Sarcoendoplasmic Reticulum Ca(2+) ATPase 2b Research Articles

Temperature-Dependent Effects of Eicosapentaenoic Acid (EPA) on Browning of Subcutaneous Adipose Tissue in UCP1 Knockout Male Mice.

Uncoupling protein 1 (UCP1) plays a central role in thermogenic tissues by uncoupling cellular respiration to dissipate energy. Beige adipocytes, an inducible form of thermogenic cells in subcutaneous adipose tissue (SAT), have become a major focus in obesity research. We have previously shown that eicosapentaenoic acid (EPA) ameliorated high-fat diet (HFD)-induced obesity by activating brown fat in C57BL/6J (B6) mice at thermoneutrality (30 °C), independently of UCP1. Here, we investigated whether ambient temperature (22 °C) impacts EPA effects on SAT browning in wild-type (WT) and UCP1 knockout (KO) male mice and dissected underlying mechanisms using a cell model. We observed resistance to diet-induced obesity in UCP1 KO mice fed HFD at ambient temperature, with significantly higher expression of UCP1-independent thermogenic markers, compared to WT mice. These markers included the fibroblast growth factor 21 (FGF21) and sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b), suggesting the indispensable role of temperature in beige fat reprogramming. Surprisingly, although EPA induced thermogenic effects in SAT-derived adipocytes harvested from both KO and WT mice, EPA only increased thermogenic gene and protein expression in the SAT of UCP1 KO mice housed at ambient temperature. Collectively, our findings indicate that the thermogenic effects of EPA, which are independent of UCP1, occur in a temperature-dependent manner.

Activation of the EGFR-PI3K-CaM pathway by PRL-1-overexpressing placenta-derived mesenchymal stem cells ameliorates liver cirrhosis via ER stress-dependent calcium

BackgroundCholesterol accumulation and calcium depletion induce hepatic injury via the endoplasmic reticulum (ER) stress response. ER stress regulates the calcium imbalance between the ER and mitochondria. We previously reported that phosphatase of regenerating liver-1 (PRL-1)-overexpressing placenta-derived mesenchymal stem cells (PD-MSCsPRL−1) promoted liver regeneration via mitochondrial dynamics in a cirrhotic rat model. However, the role of PRL-1 in ER stress-dependent calcium is not clear. Therefore, we demonstrated that PD-MSCsPRL−1 improved hepatic functions by regulating ER stress and calcium channels in a rat model of bile duct ligation (BDL).MethodsLiver cirrhosis was induced in Sprague–Dawley (SD) rats using surgically induced BDL for 10 days. PD-MSCs and PD-MSCsPRL−1 (2 × 106 cells) were intravenously administered to animals, and their therapeutic effects were analyzed. WB-F344 cells exposed to thapsigargin (TG) were cocultured with PD-MSCs or PD-MSCsPRL−1.ResultsER stress markers, e.g., eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), were increased in the nontransplantation group (NTx) compared to the control group. PD-MSCsPRL−1 significantly decreased ER stress markers compared to NTx and induced dynamic changes in calcium channel markers, e.g., sarco/endoplasmic reticulum Ca2+ -ATPase 2b (SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3R), mitochondrial calcium uniporter (MCU), and voltage-dependent anion channel 1 (VDAC1) (*p < 0.05). Cocultivation of TG-treated WB-F344 cells with PD-MSCsPRL−1 decreased cytosolic calmodulin (CaM) expression and cytosolic and mitochondrial Ca2+ concentrations. However, the ER Ca2+ concentration was increased compared to PD-MSCs (*p < 0.05). PRL-1 activated phosphatidylinositol-3-kinase (PI3K) signaling via epidermal growth factor receptor (EGFR), which resulted in calcium increase via CaM expression.ConclusionsThese findings suggest that PD-MSCsPRL−1 improved hepatic functions via calcium changes and attenuated ER stress in a BDL-injured rat model. Therefore, these results provide useful data for the development of next-generation MSC-based stem cell therapy for regenerative medicine in chronic liver disease.

MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme

ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.

