM. PI3K-C2α and -C2β specific siRNA-transfected, the fluorescent Ca2+ indicator fluo-8 loaded HASM cells were stimulated with ionomycin (0.3 µM) at 1 min, and contractile responses and changes in [Ca2+]i response were continuously monitored for 10 min .Additional file 5: Video S5. The time-lapse imaging of RhoA-FRET in ionomycin-stimulated HASM. HASM cells were transfected with RhoA-FRET probe expression vector and either control siRNA (left) or PI3K-C2α and -C2β specific siRNAs (right), and stimulated with ionomycin (0.3 µM) at 1 min. RhoA-FRET signals were monitored by confocal microscopy for 10 min . Note that the addition of ionomycin resulted in rapid activation of RhoA in control cells whereas ionomycin-induced RhoA activation was substantially reduced in C2α- and C2β-depleted cells.Additional file 6: Video S6. The time-lapse imaging of RhoA-FRET in ET-1-stimulated HASM. HASM cells were transfected with RhoA-FRET probe expression vector and either control siRNA (left) or PI3K-C2α- and -C2β specific siRNAs (right), and stimulated with endothelin-1 (1 µM) at 1 min. RhoA-FRET signals were monitored by confocal microscopy for 10 min . Note that endothelin-1 resulted in rapid activation of RhoA in control cells whereas endothelin-1-induced RhoA activation was substantially reduced in C2α- and C2β-depleted cells.


In addition, the fragmentary results from the literature were compiled to develop a harmonized, informational, and contemplative model that is intended to improve our perception of these adaptative mechanisms and set a common platform for plant biologists to identify intriguing research questions in this area.Plant stress is a real dilemma; it puzzles plant biologists and is a global problem that negatively affects people’s daily lives. Of particular interest is salinity, because it represents one of the major water-related stress types. We aimed to determine the signals that guide the cellular-related events where various adaptation mechanisms cross-talk to cope with salinity-related water stress in plants. In an attempt to unravel these mechanisms and introduce cellular events in the right context, we expansively discussed how salt-related signals are sensed, with particular emphasis on aquaporins, nonselective cation channels (NSCCs), and glycosyl inositol phosphorylceramide (GIPC). We also elaborated on the critical role Ca2 Unlike animals that can exhibit the fight or flight response, plants are sessile organisms that have only one approach when encountering stress—adaptation. Plants constantly monitor an ever-changing environment, sense the incoming signals, and then correctly integrate and decode the signals to formulate a battery of signaling molecules that ultimately enable them to cope with stress. Water-related stress  represents one of the major types of plant stressors, of which osmotic stress is considered a general component . In the 2O, but also a high diversity of small and noncharged substrates, such as H2O2, CO2, and urea ). In contrast to a steady-state condition, water-related stress raises extracellular osmolarity in conjunction with low apoplastic H2O potential. Therefore, an efflux of water out of the cell will dominate the influx process through AQP aqueous filters, leading to cytosolic osmotic disturbances and the loss of cell turgor pressure [2+” channels (MSCCs) (O]) [2+ influx [C→1]).When it comes to plant–water relations, aquaporins (AQPs) play a central role in transport and adaptation processes. AQPs are intrinsic membrane proteins that channel not only Hand urea . Based oand urea . Under wand urea . AQP gatand urea ,7,8. Forand urea , as welland urea ,11. Furtsalinity . The invpressure . These iCs) (O]) . The ope+ influx , which i121 in an ABA-dependent manner. Furthermore, transgenic Arabidopsis pip2;1-1 and pip2;1-2 mutants exhibit ABA-specific defects in their stomatal movement ), thereby enhancing the Ca2+ influx (C→1]). Of particular interest are the newly identified MSCCs termed the OSCAs , in which the osmotically induced Ca2+ signaling is impaired in the guard cells and root cells of the knock-down osca1 mutant ). Indeed, hypo- and hyperosmotic challenges promote cytoskeletal reorganization (actin “AFs” or microtubules “MTs”), which in turn results in modifying cell hydraulic conductivity . Th. Th+ cannization . Further2+]i increases 1 (moca1), that is defective in salt-induced Ca2+ spikes. The authors identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids located on the plasma membrane. The authors also showed that MOCA1 is essential for NaCl-triggering depolarization of the cell-surface potential. In essence, Na+ binds to GIPCs, and facilitates Ca2+ influx that ultimately leads to Ca2+ spikes and activation of the downstream signals , thereby depolarizing the cytosolic surface of the PM (N→1]) ) [N→2]) ) . Therefo3N→1→1]) . Therefo3N→1→1]) ,46. For ia cells ,47. Thiss (N→3]) . Further) [N→2]) . + in the cytosol is detrimental ), but promotes KORs that subsequently efflux K+ out of the cell (N→1→3]) ) ) ) ) ,53. When(N→1→3]) . Salt-toy (O→2]) ,56. This& 4O→3]) . Subsequ(C→6&9]) ,45. In aaptation .+ distribution at the systematic level through the high affinity K+ transporters (HKTs), with only one representing the gene AtHKT1;1 in the Arabidopsis genome ) ) ) ). Further research is required to advance our knowledge about the exact role of water-induced Ca2+ on AQP gating.Calcium plays a central role in all kingdoms of life, and is involved in nearly all biological processes. Despite such universality, signals of Cacapacity . These Cvacuoles . In termnditions ,70; the –200 nM) . In contis roots , and in 3N→1→1]) ,73. Thesmulation ,72. Indet manner . Similars (C→1]) . It rema+ ions. Therefore, it has to deploy parallel mechanisms to (1) arrest the additional Na+ influx, (2) remove it from the place of action (cytosol), as well as (3) reduce its side effects. The first task can be partially accomplished via cAMP or cGMP, while the Ca2+-dependent SOS pathway seems to be a central hub for Na+ exclusion and sequestration ) ) ,85. On tC→10→1]) . In factfunction ,88. Thusfunction , but thiBP8/SOS2 . Moreoveectivity .C→6-Shoot]). However, as the N-terminal myristoylation domain is missing, SCaBP8 localizes the PM by its N-terminal hydrophobic domain [237 in a NaCl-dependent manner (N→4-Shoot]) ). Interestingly, 14-3-3 proteins, the positive regulators of PM H+-ATPases, can also bind to SOS2 at the Ser294 residue under nonstressful conditions, resulting in a repression of its basal kinase activity. However, NaCl reduces this interaction, thereby activating the SOS pathway for salt adaptation ) . Recent -Shoot]) . However-Shoot]) . The faseduction . These reduction ,94. In oaptation . The inv2+ signals are also self-regulated, whether by further releasing Ca2+ from or sequestrating Ca2+ into intracellular organelles and the apoplast ). In contrast, the fou2 mutant that carries a point mutation D454N of the TPC1 gene remains active under 100-fold accumulation of vacuolar Ca2+ ). Once arriving via the cortical/endodermal route, Ca2+ signals prime the expressions of different NaCl-related genes in leaves, including JA-related genes ) ) ) ,99. In aophyll]) . This mil stress ,104. Furtoxicity . For comll cells . 