Acute challenge of adult ventricular cardiomyocytes to oxLDL reduced systolic Ca2+ transients and sarcoplasmic reticulum Ca2+ load. Moreover, diastolic spontaneous Ca2+ leak increased significantly after acute exposure to oxLDL. Thus, we demonstrate that oxLDL associates with NT-proBNP in young subjects, and can directly induce Ca2+ mishandling in adult ventricular cardiomyoyctes, predisposing cardiomyocytes to cardiac dysfunction and arrhythmogenicity.Oxidized low-density lipoprotein (oxLDL) is associated with cardiac damage and causes injury to multiple cell types. We aimed to investigate the role of oxLDL in ventricular stress. We first examined the association between circulating oxLDL and N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of myocardial stress, in young subjects (30–50 years) with or without stable coronary artery disease (SCAD). oxLDL and NT-proBNP were significantly higher in subjects at high cardiovascular risk (CVR) than in subjects at low CVR and were associated independently of traditional CVR factors and C-reactive protein. Furthermore, the levels of oxLDL and NT-proBNP were significantly lower in subjects with SCAD than in peers at high CVR. To determine the intracellular mechanisms involved in the cardiac effects of oxLDL, we analyzed the in vitro effect of oxLDL on intracellular Ca Cardiovascular disease is the leading cause of death and disability worldwide. Among cardiovascular risk (CVR) factors, one of the major contributors to atherosclerosis development and progression is raised serum levels of low-density lipoprotein (LDL) cholesterol, which can penetrate the vascular wall and accumulate in the subendothelial space. LDL is highly susceptible to oxidation in the environment of the atheromatous plaque. Oxidized LDL (oxLDL) exacerbates the atherosclerosis process by triggering pro-oxidant and pro-inflammatory pathways that lead to plaque instability through mechanisms independent of LDL ,2. Conse2+ represents a key player in cardiac EC coupling [2+ channels at the sarcolemma and allowing a small influx of Ca2+ into the cytoplasm. This increase in cytoplasmic Ca2+ concentration induces the activation of ryanodine receptors at the sarcoplasmic reticulum (SR) and the subsequent release of Ca2+ from the SR to the cytoplasmic space, elevating the intracellular concentration of Ca2+, which binds to myofilaments and activates contraction [2+ is pumped back to the SR mainly by the SR-Ca2+-adenosine triphosphatase 2a (SERCA) pump and, to a lesser extent, is extruded outside of the cell by the Na+-Ca2+ exchanger (NCX) [2+ handling are related to contractile dysfunction and arrhythmias [Cardiomyocyte electrical activity and contractility are governed by excitation–contraction (EC) coupling, which is the physiological process of converting an electrical signal into mechanical work. Cacoupling . During traction . During er (NCX) . Alterathythmias . Previouhythmias ,9,10. Hohythmias ,11.2+ handling in adult rat ventricular cardiomyocytes.The aim of the present study was to question whether oxLDL is associated with cardiac dysfunction at an early age and before the onset of overt cardiovascular disease. To do this this, we investigated the association between oxLDL and N-terminal pro-B-type natriuretic peptide (NT-proBNP), a marker of ventricular stress, in a young population with or without previous coronary events. At the cellular level, we assessed in vitro the acute effect of oxLDL on CaIn this cross-sectional study, 66 subjects aged 30–50 years were recruited from Ibermutua , a SpaniAll subjects underwent clinical examination and blood analysis. Blood samples were collected in ethylenediamine tetraacetic acid (EDTA) tubes and immediately centrifuged at 2000 rpm for 10 min at 4 °C. Plasma was stored at −80 °C. Plasma levels of NT-proBNP and C-reactive protein (CRP) were determined using commercial ELISA kits . Plasma levels of oxLDL were determined with a commercial sandwich ELISA based on the monoclonal antibody 4E6 , which binds approximately 1000 times stronger to oxLDL than to native LDL , in ordeThis study was approved by the ethics committee of the Hospital Universitario 12 de Octubre (Madrid), and was performed according to the tenets of the Declaration of Helsinki. All patients signed an informed consent.The animal study was conducted following the recommendations of the Spanish Animal Care and Use Committee according to the Guidelines for ethical care and welfare (2013/175) of experimental animals of the European Union (2010/63/EU), and was approved by the General Direction of Agriculture and the Environment at the Environment Council of Madrid (PROEX 053/16).2, 1 mg/mL type II collagenase  and 1 mg/mL bovine serum albumin. The left ventricle was then removed, cut into pieces and stirred for 1 min in Tyrode’s solution containing 0.1 mM CaCl2. The resultant cell suspension was filtered through a nylon mesh (250 μm) and centrifuged for 3 min at 500 rpm. Cells were resuspended in Tyrode’s solution containing 0.5 mM CaCl2, centrifuged again for 3 min at 500 rpm, and finally resuspended in a storage solution containing 1 mM CaCl2 [Adult ventricular cardiomyocytes were isolated from hearts of young adult male Wistar rats (150–200 g). Rats were sacrificed with sodium pentobarbital–heparin  and hearts were quickly removed and cannulated via the ascending aorta on a Langendorff perfusion system. Hearts were retrograde perfused with calcium-free Tyrode’s solution supplemented with 0.2 mM ethylene glycol tetraacetic acid (EGTA) for 3–7 min at 37 °C, followed by 3–5 min perfusion with Tyrode’s solution supplemented with 0.1 mM CaClmM CaCl2 ,17.2+ tolerant rod-shaped cardiomyocytes were included in the study. Ventricular cardiomyocytes were perfused with vehicle solution followed by 3–4 min perfusion with 10 μg/mL oxLDL  [Only excitable and CaMD, USA) ,9,10.2+ transients were recorded in cells field stimulated at 1 Hz using two electrodes for 1 min to reach a steady state. Fluorescence (F) values were normalized to the basal fluorescence (F0) to obtain the fluorescence ratio (F/F0). The decay time constant of Ca2+ transients–Tau–was obtained by fitting the decay time trace to a single exponential. The SR Ca2+ load was estimated as the amplitude of caffeine-induced Ca2+ transients (F/F0) after 10 mM caffeine administration.Isolated cardiomyocytes were preloaded with the calcium-sensitive fluorescent dye Fluo-3AM . Images were obtained with a MetaZeiss LSM 510 confocal microscope (objective w.i. 40×) by tracing a line across the longitudinal axis of the cells. Ca2+ sparks, which were defined as localized, rapid and brief elevations in Ca2+ fluorescence of at least 3 times the standard deviation of F0 after Ca2+ transient recordings. Pro-arrhythmogenic Ca2+ events were assessed by determining the frequency of Ca2+ waves, which were identified as increases in fluorescence that start locally and propagate to both sides of the cell. The fluorescence registered was corrected by the background fluorescence in each image.Quiescent Fluo-3AM-loaded cells were scanned to record spontaneous Ca2+ images were analyzed using home-made routines in Interactive Data Language  and Image J .Confocal Cat-test for normally distributed variables, or the Mann–Whitney test for not normally distributed variables. Results are expressed as mean±standard error of the mean (SEM), and p-values < 0.05 were considered significant. Analyses were performed with GraphPad Prism 6 , IBM SPSS Statistics v22 , and OriginPro 9.0 .Normality was assessed with the Kolmogorov–Smirnov test. For the human study, groups were compared using analysis of variance with Newman–Keuls correction or Fisher’s test for categorical variables. Correlations were examined using Pearson’s r correlation coefficient. Multiple linear regression analysis was performed to determine whether the association between oxLDL and NT-proBNP was independent of LDL and CRP levels, sex, body mass index (BMI) and systolic blood pressure. In the animal study, groups were compared using paired Student’s p < 0.01). BMI was also lower in the low-lifetime CVR group than in the high-lifetime CVR group (p < 0.001) and the SCAD group (p < 0.01). Hypertension was more prevalent in the high-lifetime CVR and SCAD groups than in the low-lifetime CVR group . Values for systolic blood pressure followed the same pattern, but were significantly lower in the SCAD group than in the high-lifetime CVR group (p < 0.001). The high-lifetime CVR group had greater levels of total cholesterol, LDL, and triglycerides than the low-lifetime CVR  or SCAD (p < 0.001) groups. Moreover, the SCAD group had lower levels of total cholesterol and LDL than the low-lifetime CVR group , which is likely because all subjects with SCAD were chronically treated with statins. However, high-density lipoprotein levels were significantly higher in the low-lifetime CVR group than in the other groups (p < 0.001).Subjects were stratified into three groups according to whether or not they had SCAD and according to lifetime CVR . The meap < 0.05). Conversely, the high-lifetime CVR group had higher levels of both NT-proBNP  and the SCAD  groups, and the SCAD group had significantly lower levels of oxLDL than the low-lifetime CVR group (p < 0.05). Correlation analysis demonstrated a positive correlation between oxLDL and NT-proBNP in the whole sample (p = 0.021), whereas CRP showed no correlation with NT-proBNP  or oxLDL  (not shown). Multivariate regression analysis showed that the increase in NT-proBNP levels was associated with higher levels of oxLDL independently of LDL, sex, BMI, systolic blood pressure or CRP  or oxLDL (right panel) perfusion are shown in 2+ transients (F/F0) was significantly lower in cardiomyocytes acutely exposed to oxLDL than to vehicle (p < 0.05). The decrease in the amplitude was due to a reduction in peak systolic Ca2+ transients  and not to changes in the baseline Ca2+ fluorescence  and in the time constant of Ca2+ transient decay, Tau (p < 0.05).We next evaluated the acute effects of oxLDL on Caence F0, D. Challe2+ release might be related to alterations in SR-Ca2+ load. Therefore, we studied the estimated SR-Ca2+ load after caffeine administration. Representative line-scan images of caffeine-induced Ca2+ transients in cardiomyocytes challenged with vehicle (upper panel) or oxLDL (bottom panel) are shown in 2+ load was significantly reduced following oxLDL perfusion (p < 0.001), but no differences were found in the decay time constant Tau (2+ load might be due to Ca2+ leak from the SR during diastole. Representative line-scan images of Ca2+ spark recordings after vehicle (upper panel) or oxLDL (bottom) challenge are shown in 2+ spark frequency was significantly higher after oxLDL perfusion than after vehicle perfusion (p < 0.01). Analysis of the biophysical properties of Ca2+ sparks revealed that oxLDL induced a reduction in Ca2+ spark amplitude (0F/F), with no differences in duration or width (2+ leak was increased after oxLDL perfusion relative to vehicle (p < 0.01).Alterations in systolic Catant Tau C. The reor width . The cal2+ events, we also analyzed the diastolic spontaneous Ca2+ release in the form of Ca2+ waves after oxLDL challenge in resting quiescent cells, finding a significant increase in the frequency of Ca2+ waves (p < 0.05).Finally, to determine whether oxLDL treatment of intact cells might trigger other forms of pro-arrhythmogenic Ca2+ handling. Our results suggest that an increase in the levels of oxLDL might contribute to ventricular dysfunction in individuals at high CVR before the onset of a coronary event, even from a young age, and that oxLDL predisposes cardiomyocytes to systolic Ca2+ release impairment and pro-arrhythmogenic forms of diastolic Ca2+ release as a consequence of abnormal intracellular Ca2+ cycling. Accordingly, subjects with high levels of oxLDL are at risk of cardiac dysfunction even at a young age.This study demonstrates, for