T cell exosome-derived miR-142-3p impairs glandular cell function in Sjögren's syndrome.

Sjögren's syndrome (SS) is a systemic autoimmune disease that mainly affects exocrine salivary and lacrimal glands. Local inflammation in the glands is thought to trigger glandular dysfunction and symptoms of dryness. However, the mechanisms underlying these processes are incompletely understood. Our work suggests T cell exosome-derived miR-142-3p as a pathogenic driver of immunopathology in SS. We first document miR-142-3p expression in the salivary glands of patients with SS, both in epithelial gland cells and within T cells of the inflammatory infiltrate, but not in healthy volunteers. Next, we show that activated T cells secreted exosomes containing miR-142-3p, which transferred into glandular cells. Finally, we uncover a functional role of miR-142-3p-containing exosomes in glandular cell dysfunction. We find that miR-142-3p targets key elements of intracellular Ca2+ signaling and cAMP production - sarco(endo)plasmic reticulum Ca2+ ATPase 2b (SERCA2B), ryanodine receptor 2 (RyR2), and adenylate cyclase 9 (AC9) - leading to restricted cAMP production, altered calcium signaling, and decreased protein production from salivary gland cells. Our work provides evidence for a functional role of the miR-142-3p in SS pathogenesis and promotes the concept that T cell activation may directly impair epithelial cell function through secretion of miRNA-containing exosomes.

The cell-wide web coordinates cellular processes by directing site-specific Ca2+ flux across cytoplasmic nanocourses

Ca2+ coordinates diverse cellular processes, yet how function-specific signals arise is enigmatic. We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca2+ diffusion and by nanocourse-specific Ca2+-pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca2+ flux through RyR2/3 clusters selects for rapid propagation of Ca2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.

Evidence of CB1-Receptor Antagonism in Fat-Fed Dogs Promoting Beiging of Adipose Tissue Mainly via Activation of Sarco/Endoplasmic Reticulum Ca2+ ATPase 2b and Ryanodine Receptor 2

We have previously demonstrated that 16 weeks following exposure to a high fat diet (HFD), dogs treated with the CB1-receptor antagonist (Rimonabant (RIM)) upregulated β3-adrenergic receptors, PGC1α, Prdm16, and slight increase of UCP1 in the subcutaneous (SC) and visceral (VIS) depots when compared to control treated dogs. These data suggest an improvement in metabolic flux of adipose tissue consistent with beiging. The mechanism(s) by which RIM promotes adipose tissue beiging is unknown. We hypothesize that the beiging effect might be induced via UCP1-independent thermogenic mechanisms through upregulation of sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). To explore the mechanism by which RIM enhances adipose tissue function, dogs were fed a HFD (52% fat) for 6 weeks followed by a continued 16 weeks of fat feeding with either HFD + placebo (PL) (n=9) or HFD + RIM (1.25 mg/kg per day; n=11). Biopsies from SC and VIS depots were obtained for gene expression: before HFD (pre-fat) and 16 weeks of HFD +/- RIM. RIM increased SERCA2b expression in the SC depot by 8-fold (P&amp;lt;0.05) and in the VIS depot by 2-fold (P&amp;lt;0.05) compared to the PL+HFD groups. Also, RIM increased RyR2 3-fold in the SC (P&amp;lt;0.005) and 4-fold in the VIS depots (P&amp;lt;0.05). Our data suggest that a potential mechanism by which CB1-receptor antagonism increases beiging of adipose tissues may be through upregulation of the key genes involved in an UCP1-independent pathway. Importantly, for the first time we are beginning to identify mechanisms by which CB1-receptor antagonism enhances adipose tissue function and improves energy homeostasis in the presence of a HFD. Disclosure R.L. Paszkiewicz: None. R.N. Bergman: Advisory Panel; Self; Ingredion, Inc.. Research Support; Self; AstraZeneca. Consultant; Self; GI Dynamics Inc.. Research Support; Self; National Institute of Diabetes and Digestive and Kidney Diseases. Consultant; Self; Novo Nordisk A/S. J.M. Richey: None. M.S. Iyer: None. O.O. Woolcott: None. C.M. Kolka: Research Support; Self; AstraZeneca. D. Clegg: None. M. Kabir: None.