2+ is a central signal maker at the cellular level across the whole plant. Ca2+-signals are highly intricate and are involved not only in regulating Na+, but also with other crucial secondary messengers (H+ and ROS), as well as ABA and JA. In addition, Ca2+-signals are self-regulated to guarantee the specificity of action, and to prevent Ca2+-induced cell death. Future research is required to explain more about this master signal element in both dicots and monocots.In summary, Ca+ entry takes place downstream of Ca2+ signals, probably through PM nonselective cation/anion channels, and is suggested to act as a second messenger under stress (C→2]) ) ) ,122,123.s (C→2]) . Further cytosol ,126. Undl growth . However cytosol . Moreoveg (H→1]) ,129. Indg (H→1]) ,76. It i2+ (SCaBP1/CBL2)-dependent manner. In contrast to Thr947 phosphorylation in the C-terminal regulatory domain of AHA2 that enhances interaction with, and subsequent activation by, 14-3-3 proteins, PKS5-induced phosphorylation of the Ser931 residue prevents interaction with 14-3-3 proteins ). However, elevated salinity tolerance in halophyte quinoa, saltbush, and salt-tolerant barley genotypes were attributed to prompt activation of PM H+-ATPase, rather than higher transcription of AHAs, as observed in salt-sensitive Arabidopsis under increased salinity ) ) . Moreove tobacco ,135. Addt stress . On the  residue . Interes residue . Therefo+-ATPases seems to be very intricate, since the regulatory/autoinhibitory C-terminal domain can be phosphorylated at different sites, resulting in inactive/active forms of AHAs ) ) . Subsequvacuoles . Althougexposure . Therefo2+cyt triggers the accumulation of H+ in the cytosol, as well as ROS ) ) ) . Therefoe action . Interes pattern . These r(R→2&3]) .Gossypium davidsonii leaves and roots (R→antioxidant system]) ) . Interese damage ,158,159.To understand the underlying mechanisms of water-related stress, it is important to contextualize cellular events. The current review summarizes new advances in the field that might help in formulating an integrative perspective of cellular events under salt-related stress. It is also of paramount interest that future research focuses on the puzzling points, in addition to running parallel studies that investigate the phenomenon at different plant levels. In addition, comprehensive parallel studies on tolerant and sensitive cultivars will aid in correlating signals/events to the right cellular context.


We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da—13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome. Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca Androctonus mauretanicus mauretanicus scorpion for example, have a 10% fatality rate in children [Scorpion venom typically represents a rich resource for drug discovery as it contains a complex mixture of small peptides, proteins , amino acids, biogenic amines, lipids, carbohydrates, and inorganic salts ,2,3. Thechildren . Envenomchildren . + channel blockers), or long chain peptides (often Na+ channel modulators) [+ (Nav) channels [+ (Kv) ion channels [2+ channels [- channels relatively neglected [+ and Na+ channels  ,11, or gulators) . The vaschannels ,15,16,17channels ,19,20,21channels ,23 and Ceglected . This micarriers ,26,27 an2+ mobilization and membrane conductance are broadly interwoven. Activation of several classes of Ca2+ permeable plasma membrane ion channels can affect membrane conductance directly, as well as indirectly, via activation of Ca2+-dependent K+ channels and Ca2+ store activation . Reciprocally, activation of ryanodine receptor (RyR)-gated Ca2+ stores and inositol trisphosphate (IP3) receptor-gated Ca2+ stores in the endoplasmic and sarcoplasmic reticulum can elicit ‘capacitive Ca2+ entry’ [2+ channel, and by modulation through IP3R [2+ [The dynamics of cytosolic Ca+ entry’ ,29. CICR+ entry’ . RyRs arugh IP3R ,32,33. Rugh IP3R , RyR2 wiugh IP3R , and RyRugh IP3R ,37. RyR ugh IP3R ,39. A smugh IP3R ,41. UponIP3R [2+ ,41,42,43IP3R [2+ . These cIP3R [2+ .Hormurus waigiensis (Liocheles waigiensis) [50 of 6.0 pM and 2.0 pM, respectively) [H. waigiensis to date. The venom comprises a large pool of uncharacterised peptides [2+ mobilization and impact on bio-impedance by H. waigiensis scorpion venom fractions delineated by size exclusion fast protein liquid chromatography (SE-FPLC) and characterised by liquid chromatography mass spectroscopy (LCMS). To this end, we established a highly sensitive bioreporter HEK293 cell line that stably expressed the genetically encoded Ca2+ reporter GCaMP5G and the type 1 ryanodine receptor (RyR1) (rHEK293).The SCTX φ-LITX-Lw1a from the Australian scorpion giensis)  is the mctively) . This ispeptides , which t2+ stores via the endogenous RyR expression, as evident from differences in sensitivity to caffeine [3R-gated Ca2+ signalling in these cells, as evident in many studies where the M3 muscarinic receptor agonist carbachol has been employed to drive Ca2+ store release [2+ stores, alongside Ca2+ entry, a custom-developed recombinant human embryonic kidney 293 (rHEK293) cell line stably over-expressing the rabbit RyR1 calcium-store channel , and the GCaMP5G genetically encoded Ca2+ reporter  [Untransfected human embryonic kidney 293 (HEK293) cells have been reported to have varying capacity for gating of Cacaffeine ,49. Thiso.31788)  was deveHormurus waigiensis, a rainforest scorpion with widespread distribution across South-East Asia, the Pacific, and throughout the Australian east coast [This study used the venom milked from st coast   and then diluted with 150 μl of degassed phosphate buffered saline (PBS), centrifuged for 10 min, and filtered through a 0.22 μm Millipore filter. Venom profiles were subsequently obtained by SE-FPLC  using a Superdex 75 10/300 (Tricorn) GL Column  with PBS as the carrier (500 µL/minute for 45 mL). Venom component elution was monitored at an absorbance wavelength of 280 nm. Fractions (500 µL) were collected into a 96 well-plate and stored at 4 °C (~530-fold dilution). Venom profiles were then processed and exported using UNICORN 5.20 software . Individual elution fractions (EF) obtained from SE-FPLC were then analysed by LCMS using a Shimadzu LCMS-2020 mass spectrometer coupled to a Shimadzu Prominence HPLC system  to observe the composition and determine the molecular weights of the molecules present in each fraction. Fractions (20 µL) were injected via an autosampler (Shimadzu SIL-20AC-HT) onto a reversed-phase high-performance liquid chromatography column  at 30 °C. Solvent  was delivered via Shimadzu LC-20AD pumps at a flow rate of 0.250 mL/min. Samples were washed on the column for 5 min to remove salt from the SE-FPLC PBS buffer, then eluted with a 1% gradient , and the UV absorbance observed at 214 nm and 280 nm on a Shimadzu SPD-20A detector. Mass spectra were collected in positive ion mode over a scan range of m/z 250–2000 with a detector voltage of 1.15 kV, nebulizing gas flow of 1.5 L/min, and drying gas flow of 3.0 L/min. Data were collected and analysed using the Shimadzu LabSolutions v5.80 software.H. waigiensis venom on the rHEK293 biosensor cells was undertaken using the xCELLigence RTCA Single Plate system , which provided real-time bio-impedance measurements. This cellular impedance assay is reported as the unit-less “Cell Index” (CI). CI reflects a broad range of cell responses that affect electric current flow through the cell layer overlying an array of gold microelectrodes, including effects on cell shape and