the first time to our knowledge: (i) an association between oxLDL and NT-proBNP in young adults with or without SCAD and (ii) that acute exposure of adult rat ventricular cardiomyocytes to oxLDL triggers alterations in intracellular CaCVR tends to be estimated on the basis of short-term CVR; however, young adults have low short-term CVR simply because of their young age. Traditional CVR factors such as hypertension, dyslipidemia and obesity have a cumulative impact on CVR which is reflected in lifetime CVR, which is the risk of developing a cardiovascular disease in the remaining lifespan ,18. We sIn the present study, we describe a similar trend for the levels of the marker of ventricular stress, NT-proBNP, although the levels of NT-proBNP were within the normal range in all groups. NT-proBNP above the normal range is a widely used biomarker of heart failure. Therefore, young subjects who are not susceptible to cardiovascular disease, such as those without SCAD, are not susceptible to high levels of NT-proBNP. Subjects with cardiovascular disease might have high levels of NT-proBNP, as NT-proBNP rises during the acute phase of coronary events and can remain high in the case heart failure development. Subjects in the SCAD group were in the stable phase after the coronary event, did not develop heart failure and were very well-controlled in terms of blood pressure, which could increase volume overload and consequently NT-proBNP. Moreover, all patients with SCAD were on statin treatment, which reduces the levels of NT-proBNP after a cardiovascular event such as acute myocardial infarction , and in The association between NT-proBNP and oxLDL indicates that oxLDL might be directly related to cardiac function both before and after the cardiovascular disease. oxLDL has been associated with a decrease in cardiac function in the general population  and in pA pro-inflammatory state is known to increase the levels of oxLDL, which triggers the activation of pro-inflammatory pathways in a positive feedback loop . We thus2+ handling and in the pro-arrhythmic Ca2+ release during diastole in adult rat ventricular cardiomyocytes. Acute exposure to oxLDL induced alterations in Ca2+ handling that are similar to those found in human heart failure, such as compromised systolic Ca2+ release [2+ release and SR-Ca2+ load and an increase in spontaneous SR-Ca2+ release in the form of Ca2+ sparks and waves frequency indicate that oxLDL could trigger pro-arrhythmogenicity in adult ventricular cardiomyocytes. In support of this, treatment of guinea pig adult cardiomyocytes with oxLDL induced an increase in pro-arrhythmogenic activity [2+ cycling in ventricular cardiomyoyctes.Related to the cellular effect of oxLDL, authors indicated that oxLDL induces apoptosis in neonatal rat cardiomyocytes ,38,39 an release ,11,42. Ractivity . Therefo2+ leak in the form of spontaneous Ca2+ sparks and waves suggest that it can directly induce a pro-arrhythmogenic profile. The increased decay time of Ca2+ transients after challenge with oxLDL indicates that SERCA pump activity is impaired, and therefore intracellular Ca2+ uptake to the SR might be also altered. SR-Ca2+ load is decreased both because of the worsened intracellular Ca2+ uptake and because of the increase in spontaneous SR-Ca2+ leak. The decay time of Ca2+ transients induced by caffeine, which represents the activity of NCX, is not affected by oxLDL. Supporting these data, a previous study failed to show alterations in NCX after treatment of guinea pig adult cardiomyocytes with oxLDL [The effects of oxLDL on SR-Cath oxLDL .2+ handling [ApoE-deficient mice have a higher risk of arrhythmia post-myocardial infarction than wild-type mice [Serum lipids are suggested to affect cardiac function independently of their effect on the vascular system through multiple mechanisms including electrical cardiac remodeling . Howeverhandling ,52,53. Iype mice . Accordiype mice  that, to2+ handling given the well-known pro-oxidant effect of oxLDL.This study has some limitations that should be addressed. First, the proportion of male subjects is significantly higher in the high-lifetime CVR and the SCAD groups than in the low lifetime CVR group. Women have better cardiovascular risk profiles than men prior to menopause, presenting lower blood pressure and LDL, and higher HDL, than their male peers . Therefo2+ dynamics that may contribute to arrhythmia development. Young subjects tend to underestimate their CVR because of their low short-term CVR, which can lead to poor control of CVR factors. Our results show the importance of controlling the levels of oxLDL even before the onset of the coronary event in order to prevent cardiac dysfunction and arrhythmia development.In conclusion, the present work demonstrates a direct association between ventricular stress and oxLDL in young adults with and without previous coronary events, and also a direct functional effect of oxLDL on the regulation of Ca


They are thus involved in the development and formation of the neural system. In this study, a publicly available whole transcriptome sequencing (RNA-Seq) dataset was used to examine the expression of genes coding for putative plasma membrane and organellar Ca2+-transporting proteins  during the formation of the cerebral cortex in mice. Four ages were considered: embryonic days 11 (E11), 13 (E13), and 17 (E17), and post-natal day 1 (PN1). This transcriptomic profiling was also combined with live-cell Ca2+ imaging recordings to assess the presence of functional Ca2+ transport systems in E13 neurons. The most important Ca2+ routes of the cortical wall at the onset of corticogenesis (E11–E13) were TACAN, GluK5, nAChR β2, Cav3.1, Orai3, transient receptor potential cation channel subfamily M member 7 (TRPM7) non-mitochondrial Na+/Ca2+ exchanger 2 (NCX2), and the connexins CX43/CX45/CX37. Hence, transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1), transmembrane protein 165 (TMEM165), and Ca2+ “leak” channels are prominent intracellular Ca2+ pathways. The Ca2+ pumps sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and plasma membrane Ca2+ ATPase 1 (PMCA1) control the resting basal Ca2+ levels. At the end of neurogenesis (E17 and onward), a more numerous and diverse population of Ca2+ uptake systems was observed. In addition to the actors listed above, prominent Ca2+-conducting systems of the cortical wall emerged, including acid-sensing ion channel 1 (ASIC1), Orai2, P2X2, and GluN1. Altogether, this study provides a detailed view of the pattern of expression of the main actors participating in the import, export, and release of Ca2+. This work can serve as a framework for further functional and mechanistic studies on Ca2+ signaling during cerebral cortex formation.Cytosolic calcium (Ca However, various pathophysiological conditions elevate [Ca2+]i, which, in turn, influences a myriad of biological processes, such as gene expression, neurogenesis, neuronal migration, axon growth, synaptic transmission, synaptic plasticity, and the maintenance of synaptic networks i changes reflect an entry of Ca2+ from the extracellular milieu or a release from internal stores. These two processes are often interrelated and, thus, cooperate to influence [Ca2+]i.The divalent cation calcium  are involved in the handling of Ca2+ and the shaping of its cytosolic variations. Any insult modifying the abundance, distribution, and activity of these proteins is likely to affect cell functions and survival.An influx of Ca2+]i levels play pivotal roles during the formation of the cortex i levels  protein family. Our data indicated that the protein encoded by the Sec61a1 gene was not expressed or did not contribute to the Ca2+ leak pathway, as has been reported by others [The le cells . Howeveri levels D. It alsuromycin D. Anisomy others ,100.2+ uptake routes of the cell surface at the onset of corticogenesis , ligand-gated (GluK5 and nAChR β2), voltage-gated (Cav3.1), and Mg/ATP-regulated constitutively activated channels (TRPM7). Orai3 has been described as a store-operated Ca channel; however, it also appears to be a component of arachidonic acid-regulated calcium-selective (ARC) channels.ogenesis  are chan2+ export) or backward (Ca2+ uptake) mode. These actors (CX and NCX) permit a bidirectional transport of Ca2+ through the plasma membrane (or between adjacent cells). Intracellularly, TRPML1 and the uncharacterized “leak” channels form the two prominent Ca2+ release pathways at all ages. In front of these numerous routes that contribute to elevating cytosolic Ca2+ levels, SERCA2 and PMCA1 appeared as the crucial systems to bring Ca2+ back to its resting basal levels. TMEM165 is another actor able to store Ca2+ in internal compartments; however, this contribution is less prominent at the end of corticogenesis. A more numerous and diverse population of Ca2+ uptake systems was observed in the cortical wall at the end of neurogenesis (E17 and onward). In addition to the actors listed above, certain prominent Ca2+-conducting systems emerged, including ASIC1, Orai2, P2X2, and GluN1. In addition, the connexins CX43/CX45/CX37 allow an intercellular coupling, whereas NCX2, depending on its reversal potential, can work in a forward  imaging experiments were performed to verify the presence of functional proteins at E13. Overall, numerous Ca2+ transporter genes were expressed in the embryonic cerebral cortex. Collectively, there is a large diversity of Ca2+ transport systems at the cell surface that contrasts with the paucity of prominent internal Ca2+ transport systems.The major purpose of this study was to perform a transcriptional profiling to survey the global expression patterns of genes coding for Ca2+ influx [2+-transport proteins and contributing to Ca2+ signaling were missed in the present work. Another limitation of this work is the fact that many, if not all, ion channels require dedicated and specific auxiliary subunits that are of the upmost physiological importance for their assembly/maturation/trafficking/surface expression and/or ion transport activities and properties. Although crucial for the biogenesis and appropriate activity of ion channels, the mRNA expression of these regulatory subunits was not investigated.One limitation of this work is the possibility that some transcripts were missed due to the existence of genes with unknown functions. New ion channels are continuously being uncovered. For instance, it was recently shown that the stretch-activated nonselective cation channel TMEM63B is an osmosensor that can mediate the Ca+ influx . This ex2+. For example, TRPM7 is a Ca2+-conducting pathway but it may function as a physiological Mg2+ transport system [2+ during the formation and development of the cerebral cortex. This study can serve as a valuable resource and provide a framework for further functional and mechanistic studies on Ca2+ signaling during cerebral cortex formation.It is also important to consider that some actors selected in this report may function as pathways for cations distinct from Cat system . In addi