Maresin 1 attenuates NAFLD by suppression of endoplasmic reticulum stress via AMPK–SERCA2b pathway

Maresin 1 (MAR1), which is derived from docosahexaenoic acid biosynthesized by macrophages, has been reported to improve insulin resistance. Recently, it has been documented that MAR1 could ameliorate inflammation and insulin resistance in obese mice. These findings led us to investigate the effects of MAR1 on hepatic lipid metabolism. We found that MAR1 could stimulate AMP-activated protein kinase (AMPK), thereby augmenting sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) expression. This stimulation suppressed lipid accumulation by attenuating the endoplasmic reticulum (ER) stress in hepatocytes under hyperlipidemic conditions. Attenuation was mitigated by knockdown of AMPK or thapsigargin, a SERCA2b inhibitor. We also demonstrated that MAR1 administration resulted in increased hepatic AMPK phosphorylation and Serca2b mRNA expression, whereas hepatic ER stress was reduced in high-fat diet (HFD)-fed mice. Moreover, MAR1 treatment suppressed hepatic lipid synthesis, thereby attenuating hepatic steatosis in HFD-fed mice. In conclusion, our results suggest that MAR1 ameliorates hepatic steatosis via AMPK/SERCA2b-mediated suppression of ER stress. Therefore, MAR1 may be an effective therapeutic strategy for treating non-alcoholic fatty liver disease (NAFLD) via regulation of ER stress-induced hepatic lipogenesis.

UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis.

Uncoupling Protein 1 (UCP1) plays a central role in non-shivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice, as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by the activation of α1/β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism, and pyruvate dehydrogenase activity for ATP-dependent thermogenesis by the SERCA2b pathway; beige fat thereby functions as a “glucose-sink” and improves glucose tolerance independent of body-weight loss. Our study uncovers a non-canonical thermogenic mechanism by which beige fat controls whole-body energy homeostasis through Ca2+ cycling.

Astragaloside IV protects against podocyte injury via SERCA2-dependent ER stress reduction and AMPK\u03b1-regulated autophagy induction in streptozotocin-induced diabetic nephropathy

Aberrant endoplasmic reticulum (ER) stress and autophagy are associated with diabetic nephropathy. Here we investigated the effect of astragaloside IV (AS-IV) on the progression of diabetic nephropathy (DN) and the underlying mechanism involving ER stress and autophagy in streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-incubated podocytes. The diabetic mice developed progressive albuminuria and glomerulosclerosis within 8 weeks, which were significantly ameliorated by AS-IV treatment in a dose-dependent manner. Moreover, diabetes or HG-induced podocyte apoptosis was markedly attenuated by AS-IV, paralleled by a marked remission in ER stress and a remarkable restoration in impaired autophagy, which were associated with a significant improvement in the expression of sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) and AMP-activated protein kinase α (AMPKα) phosphorylation, respectively. Knockdown of SERCA2 in podocytes induced ER stress and largely abolished the protective effect of AS-IV, but had no obvious effect on the expression of autophagy-associated proteins. On the other hand, blockade of either autophagy induction or AMPKα activation could also significantly mitigate AS-IV-induced beneficial effect. Collectively, these results suggest that AS-IV prevented the progression of DN, which is mediated at least in part by SERCA2-dependent ER stress attenuation and AMPKα-promoted autophagy induction.