number, cell-cell junctions and cell–substrate adhesion, ion channel activation, and G protein-coupled receptor (GPCR)-mediated cytosolic Ca2+ elevation [H. waigiensis venom. Venom samples  were then applied to all wells via a multi-tip pipette. Combining of the elution fractions into six venom Pools (A–F) was necessary to provide sufficient material for replication across multiple wells. The elution fractions comprising these sample pools were: Pool A ; Pool B ; Pool C ; Pool D ; Pool E ; Pool F . Recording commenced at 15 sec intervals . Data were analysed using RTCA software V.2 . A high-throughput screen to investigate the biophysical actions of levation . The 96 t-tests (two-tailed) at 2.5, 5, and 25 min time points on normalized background subtracted data to assess a difference from a predicted mean of 0. Where data were not normally distributed, as determined by Systat, a one sample Signed Rank Test was undertaken. The alpha value for significance was 0.05. Two-way repeated measure analysis of variance (ANOVA), with post-hoc Holm–Sidak multiple pairwise comparison of pooled venom components was performed for runs 10 (2.5 min), 20 (5 min), and 100 (25 min).Data are presented as mean normalised CI ± s.e.m, with CI = 1 representing the CI measurement just prior to toxin addition. GraphPad Prism (v. 7) was used to produce the graphs. SigmaPlot (Systat v. 12.5) was used for statistical analysis. This included individual one-sample H. waigiensis venom was superfused into the imaging bath containing rHEK293 cells at a final dilution of ~1: 10,000 (in 3 mL) for 500 s. This was followed by a 1000 second DMEM media washout period, 500 s of 4 mM caffeine exposure (positive control), and then a 1000 s media washout. This protocol was conducted for raw whole H. waigiensis venom (three repeats), PBS elutant control from the SE-FPLC process (three repeats), media-only control (two repeats), and in triplicate for 14 peak samples reconstituted from the 53 elution fractions (EF) comprising the 12 chromatographic SE-FPLC peaks identified  which was maintained by a pump system (Gilson minipuls 3) at a constant flow rate (360 µL/min). entified B: Peak s2+ following venom and caffeine application were assessed as changes in (F/F0), where F0 is the average of three whole field mean pixel intensity values (covering 30 sec) immediately prior to changing to the test solution, and F is the average of three whole field mean pixel intensity values immediately prior to media washout.Calcium imaging was conducted using a Zeiss 710 Laser Scanning Microscope with a W N-Achroplan 10×/0.3 W (DIC) M27 water immersion objective at 1.5x zoom; 488 nm excitation/542 nm emission, 10 s sampling rate with no averaging, using Zeiss Zen Blue 2012 software. Images  were generated for each time point. Image analysis was undertaken using Image J software  which calculated mean pixel intensity for the whole of field region of interest. Increases in cytosolic Ca2+ responses were determined using single sample t-tests (single-tailed), or one sample Signed Rank Tests if data were not normally distributed. The alpha value for significance was 0.05, assessing a difference from a predicted mean value of 1.00. With validation of normal distribution, comparison of samples against the control group was then undertaken using one-way ANOVA, with post-hoc Bonferroni-corrected pairwise comparison. T-tests (two-tailed) were used to compare the SCTX Ca2+ responses against the caffeine responses.Sigmaplot (Systat v.12.5) as used for statistical analysis. Data are presented as mean ± s.e.m. For whole venom, SE-FPLC peak samples, controls (PBS and media samples) and caffeine, CaH. waigiensis venom from 14 animals . HEK293 cells are known to be sensitive to mechanical disturbances, as exhibited by changes in cytosolic Ca2+ [n = 5) at approximately 105 s, followed by a subsequent delayed increase in impedance to a maximum CI of 1.112 ± 0.009 at 7.5 min, where normalized CI = 1 prior to sample addition. This average control profile was subsequently subtracted from the individual responses to whole venom (n = 6), reconstituted whole venom (n = 6), and pooled SE-FPLC elution fractions: Pool A (n = 8); Pool B (n = 9); Pool C (n = 7); Pool D (n = 5); Pool E (n = 7); Pool F (n = 6).Investigation of the effect of whole and fractionated venom on membrane impedance was undertaken using the xCELLigence platform. Negative controls consisting of PBS eluted from the SE-FPLC were used to establish a baseline profile with solution switching (lic Ca2+ . This reWhole venom (1:2100 dilution) elicited a clear triphasic bio-impedance response from the rHEK293 cell monolayers C, that wp < 0.05) in their response for most census time points   . Two-way2+ was undertaken via confocal Ca2+ imaging using the rHEK293 cell line. 2+  following 470 s of the treatment,/F0, the mean pixel intensity obtained for the preceding reference period ).Investigation of the capacity of whole and fractionated scorpion venom to modulate cytosolic Caline. 2+ A. A compline. 2+ B. Negatiline. 2+ C. This eline. 2+ B. As des2+, based on single sample t-tests (single-tailed), or one sample signed rank tests where data were not normally distributed .Whole venom, 9 out of the 14 SE-FPLC peak samples , and caffeine, elicited statistically significant increases in cytosolic Catributed . For exa2+ responses (p = 0.001). Associated post-hoc Bonferroni-corrected pairwise comparison t-tests showed significant differences between whole venom, peak samples 1, 9, and 11, relative to the negative control group. Two-tailed t-tests were used to compare the Ca2+ response from application of whole venom and the nine significant peak samples  to that seen following 4 mM caffeine treatment. This showed that all these significant venom-induced responses were statistically indistinguishable from caffeine, except for Peak samples 2b and 5, that produced smaller Ca2+ responses (p = 0.0214 and p = 0.0470 respectively).With validation of normal distribution, comparison of samples against the negative control group was then undertaken using one-way ANOVA revealing that overall, the application of SE-FPLC peaks to rHEK293 cells elicited significant Ca2+ imaging time point to determine whether the potency of venom-induced biophysical responses identified by xCELLigence could be related to the magnitude of the venom-induced Ca2+ response. The Ca2+ responses were derived by summing the individual SE-FPLC peak responses that matched to the venom pools in the xCELLigence study. This qualitative analysis . However, the most potent Ca2+ response activator  was the weakest bioimpedance modulator (sixth), indicating that activity of some of the scorpion venom bioactive molecules are highly selective for Ca2+ mobilization, with limited secondary action on cell membrane properties.A rank order analysis of potency was performed using the 5-minute time point for the xCELLigence measurements against the 8 minute Caanalysis  showed sH. waigiensis venom components that modulated cell bio-impedance and raised cytosolic Ca2+. This broad functional screening of the effects of scorpion venom components was enabled by development of the GCaMP5G‒RyR‒HEK293 cell Ca2+ sensor bioreporter model. SE-FPLC delineated 12 primary chromatographic peaks, which LCMS resolved as 182 distinct molecules, with 3 to 63 molecular components per peak. The screening was