Trans-2-enoyl-CoA reductase (TER) is an FA reductase present in the ER membrane and catalyzes the last step in the FA elongation cycle and sphingosine degradation pathway. Here we identify sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), an ER Ca2+ pump responsible for Ca2+ accumulation in the ER, as a TER-binding protein by affinity purification from HEK293 cell lysates. We show that TER directly binds to SERCA2b by in vitro assays using recombinant proteins. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes of HEK293 cells. Depletion of TER increases Ca2+ storage in the ER and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. Moreover, depletion of TER reduces the Ca2+-dependent nuclear translocation of nuclear factor of activated T cells 4. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). Given that FAs are the components of membranes, FA metabolic enzymes might be associated with regulation of ER membrane functions. However, it remains unclear whether there is the interplay between FA metabolic enzymes and ER membrane proteins.  Trans-2-enoyl-CoA reductase (TER) is a ubiquitous membrane enzyme that catalyzes the final reduction step of the FA elongation cycle (TSC13) is lethal in yeast because of the lack of the VLCFA synthesis (Fatty acids (FAs) are essential constituents of biological membranes, energy storage, and precursor of signaling molecules. Mammalian cells contain FAs with different carbon number, which have both structural and functional diversity. Long-chain FAs (LCFAs) have 11 to 20 carbons, among which C16 and C18 species are the most abundant. Very-long-chain FAs (VLCFAs) have >20 carbons. Although the amount of VLCFAs is generally lower than that of LCFAs, VLCFAs have various functions that are not complemented by LCFAs, such as skin barrier formation, myelin formation and maintenance, retinal function, and spermatogenesis , 4. Moston cycle . To date1–5)  metabolism. S1P, which is produced by phosphorylating sphingosine, is a potent lipid mediator that acts as a ligand for five G protein–coupled receptors (GPCR) (S1P1–5) . The bin1–5) . S1P is noyl-CoA , 12. TERholipids . TherefoTECRL, a muscle-specific paralogue of TER, are associated with inherited arrhythmias   , althoug2+ is a ubiquitous signaling molecule that regulates a wide range of cellular processes, such as muscle contraction, neuronal transmission, motility, proliferation, and transcriptional control  receptors (2+ through sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) that transport Ca2+ from the cytosol to the ER lumen with the energy obtained from ATP hydrolysis  encode SERCA proteins (SERCA1-3). SERCA2, which shows the most widespread expression pattern among three SERCA proteins, has two major splicing isoforms, SERCA2a and SERCA2b. Although SERCA2a is expressed in slow skeletal muscles and cardiac muscles, SERCA2b is expressed ubiquitously and functions as a house-keeping protein , a small transmembrane protein that directly binds to SERCA2a. Although PLN inhibits the SERCA2a activity in resting states, adrenergic stimulation relieves this inhibition. The resultant enhancement of the SERCA2a activity accelerates cardiac relaxation and increases the Ca2+ content in the SR, which then strengthens cardiac contraction . Mass spectrometry analysis identified p55, p95, p170, p180, and p400 as β-tubulin, SERCA2b, protein MON2 homologue/SERCA2b, nuclear pore complex protein Nup205, and DNA-dependent protein kinase catalytic subunit, respectively . In addition to a band with the expected molecular mass (∼100 kDa), the anti-SERCA2b antibody detected a band around 130 kDa in the Strep-TER pull-down fraction. Given that the molecular mass of Strep-TER is about 30 kDa, the 130-kDa band is probably an SDS-resistant heterodimer of SERCA2b and Strep-TER. Because SERCA2b is the only protein localized to the ER in the identified proteins, and expected to be abundant in the Strep-TER pull-down fraction, we characterized SERCA2b as a potential binding partner of TER in this study.We first sought to identify ER protein(s) that bind to TER by affinity purification/mass spectrometry. We generated HEK293 clones stably expressing TER with an N-terminal Strep-tag. The Triton X-100 extracts of these cells or parental HEK293 cells were applied to beads conjugated with Strep-Tactin, which binds to the Strep-tag with high affinity and specificity . Bound pFig. 1D), indicating the binding of endogenous TER to endogenous SERCA2b. We also tested if TER bound to SERCA2b in other cell types. The n-dodecyl-β-D-maltoside (DDM) extracts of primary human keratinocytes and HuH-7, a human hepatoma cell line, were subjected to the coimmunoprecipitation analyses. In both cell types, SERCA2b was specifically coimmunoprecipitated with TER (D). These results demonstrate that TER binds to SERCA2b not only in HEK293 cells but also in other cell types including primary cells, suggesting the physiological relevance of this interaction. Next, we examined the subcellular localization of TER and SERCA2b. HEK293 cells were immunostained with the anti-TER and the anti-SERCA2b antibodies and examined by confocal fluorescence microscopy. SERCA2b showed a reticular staining pattern throughout the cytoplasm, which is consistent with its well-established ER localization. TER was colocalized with SERCA2b in the reticular structure (E). Together, these results indicate that endogenous TER forms a complex with endogenous SERCA2b in the ER membrane.We assessed whether endogenous TER bound to endogenous SERCA2b by coimmunoprecipitation analysis. The Triton X-100 extracts of HEK293 cells were incubated with anti-TER or control antibody. SERCA2b was specifically coimmunoprecipitated with TER D, indicawith TER D. These tructure E. Togeth2+. PA-SERCA2b was specifically pulled down with FLAG–TER, indicating that TER directly bound to SERCA2b (A). Removing Ca2+ from the reaction buffer did not affect the amount of PA-SERCA2b pulled down by FLAG–TER, showing that Ca2+ was not essential for the binding of TER to SERCA2b (A).To examine if TER directly bound to SERCA2b, we performed pull-down experiments with purified recombinant proteins. Recombinant TER with an N-terminal FLAG-tag (FLAG–TER) and recombinant SERCA2b with an N-terminal PA-tag (PA-SERCA2b)  were exp SERCA2b A. Removi SERCA2b A.Figure 2+ and captures Ca2+ in the cytosol, and the E2 state has low affinity for Ca2+ and releases Ca2+ in the ER lumen (B). We next examined the effect of Tg on the binding of TER to SERCA2b in cells. Strep-TER–expressing HEK293 cells were treated with Tg before cell lysis and subjected to the pull-down experiments. Tg treatment significantly reduced the amount of endogenous SERCA2b pulled down by Strep-TER (C). Collectively, these results suggest that SERCA2b in the E2 state has less affinity to TER than that in the E1 state.During the pumping cycle, SERCA undergoes conformational changes. The conformations of SERCA are classified into two major states (E1 and E2). The E1 state has high affinity for CaER lumen . ThapsigER lumen . PretreaER lumen B. We nextrep-TER C. CollecArabidopsis orthologues, human TER is predicted to have an N-terminal ubiquitin-like domain in the cytosol, 6 transmembrane helixes, and a short C-terminal cytoplasmic region . To test if these cytoplasmic regions were involved in the binding to SERCA2b, we generated truncation constructs lacking either the N- or C-terminal region (ΔN or ΔC) and short fragments containing only one of these regions (N-term or C-term) .We searched for the region in TER responsible for its binding to SERCA2b. Based on the reported topologies of yeast and c region , 33, 34 c region A, left.  C-term) A, right.B). To validate the coimmunoprecipitation, we used 3-ketoacyl-CoA reductase (KAR), another enzyme in the FA elongation cycle . On the other hand, 3xHA-SERCA2b was not coimmunoprecipitated with N-term. These results suggest that both the N- and C-terminal regions are required for the full binding of TER to SERCA2b and that the N-terminal region by itself does not bind to SERCA2b.FLAG-tagged full-length TER, ΔN, ΔC, and N-term along with 3xHA-tagged SERCA2b were expressed in HEK293 cells, followed by immunoprecipitation with the anti-FLAG antibody. Consistent with the pull-down of SERCA2b by TER , 3xHA-SEFig. 3on cycle , as a neEscherichia coli and performed pull-down experiments. GST, GST-N-term, or GST-C-term was immobilized to glutathione beads, and the beads were incubated with the lysates of HEK293 cells expressing 3xHA-SERCA2b. GST-C-term, but not GST or GST-N-term, pulled down 3xHA-SERCA2b (C), indicating that the C-terminal region of TER binds to SERCA2b. These results show that TER directly binds to SERCA2b through its C-terminal region.We next investigated if C-term bound to SERCA2b. Because we could not express FLAG-tagged C-term in HEK293 cells, we expressed it as a glutathione-S-transferase (GST)-fusion protein in -SERCA2b C, indicaB and C), we examined the effects of Tg on the binding of the C-terminal region to SERCA2b. HEK293 cells expressing 3xHA-SERCA2b were treated with Tg before cell lysis, and the lysates of these cells were incubated with GST-C-term immobilized on glutathione beads. Tg treatment reduced the amount of 3xHA-SERCA2b pulled down with GST-C-term (D). These results suggest that the C-terminal region prefers SERCA2b in the E1 state to that in the E2 state as full-length TER, validating a role of the C-terminal region as a SERCA2b-interacting region of TER.Because Tg reduced the binding of full-length TER to SERCA2b , B and CT-C-term D. These We also tested if Tg affected the colocalization of TER and SERCA2b in HEK293 cells by immunofluorescence microscopy. TER and SERCA2b were still colocalized in Tg-treated cells . BecauseB and C) raised the possibility that TER would modulate SERCA2b activity. To test this possibility, we examined the effect of TER overexpression on endogenous SERCA2b ATPase activity. FLAG–TER was transiently expressed in HEK293 cells, and a microsomal fraction was prepared by differential centrifugation. The endogenous SERCA2b ATPase activity in the microsomal membranes was measured with a spectrophotomeric assay (A). However, FLAG–TER (full-length) expression significantly reduced SERCA2b ATPase activity compared with empty vector control. The expression of the TER mutant with little SERCA2b binding (ΔC) weakly reduced SERCA2b activity (B). These results demonstrate that TER expression reduces SERCA2b activity through its binding to SERCA2b.Tg inhibits the binding of SERCA to PLN and sarcolipin, both of which regulate SERCA activity in muscle cells . Therefoic assay . FLAG–TEic assay A. Howeveactivity B. These 2+ from the cytosol to the ER lumen. We therefore hypothesized that TER would limit the Ca2+ content in the ER lumen by suppressing SERCA2b activity. To address this, we measured the ER Ca2+ content in cells depleted of TER. Two independent siRNAs were used to deplete TER, which was confirmed by Western blotting with the anti-TER antibody (A). SERCA2b protein levels were grossly normal in TER-depleted cells when compared with control cells. To monitor the ER Ca2+ content, we treated cells with Tg in the absence of extracellular Ca2+. This treatment causes the passive release of Ca2+ from the ER lumen to the cytosol. This release can be measured as an increase in the fluorescence value of a cytosolic Ca2+ indicator dye, Fluo-4 (A). To estimate the total amount of Ca2+ released to the cytosol, we calculated the area under the curve of each Ca2+ trace. The areas under the curves were significantly increased by TER depletion (A), suggesting that TER depletion increased the ER Ca2+ content.SERCA2b ATPase activity is required for uptake of Caantibody A. SERCA2, Fluo-4 . The inc, Fluo-4 A. To estepletion A, suggesB). Consistent with the results in cells without vector transfection (A), the increase of Fluo-4 fluorescence after Tg addition and the areas under the curves were larger in cells transfected with siTER #1 and empty vector than in cells transfected with siControl and empty vector. In contrast, they were not significantly changed in cells transfected with siTER #1 and FLAG–TER (rescue) compared with those in the control cells (B). These results indicate that the increase of the ER Ca2+ content by siTER #1 transfection is rescued by reconstituting TER expression, eliminating the possibility that the effect of siTER #1 is an off-target effect.We next examined if these abnormalities in TER-depleted cells was rescued by reconstituting TER expression. Transfection of an siTER #1-resistant TER construct [FLAG–TER (rescue)] with siTER #1 clearly restored TER expression B. Consissfection A, the inol cells B. These 2+ levels, we directly measured the ER Ca2+ levels with a genetically encoded Ca2+ indicator called G-CEPIA1er, which is an ER luminal protein that enhances its green fluorescence upon Ca2+ binding  was normalized to the final fluorescence value after the depletion of the ER Ca2+ with Tg . This value was supposed to be the minimum fluorescence value of G-CEPIA1er without Ca2+ binding, therefore could be used as an internal control of the G-CEPIA1er transfection efficiency. The ΔF/Ffinal values were significantly larger in TER-depleted cells than in control cells (C), indicating that the ER Ca2+ levels in TER-depleted cells were higher than those in control cells. Taken together, these results demonstrate that TER limits the Ca2+ content in the ER lumen by suppressing SERCA2b activity.To further corroborate that TER depletion increases the ER Ca binding . HEK293 ol cells C, indica2+ uptake to the ER is one of the primary mechanisms to terminate cytosolic Ca2+ responses elicited by ligands of cell surface receptors in nonexcitable cells  that trigger phospholipase C–mediated IP3 production, which in turn releases Ca2+ from the ER via IP3 receptors . The faster decrease of Ca2+ responses was more obvious when the Fluo-4 signals were normalized to the maximum value of each trace (A). To quantify the decay kinetics, we fitted our data to single exponential curves and calculated decay time constants. The decay time constants were significantly smaller in TER-depleted cells, indicating the shorter duration of Ca2+ responses (A). To examine if these observations in TER-depleted cells were due to increased SERCA2b activity, we examined the Ca2+ responses in the presence of Tg (B). In this condition, Ca2+ response curves in TER-depleted cells were similar to those of control cells. Furthermore, decay time constants were not decreased by TER depletion. Together, these results indicate that TER depletion shortens the duration of cytosolic Ca2+ responses by increasing SERCA2b activity, suggesting a role of TER in sustaining ligand-induced Ca2+ signaling.In control cells, ATP application caused a rapid increase of Fluo-4 fluorescence, indicating a rise of cytosolic Caol cells A. The fach trace A. To quaesponses A. To exace of Tg B. In thi2+ responses after ATP application were somewhat lower in TER-depleted cells than in control cells (A) despite the increased ER Ca2+ levels in TER-depleted cells (A and C). The purinoceptor experiments were conducted in the presence of extracellular Ca2+. On the other hand, the ER Ca2+ levels were examined in the absence of extracellular Ca2+. To test if the presence of extracellular Ca2+ contributes to this discrepancy, we conducted the purinoceptor experiments in the absence of extracellular Ca2+. In this condition, the maximum amplitudes of the Ca2+ responses were much lower in TER-depleted cells than in control cells (A), indicating that TER depletion attenuated the Ca2+ responses regardless of the presence of extracellular Ca2+. Importantly, in the presence of Tg, TER depletion did not reduce the maximum amplitudes of the Ca2+ responses (B). The overall Ca2+ responses after ATP application with Tg were slightly larger in TER-depleted cells than in control cells, consistent with the increased ER Ca2+ levels in TER-depleted cells. These results suggest that the increased SERCA2b activity is involved in the attenuation of the ATP-triggered Ca2+ responses in TER-depleted cells.Notably, the maximum amplitudes of cytosolic Caol cells A despiteed cells , A and Col cells A, indicaesponses B. The ov2+ regulation by TER. One of the major functions of cytosolic Ca2+ signaling is transcriptional control through Ca2+-dependent transcription factors (2+ triggers nuclear translocation of the nuclear factor of activated T cells (NFAT) family of transcription factors (2+ responses in the presence of extracellular Ca2+ (A), we reasoned that TER would enhance the nuclear translocation of NFAT. Among four NFAT isoforms (NFAT1-4), NFAT4 has been shown to robustly translocate to the nucleus after GPCR stimulation , which has been frequently used for this purpose (A). To quantify the results, we calculated the ratio of nuclear NFAT4-GFP fluorescence intensity to cytosolic NFAT4-GFP fluorescence intensity in each cell. The obtained nuclear-to-cytosolic ratio of NFAT4-GFP fluorescence intensity was significantly lower in TER-depleted cells than in control cells (B). These results indicate that TER enhances the Ca2+-dependent nuclear translocation of NFAT4, implicating that TER plays a role in transcriptional activation by NFAT4.To monitor the subcellular localization of NFAT4, we used the N-terminal Ca purpose , 43, 44. purpose A. To quaol cells B. These 2+ pump SERCA2b. We further demonstrated that TER is a negative regulator of SERCA2b activity and implicated a role of TER in sustaining cellular Ca2+ signaling. Overall, our results pointed out a previously unrecognized function of TER in limiting Ca2+ accumulation in the ER.TER is an FA reductase in the ER membrane, which plays an essential role in the FA elongation cycle and S1P metabolism. In this study, we biochemically purified and identified TER-binding proteins and showed that TER directly binds to an ER CaA). Although most species contain SERCA-like Ca2+ pumps in their genome, Saccharomyces cerevisiae lacks such pumps . This region is conserved in various organisms, suggesting that the inhibition of the SERCA activity is an evolutionally conserved role of TER A. Althouch pumps . Interes2+ (A) and that Tg inhibits this binding (B and C). SERCA2b in the Ca2+-free buffer was probably in the E1 state because the pH of the buffer was 7.0, at which SERCA1a has been shown to be predominantly in the E1 state even in the absence of Ca2+  and reduces SERCA2b activity through its binding to SERCA2b (B), suggesting that the direct binding of TER inhibits SERCA2b. On the other hand, we cannot eliminate the possibility that TER inhibits SERCA2b indirectly through synthesizing acyl-CoAs. Of note, phosphatidylcholine with saturated FAs has been shown to inhibit SERCA2b , it is possible that this putative Ser58 phosphorylation affects the binding of TER to SERCA2b. Thus, protein kinases or phosphatases targeting Ser58 may regulate the binding of TER to SERCA2b.Whether the binding of TER to SERCA2b is constitutive or regulated is an open question. One possibility is that it is regulated by post-translational modifications of TER or SERCA2b. Notably, proteomic studies show that a serine residue (Ser58) in the N-terminal cytoplasmic region of TER is phosphorylated , 51. BecFig. 33 receptor–mediated Ca2+ release increases the cytosolic Ca2+ and reduces the ER Ca2+. Reduction of the ER Ca2+ then activates the store-operated Ca2+-entry (SOCE), which incorporates Ca2+ from the extracellular space to further increase the cytosolic Ca2+ . Therefore, one simple explanation for this is that TER would limit the SERCA2b activity after SOCE, thereby sustaining the cytosolic Ca2+ responses. On the other hand, there exists another possibility that TER would limit the SERCA2b activity during SOCE, which in turn strengthens the reduction of ER Ca2+, leading to enhanced activation of the SOCE pathway. Given that SOCE is essential for the NFAT nuclear translocation (2+ responses after ATP application was longer in the presence of extracellular Ca2+ (∼300 s) than in the absence of extracellular Ca2+ (∼100 s) in the present study . We therefore assume that SOCE contributes, at least in our experimental setup, to the ATP-triggered Ca2+ responses. The ATP-induced rise of cytosolic Ca2+ has been shown to induce secretion of other signaling molecules, such as neurotransmitters, cytokines, and hormones , implying that TECRL affects SERCA2a differently from TER. Also, because TER is coexpressed with TECRL in muscle tissues  of the ardation . This muardation . Becaused memory . Interesd memory . Therefo2+-responsive region (1–407aa) of human NFAT4  was subcloned into the pGEX-4T-1 vector . For generating recombinant baculoviruses, the cDNA encoding TER was subcloned into the pFastBacFLAGa vector which was generated by the replacement of the N-terminal 6 × His-tag in the pFastBacHTa vector (Invitrogen) with a FLAG-tag. The cDNA encoding SERCA2b was subcloned into the pFastBac1 vector (Invitrogen) with an N-terminal PA-tag.The plasmid containing a cDNA encoding human TER (ID: FXC11442), human KAR (ID: FXC08601), or human SERCA2b (ID: ORK11825) was purchased from Kazusa DNA Research Institute. A cDNA encoding the N-terminal Caan NFAT4  was amplFAT4 /F-12 supplemented with 5% fetal bovine serum, penicillin, and streptomycin. The HEK293 cell clones stably expressing Strep-TER were established as described previously . HuH-7 c2, and 1-mM CaCl2] with a protease inhibitor cocktail. Solubilization was performed by adding Triton X-100 to a final concentration of 1% and subsequent agitation at 4 °C for 30 min. The lysates were then centrifuged at 100,000g for 30 min, and the resultant supernatant was applied to a column with 100 μl of Strep-Tactin Sepharose (IBA Lifesciences). After being washed extensively with buffer A with 1% Triton X-100, bound proteins were eluted with buffer B . The eluted samples were subjected to SDS-PAGE followed by sliver staining with Silver Stain MS kit (Wako) or Western blotting. The bands of interest in the silver-stained gel were cut out and subjected to mass spectrometry analysis.Cells stably expressing Strep-TER from four 10-cm dishes were lysed by sonication in buffer A  with protease inhibitor cocktail. The cell lysates were solubilized and centrifuged as described above, except that 1% DDM (Dojindo) was used to solubilize the lysates of primary keratinocytes or HuH-7 cells. The resultant supernatant was incubated with normal rabbit IgG or rabbit anti-TER pAb at 4 °C for 16 h. The immunocomplex was then precipitated with Protein G Sepharose  at 4 °C for 1 h, extensively washed with buffer C with 1% Triton X-100 or DDM, and eluted with the SDS sample buffer. For the immunoprecipitation of FLAG-tagged protein, the indicated combination of plasmids was transfected into HEK293 cells using polyethylenimine (Polysciences). After 24 h, cell lysates were prepared and subjected to immunoprecipitation as described above with the exception that the supernatant was incubated with mouse anti-FLAG mAb at 4 °C for 2 h.For the immunoprecipitation of endogenous TER, cells were lysed by sonication in buffer C . Cells expressing PA-SERCA2b were lysed by sonication in buffer F  with protease inhibitor cocktail. Solubilization was carried out by adding DDM to a final concentration of 1% and subsequent agitation at 4 °C for 16 h. The samples were then centrifuged at 100,000g at 4 °C for 30 min. The resultant supernatants were incubated with anti-PA-tag NZ-1 Sepharose (WAKO) at 4 °C for 2 h. The resin was washed with buffer F with 1% DDM, with 0.3% DDM, and then with 0.1% DDM. The elution was performed with buffer F with 0.1% DDM and 200 μg/ml PA-tag peptide (WAKO).FLAG–TER or PA-SERCA2b was expressed in Sf21 cells using the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer’s instructions. Cells expressing FLAG–TER were lysed by sonication in buffer D [50-mM Hepes–NaOH (pH 7.0) and 500-mM NaCl] with protease inhibitor cocktail. The lysates were centrifuged at 2000E. coli BL21 (DE3). Cells were then lysed by sonication in buffer G  with 1-mM EDTA and protease inhibitor cocktail. Solubilization was performed by adding Triton X-100 to a final concentration of 1% and subsequent agitation at 4 °C for 30 min. The samples were then centrifuged at 100,000g at 4 °C for 30 min, and the resultant supernatants were incubated with glutathione Sepharose  at 4 °C for 1 h. Each GST-tagged protein on the resin was extensively washed with buffer G with 1% Triton X-100 and then with buffer C with 1-mM DTT and 1% Triton X-100.GST, GST-TER-N-term, and GST-TER-C-term were expressed in 2, 5-mM EGTA, 1% DDM, 20% (w/v) glycerol] with or without 0.77-mM CaCl2. The free concentration of Ca2+ in buffer H with 0.77-mM