achieved by pooling discrete groups of molecules based on size separation. While these molecules were not individually resolved, ~45% were in the 1.5–14 kDa range . While location ,41.2+ mobilization and a reduction in bioimpedance at 5 min (xCELLigence venom Pool F).The distinct triphasic bio-impedance response seen using the xCELLigence platform following application of whole venom , is consistent with a complex and diverse set of scorpion toxin peptide actions on the rHEK293 cells. These responses were able to be reconstructed and attributed to various pools of venom fractions, with Pool E  for example, making the predominant contribution to the increased impedance at 2.5 min, further indicating that the SCTX molecules within these mixtures have differential actions. This is expected given the molecular diversity that we identified within the venom D. Agains2+-mediated changes in cell properties, including those elicited by activation of Ca2+ channels, or Gq-type GPCRs which elevate cytosolic Ca2+, as shown in human H295R adrenoma cells [2+ mobilization . Within the timeframe of our experiments , the venom activity was reversible. This contrasts with other published applications where cytotoxic effects on cell lines have been quantified using this platform as irreversible reductions in impedance as cells died and the electrode array was exposed. An example of where this assay has been used to screen toxin activity was with the Australian box jellyfish Chironex fleckeri, where the xCELLigence showed irreversible decreases in impedance resulting from cytotoxicity on human cardiomyocytes and muscle cells following exposure to crude toxin fractions; an effect observed within the timeframe of the present study [H. waigiensis venom-induced changes in xCELLigence-measured impedance largely reflect modulation of the conductance across the cell monolayer due to changes biophysical properties. While the imaging of the cells was not at sufficient resolution to resolve direct effects on the cell membrane that would be detected as bio-impedance changes, gross changes in cell shape and number were not overtly altered.Changes in xCELLigence platform-measured impedance across the rHEK293 cell monolayer likely reflects venom-induced alterations of the cell membrane conductance properties, as well as possible effects on morphology and/or transient changes in paracellular conductivity relative to the E-plate microelectrode array. This platform is particularly sensitive to Cama cells . Howeverlization , potentint study . Convers2+ channels (Cav) were all inhibitory. For example, kurtoxin  selectively inhibited rat Cav3.1 and human Cav3.2 by raising the activation threshold (Kd 15 nM and 61 nM respectively) in the Xenopus expression system [Centruroides tecomanus) inhibit human Cav3.1 channels in tsA201-HEK293 cells [Mesobuthus tamulus, only 0.8% of the toxin elements were identified as Ca2+ channel toxins, compared with the Na+ and K+ channel toxins which were most abundant at 76.7% [2+ elicited here by many of the H. waigiensis venom components likely to have arisen from intracellular Ca2+ store release. There is also a distinct possibility that H. waigiensis venom components, as for peptides in other scorpion species, also modulate Na+ and K+ channels, typically leading to membrane depolarization  and 2+ mobilization. While it was not possible to specifically delineate scorpion venom-mediated positive modulation of RyR channels, such action has previously been reported with calcin-like molecules isolated from several scorpion venoms [2+ increases quickly in the present study (commencing within 30 sec of venom application and developing over 5–10 min), which closely matches data reported for the calcin imperacalcin , when applied at 20 nM within a sarcoplasmic reticulum heavy vesicle model using the Ca2+ reporter Arsenazio III [2+ imaging study using the fluorophore Fluo-3, IpTxa (100–300 nM) stimulated Ca2+ transients within seconds of application [H. waigiensis). While for drug discovery, the mode of action needs to be resolved at the level of individual isolated peptides, the current study clearly highlights the need to consider the interactions between activities of the bioactive molecules, with respect to the net pathophysiological outcome of envenomation. The GCaMP5G‒RyR1‒HEK293 bioreporter cell line had enhanced sensitivity for Can venoms . In suppazio III . Additiolication . Includilication , while olication ), includ2+ store release [H. waigiensis scorpions, LCMS did not identify a molecule with the molecular weight of the ϕ-liotoxin-Lw1a peptide. Our analysis of the size distribution of H. waigiensis venom components suggests that ϕ-liotoxin-Lw1a peptide would be within peak sample 2b–peak sample 6 , both the xCELLigence platform and confocal GCaMP5G fluorescence assay would likely have been able to detect the ϕ-liotoxin-Lw1a peptide–mediated RyR activation. On this basis, it is possible that the ϕ-liotoxin-Lw1a peptide was indeed absent, or at un-detectible levels, in our venom samples, possibly reflecting the reported influence of environmental context on toxin proteomic plasticity, as shown in H. waigiensis [ϕ-liotoxin-Lw1a shares structural homology with the calcin family and has been demonstrated to induce prolonged reversible subconductance states in RyR channels in vesiculated sheep cardiac sarcoplasmic reticulum and rabbit skeletal sarcoplasmic reticulum in lipid bilayers, inducing Ca release . Indeed, release ,57. Whilsample 6 . Given tigiensis . 2+ mobilization mechanisms to be resolved.While the identification of specific channel targets was precluded in the current study by the limited quantity of available venom, in the future, isolation of the individual SCTX peptides and small molecules within the peaks evident from our LCMS analysis will enable ion channel actions and CaH. waigiensis scorpion venom that modulated cell layer impedance  and raised cytosolic Ca2+ within a specifically constructed HEK293 cell line expressing the GCaMP5G fluorescent Ca2+ reporter and IP3R and RyR1 Ca2+ store-gated Ca2+ channels. The correlation of venom fraction bioactivity between bio-impedance and Ca2+ imaging platforms, despite differences in rank order potency, suggests xCELLigence can be reasonably used to screen bioactive compounds within scorpion venoms in future experiments. Our results demonstrate that the venom from this scorpion species will enable biodiscovery of peptides mobilizing cytosolic Ca2+. In particular, the study highlights the bioactivity of the smaller molecular weight molecules (<1 kDa), which fall outside the typical size of the channel blocker toxins, and are shown here to have significant potency for intracellular Ca2+ mobilization. Such biodiscovery strategies around scorpion venom components has tremendous potential for facilitating the development of novel pharmaceutical agents and therapeutics.The present study identified an unexpectedly broad bioactivity within the components of