CaCl2 was 100 nM, which was calculated by the chelator program (The binding experiment between PA-SERCA2b and FLAG–TER was performed in buffer H  with protease inhibitor cocktail on ice for 10 min. The cells were homogenized using a glass homogenizer with a Teflon pestle (10 strokes), and the homogenates were diluted with an equal volume of buffer J  with a protease inhibitor cocktail. The samples were then centrifuged at 1000g at 4 °C for 10 min and then at 6000g at 4 °C for 10 min. The resultant supernatants were centrifuged at 100,000g at 4 °C for 30 min, and the pellets were resuspended with a 1:1 mixture of buffer I and buffer J with protease inhibitor cocktail. The suspensions were centrifuged again at 100,000g at 4 °C for 30 min, and the pellets  were homogenized in a reaction buffer  with 20-μM CaCl2 by passing through 25 G needles.The preparation of microsomal fractions was performed as described previously  with mod3, 5-mM MgCl2, 0.37-mM CaCl2, 1-mM EGTA (free Ca2+ concentration is 320 nM), 1-μM A23187 (Cayman), 200-μM 2-amino-6-mercapto-7-methylpurine ribonucleoside, and 1 U/ml purine nucleoside phosphorylase, supplemented with DMSO or 1-μM Tg at 37 °C for 10 min. The ATPase reaction was then started by adding 1-mM ATP. Inorganic phosphate (Pi) generated by ATP hydrolysis caused the enzymatic conversion of 2-amino-6-mercapto-7-methylpurine ribonucleoside by purine nucleoside phosphorylase, resulting in a shift of absorbance maximum to 360 nm, which was measured with Novaspec Plus spectrophotometer . The concentration of released Pi was calculated as described previously (i release was determined by subtracting the amount of Pi released in the presence of Tg from that in the presence of DMSO.SERCA2b ATPase assay was performed using the EnzChek phosphate assay kit (Invitrogen) as described previously , 68 withSilencer select predesigned siRNAs  and Silencer select negative control siRNA #2 were purchased from Thermo. Cells were transfected with each siRNA (10 nM) using LipofectAmine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. After 48 or 72 h, cells were subjected to the subsequent experiments. For the reconstitution of TER expression in TER-depleted cells, siTER #1 (10 nM) and FLAG–TER (rescue) were transfected into HEK293 cells with LipofectAmine 2000 (Invitrogen) according to the manufacturer’s instructions. After 72 h, cells were subjected to the subsequent experiments. To check the knockdown efficiency or the reconstitution of TER expression, the whole-cell lysates prepared by sonication were subjected to SDS-PAGE followed by Western blotting.2+ content, cells were loaded with 5-μM Fluo 4-AM (Dojindo) at 37 °C for 1 h and then suspended in Ca2+-free Hank’s balanced salt solution (HBSS) . The cells were transferred to a 96-well plate, and the fluorescence values were measured using Fluoroskan Ascent FL microplate reader (Thermo) at 37 °C with 485-nm exitation and 538-nm emission. After the baseline fluorescence values were obtained, 1-μM Tg was added to each well. The fluorescence values were then measured every 5 s for a period of 10 min. The data in each independent experiment were the average value from 6 wells per condition and expressed as the change in fluorescence values relative to baseline (ΔF/F0).For the indirect measurement of the ER Ca2+ levels was carried out with G-CEPIA1er .The direct measurement of the ER CaCEPIA1er . At 48 h2+ responses, cells were plated on a 96-well plate coated with poly-L-lysine 16 h before subsequent experiments. The cells were loaded with Fluo-4 as described above and then incubated in HBSS containing Ca2+ and Mg2+ (Gibco) supplemented with 10-mM Hepes–NaOH (pH 7.5) for >30 min or in Ca2+-free HBSS for 5 min at 25 °C while measuring the fluorescence values. After the baseline fluorescence values were obtained, 100-μM ATP with or without 1-μM Tg was added to each well. The fluorescence values were then measured every 2 s for the periods indicated in the figures. The data in each independent experiment were the average values from 2 or 3 wells per condition and expressed as the change in fluorescence values relative to baseline (ΔF/F0) or the percentage of the maximum value in each trace. To determine decay time constants, the data were fitted to a single exponential function: ΔF/F0 = ΔF/F0, peak × e−t/τ, where t and τ represent the time after the peak and the decay time constant, respectively. The data from the peak until 100 s (for ATP) or 300 s (for ATP and Tg) later were analyzed with Microsoft Excel software.For the measurement of the cytosolic Cahttps://www.ebi.ac.uk/pride/archive/projects/PXD020792. All remaining data are contained within the article.Raw mass spectrometry data were deposited in PRIDE (Project ID: PXD020792) and can be accessed at The authors declare no conflicts of interest in regards to this article.


A role for L-type Ca2+ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells  displayed localised asynchronous Ca2+ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells  generated rhythmic, global Ca2+ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca Reports suggest that approximately 43% of faecal incontinence cases may be related to disturbances in IAS motility3. Unlike the majority of the gastrointestinal (GI) tract, the IAS spontaneously develops tone; a critical feature for raising pressure in the anal canal. While others have suggested that the IAS is a “purely tonic muscle” that develops tone as a result of enhanced myofilament sensitivity to Ca2+ 5, we have found that the IAS is fundamentally a phasic smooth muscle that generates tone as a result of the summation of phasic contractions10. The IAS exhibits slow wave (SW) activity, the electrophysiological events that give rise to phasic contractions in many regions of the GI tract11. SWs, phasic contractions and tone rely upon Ca2+ influx via voltage-gated L-type Ca2+ channels (CavL)14. Thus, mechanisms regulating Ca2+ entry are clearly fundamental for tone development in the IAS.The internal anal sphincter (IAS) is responsible for approximately 70% of resting anal pressure; an important property for maintaining faecal continence11. Pacemaker ICC in these regions are coupled electrically to one another and to adjacent smooth muscle cells (SMCs) via gap junctions allowing conduction of SWs from ICC to SMCs where excitation-contraction coupling occurs15. These cells are typically highly-branched stellate-shaped cells17. In contrast, another population of spindle-shaped intramuscular ICC (ICC-IM) are involved in neuromuscular transmission18. ICC are also present in the IAS of various species but their distribution and morphology differs significantly from that of non-sphincteric muscles23. Importantly, the density of ICC-MY and ICC-SM declines from rectum to IAS with only ICC-IM present in the distal IAS23. SW amplitude and frequency are greatest in the distal IAS24 and thus we hypothesise that ICC-IM could be the pacemaker cells that generate SWs in the IAS.Interstitial cells of Cajal (ICC) are present throughout the GI tract and studies of ICC in non-sphincter muscles indicate that SWs are generated by specialised ICC located predominantly at the myenteric (ICC-MY) and/or submucosal (ICC-SM) edges of the circular muscle layerL25, but IAS-SWs are inhibited by these antagonists, suggesting an essential role for CavL in these events in the IAS26. In addition, intestinal SWs have a more rapid upstroke due to the opening of T-type Ca2+ channels (CavT) which activate and deactivate at a more negative membrane potential (Em) than CavL28. The predominance of CavL as a mediator of SWs in the IAS is not surprising because “resting” Em in the IAS (−43 to −49 mV mouse IAS26) is more depolarised than intestine (−65 mV mouse jejunum29) and thus CavT channels would be substantially inactivated30.IAS-SWs differ significantly from intestinal SWs. Intestinal SWs persist in the presence of antagonists of Cav2+-activated Cl- channel, Anoctamin-1 . ANO1 is highly expressed in ICC throughout the GI tract including the IAS32, but this conductance is not resolved in SMCs or the other type of interstitial cell found in GI muscles, platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells. The cell-specific expression of ANO1 is important because antagonists of this conductance or genetic deactivation of Ano1 can be used to examine the functional role of ICC in intact muscles. ANO1 antagonists greatly reduce or abolish SWs29, and SWs fail to develop in ANO1 deficient mice35. Localised intracellular Ca2+ transients in ICC activate ANO1 channels, causing depolarisation, activation of voltage-gated Ca2+ channels and SW generation38. Since CavL and ANO1 antagonists block SWs as well as tone in the IAS we have proposed that IAS-SWs are important for tone generation26.SWs in the IAS and other GI muscles are dependent upon the Ca2+ indicator, GCaMP6f, in a cell-specific manner to visualise intracellular Ca2+ events in ICC-IM in the distal IAS in situ. The spatiotemporal properties of Ca2+ transients were measured in order to determine if ICC-IM generate pacemaker activity in the IAS. The effects of inhibition of CavL, hyperpolarisation of the Em and blockade of ANO1, were examined as these characteristics have previously been shown to alter the electrical activity, phasic contractions and tone in the IAS.The current study utilised transgenic mice that express the genetically encoded Ca2+ transients in ICC-IM in the distal IAS were imaged in situ using a spinning-disk confocal microscope. Initial experiments revealed two distinct patterns of Ca2+ transients in different spindle-shaped cell populations within the same field of view . In contrast, Type II cells generated synchronised, rhythmic Ca2+ transients that spread globally throughout the whole cell . By superimposing the plot profiles of Ca2+ transients from adjacent cells it is apparent that Type I cell activity was not synchronised within or between cells , mean time to peak (P = 0.006), and mean spatial spread (P = 0.0001) of Ca2+ transients in Type II cells whereas the modal interval between Ca2+ transients was not significantly different (P = 0.441) between Type I and Type II cells ; a value that is well within the range reported for SWs at the distal end of the IAS (69.7 ± 3.2 cpm23). These data are consistent with the hypothesis that a relationship exists between Ca2+ transients in ICC-IM and SWs in the IAS.The rhythmic, highly synchronised behaviour of Type II cells makes it possible for these cells to be the pacemaker cells that generate IAS-SWs. To provide further insight into this relationship we determined the average frequency of Type II cell CaP = 0.0001), which represents a novel distribution for pacemaker cells compared to other regions of the GI tract, i.e., ICC-MY and ICC-SM form networks along the margins of the circular muscle layer in other GI regions11. Although this study focused upon the distal end of the IAS, additional measurements were also undertaken to evaluate whether cell numbers within the muscle interior changed in the proximal direction  completely abolished Ca2+ transients in both cell types indicating their dependence on Ca2+ release from the endoplasmic reticulum .The dependence upon extracellular CaL26. Thus, we compared the sensitivity of Ca2+ transients in Type I and Type II cells to blockade of CavL. Ca2+ transients were recorded from Type I and Type II cells before and during superfusion with the CavL antagonist nifedipine (1 µM). Nifedipine caused a small but significant decrease in the time to peak (P = 0.015) of Ca2+ transients in Type I cells but did not significantly change the amplitude (P = 0.204), spatial spread (P = 0.279) or the mean modal interval (P = 0.999) between Ca2+ transients  of Ca2+ transients and a significant increase in the mean modal interval (P = 0.001) between Ca2+ transients  had no significant effect on any of the Ca2+ transient parameters measured in Type I cells in the IAS  and spatial spread (P = 0.0001) was noted  between Ca2+ transients  on Type I and Type II cells. Previously we have shown that 10 µM CaCCinh-A01 reduces spontaneous contractions in the mouse IAS by 83% while the contraction elicited by raising [K+]o to 66 mM is reduced by only 14%14. Since the K+-induced contraction is entirely abolished by nifedipine but is largely intact following CaCCinh-A01, this concentration of CaCCinh-A01 appears to have limited direct effects upon CavL. CaCCinh-A01 (10 µM) had no effect on Type I cells  were GFP+/c-Kit+ while 34% were GFP+/c-Kit− . We reasoned that the reduced expression of c-Kit in GFP+ cells may occur as a result of the methodology used to achieve specific GCaMP6f expression in ICC, i.e., reduction of Kit alleles from two to one (see methods). Therefore, we compared the density of c-Kit+ cells in wildtype and Kit-Cre-GCaMP6f mice  than that of wildtype mice whereas GFP+ density in Kit-Cre-GCaMP6f mice was similar (P = 0.957) to c-Kit density in wildtype mice. In summary, (1) GCaMP6f expression is coupled to c-Kit, (2) GCaMP6f cells are neither SMCs nor PDGFRα+ cells, and (3) GCaMP6f cells have a similar distribution and density as c-Kit+ cells in wildtype mice. These data suggest that GCaMP6f cells are ICC-IM even though c-Kit expression in some cells is low.We tested whether GCaMP6f is expressed selectively in ICC using dual labelling immunohistochemistry. In the GI tract, SMCs, ICC and PDGFRαFRα Fig.  or smMHCFRα Fig.  indicatiice Fig. . The cel10. The present study examined the possible link between ICC-IM and pacemaker activity  in the IAS. To this end, Cre-lox methodologies were used to generate mice containing the genetically encoded Ca2+ indicator GCaMP6f in ICC (Kit-Cre-GCaMP6f). With the aid of these mice Ca2+ transients were examined in ICC-IM in situ within the IAS. An interesting outcome of this study was that two distinct sub-populations of cells were present. The first population (Type I ICC-IM) exhibited asynchronous Ca2+ transients similar to the ICC-IM that are involved in neuromuscular transmission in other GI muscles48. The second population (Type II ICC-IM) was novel and displayed spatiotemporal and pharmacological properties consistent with these cells generating pacemaker activity in the IAS.The IAS generates SWs that initiate phasic contractions which can summate to produce tone23. The present study supports this assessment since GCaMP6f expression in the distal IAS was limited to spindle-shaped intramuscular cells. Immunohistochemical examination of Kit-Cre-GCaMP6f tissues revealed that GCaMP6f (GFP+) cells. were smMHC−/PDGFRα− indicating they were neither SMCs nor PDGFRα+ cells. As described in Methods, the Kit-CreERT2 mice used to produce Kit-Cre-GCaMP6f mice were generated by inserting CreERT2 in exon 1 of the Kit gene on a single allele46. Since, CreERT2 is expressed in place of Kit on one allele, Kit-Cre-GCaMP6f mice may have reduced Kit gene expression. None-the-less, the majority of GCaMP6f cells were c-Kit+ and the density of GCaMP6f cells in Kit-Cre-GCaMP6f mice was equivalent to the density of c-Kit+ cells in wildtype mice. Taken together, these data suggest that GCaMP6f expressing cells are ICC-IM.Immunohistochemical studies of the mouse rectoanal region have shown that ICC-MY and ICC-SM populations decline aborally reaching undetectable levels at the distal IAS whereas spindle-shaped ICC-IM are found throughout2+ imaging of GCaMP6f cells in the distal IAS revealed two distinct functional populations, i.e., Type I and Type II cells. Type I cells exhibited localised Ca2+ transients originating from multiple active sites each spreading a short distance. These Ca2+ transients were asynchronous both within and between neighbouring cells. Ca2+ transients in Type I cells were also smaller in amplitude and reached peak intensity faster than Ca2+ transients in Type II cells. The asynchronous activity described here for Type I cells is similar to that previously described for ICC-IM in the mouse colon and small intestine47. A large body of evidence from other GI muscles suggests that ICC-IM  serve as post-junctional targets for excitatory cholinergic and inhibitory nitrergic neuromuscular transmission48. It is likely that the Type I cells described here serve a similar role in the IAS. Indeed, functional nitrergic and cholinergic pathways are present in the mouse IAS13 and we have previously identified expression of guanylate cyclase and cGMP-dependent protein kinase in ICC-IM49. Ca2+ release events were independent of ANO1 but it is still possible that neurotransmitter-induced changes in Ca2+ release modify ANO1 activity giving rise to junction potentials  that change the contractile behaviour of adjacent SMCs.Ca2+ transients. Furthermore, the activity of neighbouring Type II cells was synchronised. In other GI regions, pacemaker ICC form networks at the myenteric or submucosal edges of the circular muscle, and SWs spread actively through these networks conducting to coupled SMCs as the wave of activity progresses11. In the IAS, synchronisation of Type II cells occurs in spite of the fact that these cells do not form a network with one another. However, ICC-IM are electrically coupled to SMCs via gap junctions51. Thus, SMCs may act as a bridge in the propagation of SW activity between ICC-IM. This is a novel model of SW propagation that may be unique to the IAS.In contrast, the activity of Type II cells consisted of rhythmic, global Ca23 and SMC Ca2+ transients52 are greatest at the distal IAS and decline proximally. Thus, the anatomical location of Type II cells in the anorectum coincides with the functional distribution of SWs; an observation consistent with a role for Type II cells in the generation of SWs.Type I and Type II cells were distributed across the thickness of the distal IAS but the changes occurring in their density in the oral direction differed. Type I cell density increased moderately in the proximal direction. In contrast, Type II cell density declined proximally reaching negligible levels 2 to 3 mm from the distal edge indicating that these cells are unique to the IAS. In like manner, previous functional studies have shown that the amplitude and frequency of SWsL plays a critical role in the generation of SWs and tone in the IAS26. In a similar manner, rhythmic, global Ca2+ transients in Type II cells were abolished by blocking CavL with nifedipine or by hyperpolarisation of the Em with the KATP agonist pinacidil. In contrast, asynchronous, localised Ca2+ transients of Type I cells persisted following both procedures. Interestingly, following CavL blockade, the rhythmic, global Ca2+ transients observed in Type II cells were replaced by smaller amplitude asynchronous, localised Ca2+ transients similar in time course and amplitude to those of Type I cells indicating a common underlying feature of Type I and Type II cells.CavL the primary mediator of SWs rather than CavT30. The depolarised status of Em is likely the result of low resting K+ conductance (GK) since increasing GK with pinacidil hyperpolarises Em by more than 20 mV, blocking SWs53 and global Ca2+ transients in ICC (present study). In like manner, purinergic neuromuscular transmission, which activates small conductance Ca2+-activated K+ channels on PDGFRα+ cells57, also hyperpolarises Em in the mouse IAS by more than 20 mV, again abolishing SWs26. Although nerve stimulation can dramatically alter the electrical and contractile activity of the in vitro mouse IAS58, blockers of cholinergic, purinergic and nitrergic transmission as well as the neurotoxin TTX have little or no effect upon the basal electrical and contractile activity in the mouse IAS58. Hence, the properties of Ca2+ transients described in this study are intrinsic to the Type I and Type II ICC-IM examined rather than to neural inputs.As mentioned in the introduction, “resting” Em  is more depolarised in the IAS than intestine making Cav2+ from stores followed by activation of ANO1, depolarisation, activation of voltage-gated Ca2+ channels and finally a fully developed SW61. To examine the role of Ca2+ release in Type I and Type II cells, extracellular Ca2+ was removed or the Ca2+-ATPase inhibitor CPA was added. Ca2+ removal abolished all Ca2+ transients in Type I and Type II cells although the time required to produce full inhibition was greater for Type I cells (10.2 min) than Type II cells (6.4 min). This difference in time course may reflect the greater ongoing entry of Ca2+ into Type II cells via CavL and/or difference(s) in the mechanism(s) contributing to the uptake and storage of Ca2+ by the endoplasmic reticulum (ER). Asynchronous Ca2+ transients in Type I and Type II cells were also abolished by addition of the SERCA pump inhibitor CPA providing evidence they are due to Ca2+ release from the ER. The characteristics observed for asynchronous Ca2+ transients in Type I and Type II cells in the IAS are similar to those described for ICC-IM and ICC-DMP in the mouse colon and small intestine47. Furthermore, both studies conclude that Ca2+ release mechanisms involve inositol triphosphate receptors (InsP3Rs) as well as ryanodine receptors (RyRs) with ongoing refilling of the endoplasmic reticulum via Ca2+-ATPase.Substantial evidence indicates that SW generation in ICC begins with localised release of Ca2+ transients in Type I cells while rhythmic, global Ca2+ transients in Type II cells were replaced by asynchronous localised Ca2+ transients. These data indicate that CavL, Ca2+ release and ANO1 each contribute to the generation of synchronised rhythmic Ca2+ transients in Type II cells; observations consistent with SW generation in ICC  initiated by ANO1 in ICC-IM contribute to regulating resting Em and modulation of Em by nerves63. However, since nifedipine, pinacidil and CaCCinh-A01 all have minimal effects on Ca2+ transients in Type I cells it supports the general consensus that Ca2+ transients in ICC-IM (other than Type II cells in the IAS) are voltage-independent events.Blockade of ANO1 with CaCCinh-A01 had similar effects on Type I and Type II cells to those observed with nifedipine or pinacidil. CaCCinh-A01 failed to block asynchronous localised Casee Fig. . The ver10. CavL channels play a fundamental role in this process since tone is blocked by manoeuvres which inhibit CavL activity66. A common feature of the IAS in all animal species studied is SWs10. Like tone, SWs are abolished by inhibitors of CavL activity67 indicating an important link between SWs and tone. One possible way in which phasic SWs can give rise to tone is via a partial (incomplete) tetanus type mechanism like that first described for skeletal muscle. Partial tetanus occurs as stimulus frequency is increased beyond a certain level, leading to an inability of Ca2+ removal to keep up with Ca2+ delivery and thus resulting in increased basal cytoplasmic [Ca2+]68. This “partial tetanus” consists of phasic contractions superimposed upon tone. SW frequency in the mouse IAS is quite high  and we have recently provided evidence for a partial tetanus type mechanism in studies of the SM-GCaMP mouse IAS52. In larger animal species  the IAS is composed of numerous muscle bundles separated by wide connective tissue septa21. The degree to which these muscle bundles are electrically coupled is still unclear. Thus, an additional mechanism by which SWs may give rise to tone in larger animals is via summation of asynchronous phasic events arising from poorly coupled muscle bundles. In like manner, skeletal postural muscles can maintain tone through the asynchronous firing of motor units69. Finally, the overall depolarised level of Em in the IAS likely results in some Ca2+ entry via CavL that is time-independent 10.The IAS differs from the majority of GI muscles in that it generates tone to raise pressure in the anal canal thereby facilitating faecal continence2+ transients in ICC-IM were