The aim of this review is to revisit this age-old literature and collate it with recent findings to provide a pathophysiological context for the growing body of metabolomic association studies that link circulating LCACs with CVD.A growing number of metabolomic studies have associated high circulating levels of the amphiphilic fatty acid metabolites, long-chain acylcarnitines (LCACs), with cardiovascular disease (CVD) risk. These studies show that plasma LCAC levels can be correlated with the stage and severity of CVD and with indices of cardiac hypertrophy and ventricular function. Complementing these recent clinical associations is an extensive body of basic research that stems mostly from the twentieth century. These works, performed in cardiomyocyte and multicellular preparations from animal and cell models, highlight stereotypical derangements in cardiac electrophysiology induced by exogenous LCAC treatment that promote arrhythmic muscle behavior. In many cases, this is coupled with acute inotropic modulation; however, whether LCACs increase or decrease contractility is inconclusive. Linked to the electromechanical alterations induced by LCAC exposure is an array of effects on cardiac excitation-contraction coupling mechanisms that overload the cardiomyocyte cytosol with Na Metabolomic studies have identified numerous circulating metabolites that correspond to an enhanced risk of cardiovascular disease (CVD) . One of LCACs accumulate intracellularly and embed within membrane compartments of the ischemic myocardium . AdditioA detailed description of LCAC biogenesis is beyond the scope of this review and has been done previously ; howeverA growing body of evidence has correlated circulating LCACs with total CVD risk and CVD prognosis . LCACs hTaken together, metabolomics has provided compelling evidence for an independent association of circulating LCACs with ischaemic and non-ischaemic CVD risk and severity. To date, determination of blood LCAC levels has both diagnostic and prognostic value for manifestations of HF and CAD, and there is promising evidence for a similar LCAC association with cardiac arrhythmias. The following sections detail what is understood about the pathophysiological effects of LCACs on cardiac electrophysiology and contractility.max of phase 0) and, often, a depolarization of the resting membrane potential (RMP). Furthermore, these alterations can establish a substrate for electrical arrhythmias  and with an effect latency analogous to that found in the ischaemic myocardium (2–10 min). These findings have been replicated by many groups in canine, guinea pig, and rabbit isolated cardiomyocyte and multicellular preparations exposed to similarly high LCAC doses and to lower LCAC doses (0.1–50 μM). Moreover, as with endogenous LCAC upregulation, exogenous LCAC delivery also induces spontaneous electrical arrhythmias, inhibits gap junction conductance, and promotes in vivo ventricular extra-systoles . LCACs have previously been investigated as modulators of many key enzymatic and channel ion fluxes. The pleiotropy of effects spans mechanisms that alter myocyte Cahandling . These eLCAC membrane incorporation has been modeled in purified phospholipid vesicles, with findings suggesting that LCACs alter the biophysical properties of cardiomyocyte membranes in a manner that could be linked to electrophysiological derangements , b. LCAC2+ overload within cardiomyocytes via release of sarcoplasmic reticulum (SR) Ca2+ and inhibition of mitochondrial Ca2+ uptake  facilitate Ca2+ influx during late myocyte depolarization, provoking the Ca2+-induced Ca2+ release that sustains the action potential plateau and catalyzes myofilament interaction .L-type Caeraction . LCAC sueraction . This ineraction . A biphaeraction . Paradoxmulation . Furthermulation . In contlar Ca2+ . This di2+ release and uptake mechanisms facilitate the majority of systolic intracellular Ca2+ increases and diastolic decreases in the mammalian myocardium, respectively (2+ release channel is the cardiac ryanodine receptor (RyR2), while Ca2+ is re-sequestered by the action of the SR Ca2+-ATPase (SERCA2a) (Sarcoplasmic reticulum (SR) Caectively . In cardSERCA2a) . LCAC su2+ leak, resulting in reductions in the SR Ca2+ content and cytosolic Ca2+ transients. Additionally, this Ca2+ leak enhanced spontaneous Ca2+ spark and Ca2+ wave propensity, which was linked to LCAC-mediated in vivo premature ventricular complexes. These findings support earlier reports of rapid, dose-dependently enhanced 45Ca2+ release from cardiac  of ryanodine, which depletes SR Ca2+  open probability  contributes to cytosolic Ca2+ clearance in diastole, while the ubiquitous Na+/K+-ATPase (NKA) energetically maintains the stable cardiac RMP , accounting for LCAC-mediated reductions in action potential Vmax and APA (+ current (INa(L)) that exhibits a markedly prolonged channel inactivation period and drives an increase in intracellular Na+ concentration (Na(L) has been found to occur spontaneously within 2 min of LCAC exposure and to activate a secondary transient inward current (Iti) (2+ chelator) or extracellular NiCl2 (NCX1 inhibitor) application revealed that this Iti is dependent on intracellular Ca2+ and sarcolemma Na+/Ca2+-exchange. Furthermore, INa(L) inhibition prevented the formation of Iti and spontaneous afterdepolarizations induced by LCACs, thereby validating the temporal association of Na+ accumulation with Na+/Ca2+-exchange and Ca2+ overload in arrhythmogenesis  in LCAC-mediated arrhythmias, treatment with INa(L) inhibitors lidocaine, ranolazine, or TTX prevents LV diastolic dysfunction in Langendorff rat hearts (Na(L) is also suggested to reduce the metabolic deficit developed with LCAC treatment (LCAC superfusion of rodent myocytes induces a dose-dependent inhibition of the early inward Na and APA . Howeverntration . This INnt (Iti) , 1995. Iogenesis , 1995. Tmiloride . Besidest hearts . Blockadreatment . Moreovereatment .3H]ouabain binding with LCAC exposure. This inhibition exhibits dose-dependency ranging from 2.5 up to 600 μM for onset of inhibition – the wide variation likely due to preparation differences and the inter-relatedness of NKA activity with other ion-handling proteins  induced by LCACs.Near consensus exists in the literature regarding LCAC inhibition of the sarcolemma NKA. Studies using partially purified canine and bovine NKA , b, sarcproteins . Like thmulation . Functiomulation . In cont+-handling controls action potential repolarization kinetics and maintenance of resting electrochemical gradients; therefore, modulations of K+-handling predominantly alter APD and the RMP (+ currents.Cardiac K the RMP . In conj+ current (IK1) is tightly coupled with the NKA in the polarization of the RMP  amplitude (responsible for rapid delayed rectifier K+ current). This was associated with an enhancement in deactivation kinetics that, when fitted to computational models, was consistent with an attenuated APD. Furthermore, the authors reported subtle differences in IhERG stimulation depending on LCAC chain length     and ATP- , which t  and, in d gating .INa(L), thereby increasing the intracellular Na+ concentration and driving arrhythmogenic Na+/Ca2+ exchange , due to its relative abundance in the ischaemic myocardium (A limitation of the LCAC literature is that all research was performed using non-human models of the mammalian myocardium. Exogenous LCACs affect multiple proteins and mechanisms of cardiac ECC, many of which are differentially expressed between animal models and/or function with a divergent ionic stoichiometry . Such diocardium . In addidiabetes . Severaldiabetes . Therefodiabetes  and atridiabetes  it is pediabetes , is thatdiabetes . Furtherdiabetes . Therefodiabetes . As amphocardium . In