imaged in situ in Kit-Cre-GCaMP6f mice to examine the possibility that these cells are responsible for SW generation in the IAS. Two distinct sub-populations of ICC-IM were identified. The first, Type I ICC-IM, exhibited asynchronous Ca2+ transients with properties resembling those of ICC-IM in other GI muscles that participate in enteric neuromuscular transmission. The second, Type II ICC-IM, exhibited Ca2+ transients with spatiotemporal and pharmacological properties novel for ICC-IM but very consistent with the behaviour of pacemaker ICC. Type II ICC-IM exhibited rhythmic, global Ca2+ transients that were synchronised with adjacent Type II cells. These events were dependent upon CavL as well as ANO1; a Ca2+-activated Cl− channel that is highly expressed in ICC-IM of the mouse IAS14. From these data we conclude that Type II ICC-IM are a viable candidate for the generation of pacemaker activity in the mouse IAS. Since tone in the IAS is also dependent upon CavL and ANO114, it follows that Type II ICC-IM may also play an important physiological role in the regulation of anal pressure and maintenance of faecal continence.In summary, Ca2+ activity in ICC, the genetically encoded Ca2+ indicator GCaMP6f was expressed specifically in ICC. GCaMP6f-lox+ mice 26S°rtm95.1(CAG-GCaMP6f)Hze/J; The Jackson Laboratory; strain number: 024105) were crossed with Kit+/Cre-ERT2 mice  to generate Kit-Cre-GCaMP6f mice. As previously described, Kit-CreERT2 mice were generated by inserting CreERT2 into exon 1 of the Kit gene on a single allele46 thus confining Kit expression to one allele. To activate Cre-recombinase and induce GCaMP6f expression, mice were injected with tamoxifen at 6–8 weeks old (2 mg for three consecutive days) as previously described47. Animals were sacrificed 10–30 days after tamoxifen injection. For cell density studies, wildtype C57Bl/6 mice  were also used. Mice were euthanised with isoflurane (Baxter) inhalation and cervical dislocation. All procedures were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno. Animals used and experiments performed were also in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.In order to study intracellular Ca2, 23.8 NaHCO3, 1.2 KH2PO4, 11 dextrose. KRBS had a pH of 7.4 after bubbling to equilibrium with 95% O2/5% CO2. Flat sheet preparations of the rectoanal region were prepared for Ca2+ imaging experiments by clearing the tissue of surrounding skeletal muscle including the external anal sphincter and fat, cutting the tissue open from rectum to anus and removing the mucosal layer to expose the inner circular layer of the muscularis externa. Tissues were pinned flat, with the circular muscle facing upwards in a 60 mm Sylgard/charcoal-lined dish. To rule out the possible excitatory effects of cholinergic motor neurons all experiments were carried out in the presence of atropine (1 µM).Rectoanal tissues were removed and placed in a Sylgard-lined dissection dish with ice cold Krebs-Ringer bicarbonate solution (KRBS) of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2+ the tissue was perfused with Ca2+ free KRBS plus 0.5 mM EGTA. Images were obtained with an upright confocal microscope (Olympus) equipped with a spinning disc  and an Andor iXon EMCCD camera  to detect images. GCaMP6f was excited using a 488 laser (Coherent OBIS) and all recordings were acquired with a 20 × 1.00 NA XLUMPlanFL N lens  at a frame rate of 30 frames per second. All recordings, unless otherwise indicated were made from GCaMP6f cells at the distal extremity of the IAS.Tissues were continually perfused with oxygenated KRBS at 37 °C and left to equilibrate for 1 hour. For experiments determining the reliance of each cell type on extracellular Ca2+ activity in ICC-IM of the IAS were imported into custom analysis software . If necessary, recordings were motion-stabilised to ensure accurate Ca2+ transient analysis. De-bleaching techniques were applied where appropriate in order to remove background fluorescence. Spatio-temporal (ST) maps were created for specific cells of interest by rotating image stacks so that ICC-IM were orientated vertically and drawing a region of interest (ROI) around the visible part of the cell. Further Ca2+ transient analysis was carried out by importing the ST maps into Image J  as previously described71. Briefly, ST maps were normalised to obtain F/F0 by dividing the image by the mean intensity of the cell between Ca2+ transients. Active sites of Ca2+ activity, defined as sites that Ca2+ transients consistently originated from were analysed over a 10 s period . The amplitude (expressed as Δ F/F0), time to peak (time from trough to peak of a Ca2+ transient), spatial spread  and interval  were measured.Recordings of CaThe cellular distribution within the circular muscle layer was calculated using confocal microscopy at 20x magnification (400 µm × 400 µm) and focusing upon either the myenteric, middle, or submucosal third of the circular muscle layer. All cells in focus within that region were then assigned according to established behaviours (see further description in the results section).23. Tissues were fixed in ice-cold Zamboni’s fixative  for 20 min at 20 °C before washing in 0.1 M PBS, dehydrated in graded sucrose solutions (5–20%), and frozen as previously described22. Frozen tissues were sectioned perpendicular to the circular muscle layer at a thickness of 10–12 µm with a Leica CM 3050 cryostat  and labelled with primary and secondary antibodies as previously described14. Sections were blocked using 1% bovine serum albumin (BSA) for 1 hr at 20 °C before incubation in the first primary antibody  for 16 hours at 4 °C. Sections were washed with 0.1 M PBS before incubation in secondary antibody . Subsequently, sections were washed in 0.1 M PBS prior to incubation with the second primary antibody , Biomedical Technologies Inc., Stoughton, MA, USA; 1:100 dilution). Following incubation with the second primary antibody, sections were washed and incubated with secondary antibody  or Alexa Fluor anti-rabbit 594 (smMHC), Invitrogen, Carlsbad, CA, USA; 1:1000 dilution) as described above. After washing with 0.1 M PBS, slides were covered with coverslips using Aquamount mounting medium . Wildtype mouse rectoanal tissues were dissected, fixed and labelled as described above. In this case, preparations were labelled with anti-c-Kit antibody (mSCFR) followed by Alexa Fluor anti-goat 594.The distal GI tract was kept in a tube and the mucosa removed as described previouslyConfocal images of tissue sections were imaged using a Zeiss LSM 510 Meta confocal microscope . Images shown in figures are composites of Z-series stacks taken through a depth of 1–13 µm. Figures were prepared using Zeiss LSM 510 Images Examiner Software, Adobe Photoshop CS5 Software and CorelDraw 2019 Software.2.Cell density was determined from cross sections of the distal IAS circular muscle layer of wildtype (C57Bl/6) and Kit-Cre-GCaMP6f mice. A line was drawn around the edge of the circular muscle layer using CorelDraw 2019 Software and then all green, red and yellow cells were counted within this area. Total area was determined from images in Adobe Photoshop CS5 Software. Cell density was then expressed as number of cells per 100 µmpost-hoc test was used to compare Type I and Type II cell distribution in Ca2+ imaging studies. One-way ANOVA with Tukey’s post-hoc test was used in comparing c-Kit+ and GFP+ cell numbers in immunohistochemical studies. A P-value of <0.05 was considered statistically significant. n= number of cells, N = number of animals.Data is expressed as the mean ± SD. Statistical analysis was carried out using GraphPad Prism (version 7.0). Student’s paired or unpaired two-tailed t-test was used to compare two groups, i.e., control conditions versus drug (paired) or various parameters in Type I versus Type II cells (unpaired). Two-way ANOVA with Šidák −1.Nifedipine, pinacidil, CPA (cyclopiazonic acid), EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), atropine and tamoxifen were purchased from Sigma-Aldrich . CaCCinh-A01 was purchased from Tocris Bioscience . Stock concentrations were made by dissolving in de-ionized water (pinacidil), ethanol (nifedipine) or DMSO (CaCCinh-A01). Final concentrations were achieved by further dilution in KRBS. EGTA was dissolved to final concentration in KRBS. Tamoxifen was first dissolved in ethanol before addition of safflower oil to make a final concentration of 20 mg mlSupplementary Information.Supplementary Information2.


Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca2+ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+ signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+. The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca2+ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca2+ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca2+ release. We conclude that Ca2+ signals evoked by GPN most likely derive from acidic organelles.Lysosomes are acidic Ca Summary: Methods of releasing calcium from lysosomes are limited but characterization of the effects of GPN in primary cultured human fibroblasts confirmed that it probably targets acidic organelles. Owing tevations . Reciprolysosome . Such bilysosome . Local arphology  and trafrphology  through rphology  and funcrphology . Importaisorders , undersc2+ stores in live cells is challenging. Lysosomal Ca2+ is difficult to measure directly due to the extreme pH and proteolytic environment. Additionally, there are few cell-permeable activators of lysosomal Ca2+ release channels available. In this context, the modified dipeptide glycyl-L-phenylalanine 2-naphthylamide (GPN) has long been used to probe lysosomal Ca2+ content , IP3 and thapsigargin (2+ uptake by lysosomes. Ca2+-H+ exchangers (CAX) have been molecularly identified and characterised in plants, microorganisms (such as yeast) and more recently in select animals  incubation with bafilomycin A1. This potentiation is consistent with recent findings  evoked robust Ca2+ signals in the absence of extracellular Ca2+   . But, tw cytosol J. The senot Ca2+ D-F. NH4Cme cells  but not me cells  pointing2+ signals  is unknown. If leak of Ca2+ from the lysosome is also slow then this might explain why only prolonged bafilomycin A1 treatment inhibits GPN-evoked Ca2+ signals. It might also explain why neither bafilomycin A1 nor NH4Cl acutely induce a Ca2+ signal. Interestingly, incubation of cells with bafilomycin A1 for 2 h only modestly increased lysosomal pH when the incubation included a 1 h culture period at 37°C relative to cells that were maintained at room temperature throughout. Yet, these treatments had reciprocal effects on GPN-evoked Ca2+ signal such that inhibition was only noted in the former conditions. This implies that the effects of bafilomycin A1 are temperature-dependent, perhaps pointing to accelerated lysosomal Ca2+ leak at elevated temperature. Of relevance here is the original study identifying lysosomal-like Ca2+ stores as NAADP targets P2 (2+ mobilizing activity of the NAADP-like agonist (TPC2-A1-N) was abolished by prior treatment of cells with GPN P2 . Notablywith GPN . This is, ML-SA1 . In cont2+ release by targeting bafilomycin A1-sensitive lysosomal Ca2+ stores. Exactly how GPN releases Ca2+ from lysosomes requires further work given that GPN-evoked Ca2+ signals are reportedly independent of cathepsin C and amplification by IP3 receptors  or following trypsin treatment (HeLa cells), and plated onto coverslips for imaging. For experiments with HeLa cells, coverslips were coated with poly-L-lysine. Cells were mycoplasma-negative.Primary cultured human dermal fibroblasts or HeLa cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin  at 37°C in a humidified atmosphere with 5% CO2+ and pH were measured independently using the fluorescent ratiometric indicators Fura-2 and BCECF, respectively. All experiments were performed in HEPES-buffered saline (HBS) comprising 1.25 mM KH2PO4, 2 mM CaCl2, 2 mM MgSO4, 3 mM KCl, 156 mM NaCl, 10 mM glucose and 10 mM HEPES . For measurement of Ca2+, cells were incubated with Fura-2 AM (2.5 µM) and 0.005% (v/v) pluronic acid (from Invitrogen) for 1 h in HBS. Fura-2 was excited at 340/380 nm and emitted fluorescence was captured using a 440 nm long pass filter and a 20× objective. For the measurement of pH, cells were incubated with BCECF-AM (5 µM) and 0.005% v/v pluronic acid (Invitrogen) for 30 min in HBS. BCECF was excited at 490/440 nm and emitted fluorescence was captured using a 515 nm long pass filter and a 20× objective.Cytosolic CaLysosomal pH was measured using the fluorescent acidotrope LysoTracker Red and the ratiometric fluorescent indicator fluorescein. Cells were labelled with LysoTracker Red  for 15 min at room temperature in HBS. The dye was excited at 568 nm and emitted fluorescence was captured using a 590 nm filter and a 20× objective. This was used as a proxy for lysosomal pH given that LysoTracker Red is a hydrophobic weak base that accumulates in acidic compartments in a pH-dependent manner . To measTPC2 activity was measured using TPC2-GCaMP6s as described by 2+ was omitted from the HBS.After dye labelling or transfection, cells were washed with HBS and mounted in a 1 ml imaging chamber (Biosciences Tools) prior to microscopy. Epifluorescence images were captured every 3 s with a cooled coupled device camera (TILL photonics) attached to an Olympus IX71 inverted fluorescence microscope fitted with a monochromator light source. Cells were stimulated with 200 µM GPN (Santa Cruz Biotechnology), 1 µM thapsigargin (Santa Cruz Biotechnology) or 30 µM TPC2-A1-N  during recording, or preincubated for up to 2 h at room temperature. For extended bafilomycin A1 treatments, cells were incubated for up to 4 h in culture followed by an additional 1 h at room temperature in HBS in the continued presence of bafilomycin A1. For Fura-2 and LysoTracker Red measurement, the dyes were loaded in the presence of bafilomycin A1 at room temperature. For fluorescein-dextran measurements, bafilomycin A1 was added after the chase period.0) upon stimulation. Basal ratios and intensities were averaged for 60 to 90 s of recording or for shorter periods if there were spontaneous fluctuations, but this was rare. For fluorescein-dextran, the steady-state fluorescence ratio was presented. Data are collated as individual means from independent experiments or as mean±s.e.m. Statistical analyses were performed using Prism 9. Independent-samples t-tests, paired-samples t-tests or Mann–Whitney U-tests were applied. P<0.05 was considered statistically significant.Fluorescence signals were quantified on an individual cell basis and averaged for all cells in a given field of view (up to 42 cells). For Fura-2 and BCECF, the maximal fluorescence ratio increase was calculated by subtracting the basal ratio from the peak ratio obtained in response to a given stimulus. In some Fura-2 experiments, the area under the curve was calculated by integrating the fluorescence ratios following subtraction of the basal ratio. For LysoTracker Red and TPC2-GCaMP6s recordings, signals were quantified as the maximal fractional fluorescence change relative to the basal intensity (F/F


N-methyl-D-aspartate receptor (NMDAR) provides a pathway for glutamate-mediated inter-cellular communication, best known for its role in the brain but with multiple examples of functionality in non-neuronal cells. Data previously published by others and us provided ex vivo evidence that NMDARs regulate platelet and red blood cell (RBC) production. Here, we summarize what is known about these hematopoietic roles of the NMDAR. Types of NMDAR subunits expressed in megakaryocytes (platelet precursors) and erythroid cells are more commonly found in the developing rather than adult brain, suggesting trophic functions. Nevertheless, similar to their neuronal counterparts, hematopoietic NMDARs function as ion channels, and are permeable to calcium ions (Ca2+). Inhibitors that block open NMDAR (memantine and MK-801) interfere with megakaryocytic maturation and proplatelet formation in primary culture. The effect on proplatelet formation appears to involve Ca2+ influx-dependent regulation of the cytoskeletal remodeling. In contrast to normal megakaryocytes, NMDAR effects in leukemic Meg-01 cells are diverted away from differentiation to increase proliferation. NMDAR hypofunction triggers differentiation of Meg-01 cells with the bias toward erythropoiesis. The underlying mechanism involves changes in the intracellular Ca2+ homeostasis, cell stress pathways, and hematopoietic transcription factors that upon NMDAR inhibition shift from the predominance of megakaryocytic toward erythroid regulators. This ability of NMDAR to balance both megakaryocytic and erythroid cell fates suggests receptor involvement at the level of a bipotential megakaryocyte-erythroid progenitor. In human erythroid precursors and circulating RBCs, NMDAR regulates intracellular Ca2+ homeostasis. NMDAR activity supports survival of early proerythroblasts, and in mature RBCs NMDARs impact cellular hydration state, hemoglobin oxygen affinity, and nitric oxide synthase activity. Overexcitation of NMDAR in mature RBCs leads to Ca2+ overload, K+ loss, RBC dehydration, and oxidative stress, which may contribute to the pathogenesis of sickle cell disease. In summary, there is growing evidence that glutamate-NMDAR signaling regulates megakaryocytic and erythroid cells at different stages of maturation, with some intriguing differences emerging in NMDAR expression and function between normal and diseased cells. NMDAR signaling may provide new therapeutic opportunities in hematological disease, but in vivo applicability needs to be confirmed.The  N-methyl-d-aspartate receptor (NMDAR) in megakaryocytic and erythroid cells. NMDARs are best known for their functions as glutamate-gated cation channels in the central nervous system  proteins that are activated by Ca2+ entry through voltage-gated Ca2+ channels. Following release, glutamate concentrations in the synaptic cleft increase markedly, from 2–5 μM to approximately 1.1 mM. While in the synaptic cleft, glutamate activates ionotropic and metabotropic receptors located on the post-synaptic plasma membrane , and metabotropic receptors activate G-proteins that modulate ion channels directly and indirectly. The main purpose of the ionic flux is to generate and propagate action potentials characteristic of excitable tissues. The synaptic glutamate signal is terminated by the excitatory amino acid transporters (EAAT) present on astrocytes that remove glutamate from the synaptic cleft . NMDAR-mediated Ca2+ influx contributes little to membrane depolarization but modifies synaptic strength through molecular events related to the Ca2+ role as “second messenger” , and kainate receptors, each named after a distinct, synthetic agonist that activates them . AMPA anssenger” .l-glutamate on each of the GluN2 subunits, as well as glycine (co-agonist) on the GluN1 and GluN3 subunits. The alternative NMDAR ligands include d- and l- aspartate, homocysteine, homocysteic acid, and d-serine. NMDAR subunit composition varies substantially in different areas of the brain, and changes during development  or GluN3 (A or B) subunits, in various combinations. It is believed that GluN1 subunit is an essential component of all NMDARs, and variable GluN2 and GluN3 subunits are modulatory. NMDAR activation requires binding of elopment . NMDAR sng NMDAR . In addirization .50) to agonists is high, ranging from 0.4 to 1.7 μM for glutamate , and 0.1 to 2.1 μM for glycine  (50 around 7.4 μM for most of the subunits)  . These cubunits) , and Zn2ectively ].2+ entry activates a number of intracellular signaling pathways, including Ca2+/calmodulin-dependent kinase (CaMK), mitogen-activated protein kinase (MAPK) , and phosphoinositide 3-kinase (PI3K) (NMDAR-mediated Cae (PI3K) . NMDARs e (PI3K) . Other me (PI3K) .During the past 10–20 years, NMDARs have been reported in multiple non-neuronal cell types, including hematopoietic , which r+ influx to membrane depolarization, but not Ca2+ influx (2+ influx into platelets but inhibit platelet function in the presence of adenosine diphosphate (ADP) and arachidonic acid  counteract this effect, indicating that NMDARs operate as Ca2+ channels in these cells , mouse megakaryocytes also express GluN2C and GluN3B . Small nBCR-ABL1 gene fusion  carry all possible NMDAR subunits, including GluN2B, GluN3A, and GluN3B . Meg-01 e fusion . Both Mee fusion  and eryte fusion , thus prke cells . BiologiWe found that Meg-01 cells are better suited for studies of NMDAR function than K-562 and Set-2 cells, mostly because of their higher levels of NMDAR expression. Upon differentiation with phorbol-12-myristate-13-acetate (PMA), Meg-01 cells up-regulate NMDAR expression further, providing a model in which to examine NMDAR involvement in megakaryocytic differentiation .2+ block and Ca2+ entry  in megakaryocytic cells, normal and leukemic, suggests that NMDAR generates weaker but more sustained Catype “a” . GluN1a type “a” . Becausetype “a” , this foMouse and human megakaryocytes express a range of molecules for glutamate re-uptake, storage, and release, including VGLUT1, VGLUT2, SNARE, and the high-affinity glutamate re-uptake system, EAAT1, shown at both transcript and protein levels . Fluorimvia local pH changes would be more likely in tissues with higher, plasma-like glutamate concentrations. Different types of NMDAR subunits will also affect cellular sensitivity to glutamate, and modulate the receptor response .Unfortunately, there is no information on glutamate concentrations in the interstitial fluid of the bone marrow. In peripheral blood plasma, physiological glutamate levels are usually maintained between 20 and 100 μM , but varNMDAR channel blockers (memantine and MK-801) induce two types of apparently opposing effects in cultured megakaryocytic cells: inhibition of differentiation in normal megakaryocytes, but induction of differentiation in megakaryocytic leukemia cell lines .2+ influx is required for the cytoskeletal remodeling that underlies proplatelet formation , nuclear ploidy and proplatelet formation; however, progenitor proliferation is unaffected . Similarormation . This prormation . NMDAR lormation .In contrast to normal megakaryocytes that utilize NMDAR function to assist differentiation, leukemic cell lines  appear to divert NMDAR activity to increase proliferation . In the GRIN1 gene that encodes the obligate, GluN1 subunit of the NMDAR  stress and induced autophagy. Prominent cytoplasmic vacuoles accumulated in Meg-01-GRIN1−/− cells and were found to represent dilated ER and lysosomal organelles , implying the ErbB4-NMDAR link is maintained during megakaryocytic-erythroid differentiation  and a scatter of low signals for GRIN1, GRIN2A, GRIN2C, and GRIN2D (GRIN transcripts were detected at very low levels.The interrogation of publicly available transcriptomic data obtained from human megakaryocyte-erythroid progenitors at a single cell level demonstrated the presence of d GRIN2D . Deep se3H]MK-801) binding assay, basal activity of NMDARs in RBCs suspended in plasma was shown  and the presence of GluN2C, GluN2D, and GluN3 subunits  triggered vacuolization and apoptosis, with maximal cell death observed at the early differentiation stages , 2015. Tor cells .2+  is reduced by half; MAPK, mitogen-activated protein kinase; MEP, megakaryocyte-erythroid progenitor; MK, megakaryocyte; NMDAR, N-methyl-d-aspartate receptor; PI3-K, phosphoinositide 3-kinase; PMA, phorbol 12-myristate 13-acetate; PSD, post-synaptic density; RBC, red blood cell; SNARE, soluble N-ethyl maleimide-sensitive factor attachment protein receptor; TPO, thrombopoietin; VGLUT, vesicular glutamate transporter.ADP, adenosine diphosphate; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AP5, D-2-amino-5-phosphonopentanoate; CaMK, Ca