turnocardium ; therefo2+ overload mediated by the acylcarnitines [reviewed by In accordance with these limitations, many prospects for future basic research arise that could enhance the translation to metabolomic CVD associations. Firstly, studies utilizing human myocardium should be performed in conjunction with metabolomic investigations. This could be in the form of trabeculae from atrial appendages of cardiac surgery patients or myocardial preparations isolated from transplanted human hearts. Furthermore, studies in human atrial- and ventricular-programmed induced pluripotent stem cells or engineered heart tissues will allow for more long-term studies and higher throughput in elucidating LCAC pathophysiology and potential targeting strategies. Secondly, continued investigation of effects attributable to specific LCACs is required for understanding the pathophysiological aetiologies of arrhythmias and cardiomyopathies associated with LCACs. In the clinic, this could translate to patient-specific CVD diagnosis and risk stratification based on an individual’s metabolomic profile. Moreover, because circulating LCAC levels often correlate with disease severity, NYHA class, and are reduced by surgical intervention, continued monitoring of a CVD patient’s metabolome could provide insight into the progression or regression of the disease . ThirdlyBesides CVD, developing a greater understanding of cellular LCAC pathogenesis will be informative for the disease processes of rare, but often lethal, fatty acid oxidation and carnitine metabolism disorders . MetabolThis review addressed the existing basic science investigations into the direct effects of the amphiphilic metabolites, LCACs, on cardiac function. LCACs induce a pleiotropy of effects on key ion-handling processes within the mammalian myocardium, manifesting in stereotypical alterations to the cardiac action potential that enhance the propensity for arrhythmic muscle activity. These metabolites also alter myocardial contractility; however, whether the effect is positively or negatively inotropic is inconclusive. Despite an extensive body of work establishing LCACs as effectors of cardiac muscle, several key limitations exist that form an incomplete understanding of the role of LCACs on the human heart. The evidence presented highlights how these intermediates of metabolism can affect the function of the ischaemic and non-ischaemic myocardium and provides a pathophysiological context to the growing body of metabolomic implications of LCACs in CVD.HA-B reviewed the literature. HA-B and RL wrote the first version of the manuscript and prepared the figures. JK, TZ, and PJ reviewed and edited the manuscript. All authors read and approved the final 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.


However, its gating characteristics and role in primary neural cells remain unclear. Here, we characterized its Ca2+- and voltage-dependent components in rod bipolar cells using dissociated and slice preparations of the mouse retina. The I-V curves of Ca2+-dependent ANO1 tail current and voltage-gated Ca2+ channel (VGCC) are similar; as ANO1 is blocked by VGCC inhibitors, ANO1 may be gated by Ca2+ influx through VGCC. The voltage-dependent component of ANO1 has outward rectifying and sustained characteristics and is clearly isolated by the inhibitory effect of Cl− reduction and T16Ainh-A01, a selective ANO1 inhibitor, in high EGTA, a Ca2+ chelator. The voltage-dependent component disappears due to VGCC inhibition, suggesting that Ca2+ is the essential trigger for ANO1. In perforated current-clamping method, the application of T16Ainh-A01 and reduction of Cl− extended excitation periods in rod bipolar cells, revealing that ANO1 induces repolarization during excitation. Overall, ANO1 opens by VGCC activation during physiological excitation of the rod bipolar cell and has a voltage-dependent component. These two gating-modes concurrently provide the intrinsic characteristics of the membrane potential in rod bipolar cells.TMEM16A/anoctamin1 (ANO1), a calcium (Ca The retina is a specialized tissue that receives and converts the light into neural signals that travel to the central nervous system (CNS) where visual information, such as shape, color, contrast, etc., is processed and encoded. To perform this complex processing, the retina forms and uses multiple parallel pathways ,2,3. The2+)-activated chloride (Cl−) channels (CaCCs) have been identified in various cells where they play manifold roles in cell physiology o to 10 mM and lowering [EGTA]i to the range of 0–0.5 mM o (2.5 mM) and EGTA (1 mM) in retinal slices. From tail slowly declined inward at the end of the stimulation and was effectively inhibited by two common ANO1 inhibitors, namely T16Ainh-A01 (40 μM) and CaCCinh-A01 (40 μM) [n = 7, p < 0.05, tail was reduced by 5 mM BAPTA  ,42, folltail are presented in n = 7). Interestingly, Itail started to appear at −40 mV and was maximized at the range between −30 and −20 mV. The voltage profile of Itail was similar to that of VGCC elucidated previously using dissociated rod bipolar cells [tail and VGCC, we perfused 40 μM mibefradil and 40 μM nifedipine, which are T-type and L-type VGCC inhibitors, respectively. Itail was successfully inhibited by both and was almost completely inhibited upon their simultaneous application  is not elicited  solution, suggesting that a Cl− component exists in the outward current. The amplitude was measured after stimulation in each step and was plotted in − concentration was not significant at low voltage, whereas it was significantly amplified by the increase in membrane potential . The inhibitory effect was most prominent in T16Ainh-A01, and the two types of VGCC inhibitors also both reduced the Cl− current. The current difference between control and each inhibitory state was widened at 200 ms after the pulse onset, compared with that at 10 ms after the pulse onset, which confirms that the outward rectifying current was mediated by ANO1  intracellular solution to minimize Ca2+-dependency and maintain the physiological intracellular Cl− proportion (tail from four consecutive control recordings (boxed gray waveforms), the extracellular solutions were changed in this order: low Cl−, control, and with T16Ainh-A01. In each state, we confirmed that the outward current was maximized in the control solution and was decreased by Cl− reduction or ANO1 inhibition , whereas spike amplitude was not affected (p > 0.05). These results indicate that the voltage-dependent component of ANO1 exists in mouse retinal rod bipolar cell and accelerates the repolarization of the membrane potential without changing its amplitude.For the quantitative analysis of the waveforms in 2+- and voltage-dependent gating-modes operated during the excitation of rod bipolar cells, and these two modes accelerated repolarization in the current-clamp mode. Regarding ANO1-specific inhibitors, it is worth mentioning that both CaCCinh-A01 and T16Ainh-A01 reportedly do not pharmacologically discriminate between ANO1 and ANO2 [Applying the patch-clamp method, we discovered that ANO1 physiological activity was strongly associated with VGCC and that, in rod bipolar cells of the mouse retina, ANO1 showed a voltage-dependent behavior. We also demonstrated that both Caand ANO2 . Neverthand ANO2 ,42,45. Mand ANO2 , while tand ANO2 . TherefoCl(Ca) using immunohistochemistry and the patch-clamp method [Cl(Ca) in primary central neurons. However, the channel activity was not observed under experimental physiological conditions, and the gating mechanism of its distinct feature, which has voltage- and Ca2+-gating-modes as elucidated in transfected cell lines, was not determined [2+ concentration with 1 mM EGTA, Itail was apparently evoked by cell stimulation, and it was inhibited by 40 μM each of CaCCinh-A01 and T16Ainh-A01. As Ca2+ signaling is important for the excitation and synaptic transmission in mammalian rod bipolar cells [tail in Previously, we reported the ANO1 expression in rod bipolar cells of mouse retina and identified ANO1-mediating Ip method . We focutermined ,37,38,39ar cells ,48, we h tail in ,44,47.2+ chelator, and nifedipine, an L-type VGCC inhibitor, significantly inhibited ANO1 currents in photoreceptors [2+ source for ANO1. However, because T-type VGCC also contributes to the synaptic transmission in rod bipolar cells [2+ via T-type VGCC could affect the gating mechanism of ANO1. Here, mibefradil, a T-type VGCC inhibitor, effectively reduced ANO1 current (2+.Previous studies reported that ANO1 is localized close to L-type VGCC in the photoreceptors ,15,49. Peceptors ,15,49 aneceptors ,15,49, sar cells , we hypo current C. These 2+ to ANO1 has been studied in various transfected and primary cells [2+ signaling in rod bipolar cells using intracellular EGTA [tail reduction, the outward rectifying current was isolated upon high voltage stimulation (2+ and voltage can gate the ANO1 channel. Besides, the initial gating factor is increased by intracellular Ca2+ through VGCC, and afterward, membrane voltage sustains ANO1 opening. Our findings are corroborated by previous studies in ANO1-transfected HEK cells [2+ gates ANO1 similar to that in a ligand-gated channel and that the voltage-dependent mode is not activated without Ca2+.Although the contribution of Cary cells ,52,53, tlar EGTA . In thismulation . The fasmulation D,E. Thismulation . OverallEK cells ,39, whic− reduction nor ANO1 inhibition, whereas the decay after a spike to the plateau potential was significantly affected, and plateau potential thus disappeared. These results suggest that ANO1 contributes to the rapid repolarization process. It is consistent with previous studies about modulation of the action potential by ANO1 or ANO2, which showed elongation of the excitation by Cl− inhibition [To investigate the cellular function of ANO1 in rod bipolar cells of the retina, we examined its role during cell excitation using the current-clamp method. The peak amplitude of the single spike was neither affected by Clhibition ,55.2+ has a crucial role in the excitation and synaptic transmission in the mammalian retina [2+-induced excitation [During neural excitation, the balance between excitatory and inhibitory signals is essential for proper synaptic transmission. In rod bipolar cells, Can retina ,56,57. Hcitation ,59. Howecitation ,61. Althcitation . We foun2+ through VGCC rapidly opens ANO1. The ANO1 current is sustained by the synergistic effects of the membrane potential and Ca2+, thereby accelerating cell repolarization. These results suggest that ANO1 acts as a rapid intrinsic inhibitory modulator during excitation in rod bipolar cells, similar to BK and SK channels in other neural cells.The present study elucidated the dual gating mode of ANO1 in primary CNS neurons. Although the retinal rod bipolar cell, being a non-spiking neuron, is not typical, it uses graded changes in the membrane potential and contributes to repolarization during cell excitation. As illustrated in


Furthermore, quantification by immunoblotting showed that the main flippase molecule in erythrocytes, ATP11C, was partially lost in the senescent cells. Collectively, these results suggest that multiple factors, including altered intracellular substances and reduced ATP11C levels, contribute to decreased flippase activity in senescent erythrocytes in turn to, present phosphatidylserine on their cell membrane. The present study may enable the identification of novel therapeutic approaches for anaemic states, such as those in inflammatory diseases, rheumatoid arthritis, or renal anaemia, resulting from the abnormally shortened lifespan of erythrocytes.Mature human erythrocytes circulate in blood for approximately 120 days, and senescent erythrocytes are removed by splenic macrophages. During this process, the cell membranes of senescent erythrocytes express phosphatidylserine, which is recognized as a signal for phagocytosis by macrophages. However, the mechanisms underlying phosphatidylserine exposure in senescent erythrocytes remain unclear. To clarify these mechanisms, we isolated senescent erythrocytes using density gradient centrifugation and applied fluorescence‐labelled lipids to investigate the flippase and scramblase activities. Senescent erythrocytes showed a decrease in flippase activity but not scramblase activity. Intracellular ATP and K During this process, senescent erythrocytes are expected to expose phosphatidylserine (PS), which is usually localized asymmetrically in the inner leaflet of the cellular lipid bilayer,ATP11C resulted in ~90% reduction in PS transportation activity+ concentration decreased flippase activity,2+ concentration increased scramblase activity+ concentration is regulated by four reported channels and transporters, including the Ca2+‐sensitive K+ channel termed Gardos channel (KCNN4); Na+ K+ ‐ATPase, K+/Cl‐ cotransporter and Na+/K+/2Cl‐ cotransporter.+ concentration depends on intracellular Ca2+ concentration by the Gardos channel, the influence of Ca2+ and K+ concentration on PS transportation should be examined individually.Recently, we identified ATPase class VI type 11C (ATP11C) as an important flippase in human erythrocytes and demonstrated that point mutation in 2+ and K+ on lipid transportation activities in senescent erythrocytes. Moreover, we highlight the potential of ATP11C molecule, as a novel factor affecting flippase activity in senescent erythrocytes.In this study, to clarify the mechanisms of PS exposure, we investigated the flippase and scramblase activities in senescent erythrocytes isolated using density gradient centrifugation and analysed the effect of intracellular concentrations of ATP, Ca22.1A23187 and valinomycin were purchased from Sigma‐Aldrich Inc ; nitrobenzoxadiazole‐labelled PS (NBD‐PS) and NBD‐labelled PC (NBD‐PC) from Avanti Polar Lipids, Inc ; dimethyl sulfoxide (DMSO) from Wako Pure Chemical ; bovine serum albumin (BSA) from Nacalai Tesque ; rat IRDye 680RD and mouse IRDye 800 CW from LI‐COR Biosciences ; and Percoll from GE healthcare .2.22+ clamped conditions or phosphate‐buffered saline  for other experiments. Senescent erythrocytes were separated using density gradient centrifugation  using 89% Percoll in 10 mM Tris‐HCl, pH 7.4, 120 mM KCl and 0.25 M sucrose, as described previously.This study was approved by the Ethics Committee of Tokyo Women's Medical University (#3835R). Written informed consent was obtained from all the study participants. Venous blood samples were collected from four healthy donors in blood collection tubes containing 1.5 mg/mL of EDTA‐2Na, and the samples were washed three times with Tris‐buffered saline  for investigating Ca2 and 5 µM A23187, a Ca2+ ionophore, for 30 min at 37°C to obtain an intracellular Ca2+ concentration of 2 mM. After gentle centrifugation at 15,000 g for 30 s, the resultant pellets and supernatants were collected separately; the pellets were washed twice with PBS and used as residual erythrocytes (R), while the supernatants were re‐centrifuged at 15,000 g for 30 min, and the resultant precipitates collected as microvesicles (Mv).Artificial microvesicles from the washed erythrocytes were prepared as follows. The cells were incubated in TBS, 20 mM Glucose, 2 mM CaCl2.3Washed erythrocytes at 5% of the haematocrit were pre‐incubated in 0.5 mL PBSG (PBS containing 20 mM glucose) at 37°C. Then, 0.5 µL NBD‐PS or NBD‐PC (1 mg/mL in DMSO) was added, and 20 µL of suspension was mixed with PBS containing 1% BSA at every 10 min until 20 min to remove the remaining NBD‐PS/PC in the outer leaflet. NBD‐PS/PC loading was determined by observing the fluorescence of the mixture treated without BSA using a flow cytometer . The proportion of PS/PC transported was calculated by dividing the fluorescence intensity of the inner leaflet by that of the loaded NBD‐PS/PC.2.4+ concentration was measured using the LAQUAtwin potassium ion meter . Intracellular ATP was measured using the ATP Assay Kit for blood  and a luminometer 　before and after removing haemoglobin by protein‐precipitating reagents, 0.6 M HClO4 and 2.5 M K2CO3.Washed erythrocytes and senescent cells were haemolysed with the same volume of double‐distilled water and stored at −20°C. The intracellular K2.52+ into the erythrocytes, washed erythrocytes were incubated with 5 µM A23187 and Ca2+‐loading buffer  at 37°C for 30 min.2+ was determined by 1 mM EGTA, and various concentrations of CaCl2 were calculated by Calcon free software . The cells were then washed with 1% BSA in Ca2+‐loading buffer without CaCl2 to remove A23187. The intracellular and extracellular Ca2+ concentrations were expected to be equal. BSA was removed by washing the cells three times with Ca2+‐loading buffer. K+ was introduced into erythrocytes using valinomycin, a potassium ionophore, in Tris buffer containing various concentrations of K+  at 37°C for 30 min.To introduce Ca2.6®‐80i analyzer . The MCV of treated erythrocytes with artificially altered intracellular Ca2+ (under extracellular K+ of 4.15 mM) or K+ concentrations was also analysed. To resolve the inter‐individual differences in MCV, we converted the MCV to relative MCV (rMCV), which represents a percentage relative to the MCV of the treated erythrocytes at intracellular 0 µM Ca2+ or 100 mM K+.The MCV of control and senescent erythrocytes was measured using Sysmex pocH2.7Washed erythrocytes were haemolysed by mixing with 1 mM phenylmethylsulfonyl fluoride and 5P1E buffer  on ice and centrifuged at 20,400 g, 4°C, for 15 min. The pellets were washed three times with 5P1E buffer to remove the haemoglobin and obtain the erythrocyte membrane fraction. After ensuring equal protein concentrations by measuring the absorbance using the Bradford reagent  and the U‐5100 spectrophotometer , the erythrocyte membranes were mixed with the sample buffer, electrophoresed on 8% SDS‐polyacrylamide gel and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk in TBS and incubated with the following primary monoclonal antibodies separately in T‐TBS (TBS and 0.1% Tween 20): rat anti‐ATP11C antibody  and treated with 1 µL Annexin V‐FITC . The fluorescence levels of erythrocytes were quantified using flow cytometry. The erythrocytes with fluorescence values over 4000 were considered as erythrocytes with exposed PS.One microlitre of packed control or senescent erythrocytes with different concentrations of calcium was suspended in 1 mL Annexin binding buffer  or R v3.3.2.33.1P < 0.05, n = 5) than that in the control erythrocytes  for control and senescent erythrocytes, respectively . Thus, raising intracellular Ca2+ levels may inhibit flippase activity.Next, to estimate the effect of intracellular Ca7 Figure .26 Inty Figure ; therefon Figure . Increasn Figure . In cont2+ concentration. Instead, we estimated the Ca2+ concentration in the senescent erythrocytes by comparing the MCV obtained from erythrocytes with artificially altered intracellular Ca2+ and K+ concentrations. Because of the high inter‐individual differences in MCV, we expressed relative MCV (rMCV) as a percentage relative to the MCV of erythrocytes at 0 µM Ca2+ and 100 mM K+. rMCV decreased with increasing intracellular Ca2+ concentration and decreasing K+ concentration  for ATP (0.23 mM) in erythrocytes,+ concentration in senescent erythrocytes contributes to reducing flippase activity by ~30%. Thus, reduction of intracellular ATP and K+ may collectively decrease flippase activity in senescent erythrocytes. However, the flippase activity in senescent erythrocytes decreased by approximately 50‒70% from control erythrocytes  were lower than those in control erythrocytes (0.57 ± 0.03 mM).s Figure . This re2+‐treated residual membranes as shown in Figure We assessed the amount of the flippase ATP11C, which accounts for 90% of the flippase activity in erythrocytes.2+ caused increased PS exposure in both control and senescent erythrocytes, suggesting that PLSCR1 levels do not decrease in senescent erythrocytes. The reason for the presence of ATP11C and absence of PLSCR1 in microvesicles may be the difference in their association with the cytoskeleton; PLSCR1 may be tightly bound to the cytoskeleton, which is absent in microvesicles.Recently, we identified that ATP11C T418N mutation results in mild haemolytic anaemia due to improper membrane trafficking.2+ concentration using fluorescent indicators, probably due to our technical limitations in dealing with a high level of haemoglobin as a pigment interfering the measurement. Another possible reason is that the reported erythrocyte Ca2+ concentration is 30‒60 nM,2+ concentration from the alteration of MCV and assumed that intracellular Ca2+ is approximately 100 nM in senescent erythrocytes , Na+/K+‐ATPase, K+/Cl‐ cotransporter and Na+/K+/2Cl‐ cotransporter were expected.2+ is estimated to be 4.7 µM,2+ in senescent erythrocytes would not activate the channel. As the Km of Na+/K+‐ATPase for ATP in rat is reported to be 0.59 mM,+, resulting in the reduction of K+. The K+/Cl‐ cotransporter is reported to be involved in K+ leakage in young erythrocytes.+/K+/2Cl‐ cotransporter is reported to regulate steady‐state volume of erythrocytes+ concentration in senescent erythrocytes might reflect other mechanisms such as the decreased activity of Na+/K+‐ATPase and/or Na+/K+/2Cl‐ cotransporter.We attempted but failed to measure intracellular Cas Figure . Based os Figure . Thus, as Figure , the act+ levels and ATP11C expression reduces flippase activity in senescent erythrocytes ; Data curation (lead); Formal analysis ; Funding acquisition ; Investigation (lead); Resources ; Validation ; Visualization ; Writing‐original draft (lead). Nobuto Arashiki: Conceptualization (lead); Formal analysis ; Funding acquisition ; Investigation ; Methodology (lead); Project administration (lead); Writing‐review & editing . Yuichi Takakuwa: Conceptualization ; Formal analysis (supporting); Funding acquisition . Kosaku Nitta: Supervision ; Writing‐original draft . Fumio Nakamura: Conceptualization ; Formal analysis (lead); Funding acquisition ; Methodology (supporting); Project administration ; Supervision (lead); Writing‐review & editing (lead).