A natural compound jaceosidin ameliorates endoplasmic reticulum stress and insulin resistance via upregulation of SERCA2b

Increased endoplasmic reticulum (ER) stress has emerged as a vital contributor to dysregulated glucose homeostasis, and impaired function of sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) is one of the central mechanisms underlying ER stress. In this study, we reported that SERCA2b upregulation contributed to the amelioration of ER stress and insulin resistance by a small natural compound jaceosidin. In a model of differentiated C2C12 myotubes, jaceosidin-triggered SERCA2b upregulation enhanced insulin sensitivity and decreased ER stress. Moreover, the activity of Ca2+-ATPase in thapsigargin-treated myotubes was also augmented by jaceosidin. Furthermore, jaceosidin significantly suppressed blood glucose levels, improved glucose tolerance and lowered body weight, but did not alter food intake in insulin-resistant obese mice. In addition, this compound markedly reduced lipid accumulation, suppressed the expression of lipogenic genes in liver and ameliorated liver injury. The ameliorative effects of jaceosidin were due to its ability to reduce ER stress via increasing the expression of SERCA2b in the muscles of obese mice. Taken together, jaceosidin could improve ER stress and attenuate insulin resistance via SERCA2b upregulation in mice skeletal muscles.

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

The sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl l-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-d,l-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca2+ imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca2+ and decreased ER Ca2+ levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.

The non-genomic effects of high doses of Rosiglitazone on cell growth and apoptosis in cultured monocytic cells

Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a ligand-activated transcription factor which belongs to the nuclear hormone superfamily and has multiple pharmacological ligands called Thiazolidinediones (TZDs). TZDs are a class of drugs used in the treatment of type 2 diabetic patients. Rosiglitazone is one such TZD, and is used clinically to treat type 2 diabetes. In this study, the effect of Rosiglitazone on cell growth and apoptosis in cultured monocytic monomac 6 (MM6) cells was investigated. Over a 14 day period, MM6 cells were cultured in vitro and treated with 1μM and 10μM Rosiglitazone. Cell viability and proliferation were evaluated by Haemocytometer cell count and MTS assay respectively. Turbidity due to cell density was assessed spectrophotometrically. Apoptosis was determined by Caspase-Glo 3/7 assay. Expression of the endoplasmic reticulum (ER) stress-inducible protein sarco-endoplasmic reticulum Ca2+ATPase-2b (SERCA2b) was determined by Western blot. Neither 1μM nor 10μM Rosiglitazone exerted statistically significant inhibitory effects on cell proliferation, turbidity due to cell density, or cell viability (p > 0.05 in all cases). In contrast, Rosiglitazone induced increased apoptosis, but a significant difference was only observed in 10μM-treated cells compared with control cells (3.04 ± 0.52 control; p < 0.05) while 1μM-treated cells showed a non-significant increase (1.50 ± .06 control; p > 0.05). Meanwhile the expression of SERCA2b was up-regulated significantly in cells treated for >4hrs (e.g 2.45 ± 0.06 control at 24 hrs; p < 0.05) with 10μM Rosiglitazone. It was concluded that high doses (10μM) of Rosiglitazone up-regulate SERCA2b expression and induce apoptosis of MM6 cells by activating an ER stress response via a PPARγ-independent mechanism. The therapeutic relevance of these observations is a matter for further investigations. Key words: Rosiglitazone, PPARγ, Monocytes, ER Stress, SERCA2b, Apoptosis

Phe27Cys polymorphism of the human delta opioid receptor predisposes cells to compromised calcium signaling

A quarter of the human population with European background carries at least one allele of the OPRD1 gene that encodes the delta opioid receptor with cysteine at the amino acid position 27 (hδOR(Cys27)) instead of the evolutionary conserved phenylalanine (hδOR(Phe27)). The two variants have indistinguishable pharmacological properties but, importantly, hδOR(Cys27) differs from hδOR(Phe27) in having low maturation efficiency, lower stability at the cell surface and pronounced intracellular location. Both variants were previously shown to interact with the Sarco(endo)plasmic reticulum Ca²+ ATPase (SERCA) 2b in the early phase of their biosynthesis. We analyzed by pulse-chase assays, whether cellular signaling can affect hδOR(Cys27) maturation. Neither activation of the receptor by a δOR-specific agonist Leu-enkephalin, induction of intracellular calcium (Ca²+) release by ATP nor the direct stimulation of SERCA 2b by protein kinase C activation affected receptor maturation in HEK-293 cells. No signaling-mediated regulation of receptor maturation could therefore be demonstrated. Instead, we found by using single cell Ca²+ measurements that over-expression of hδOR(Cys27), but not hδOR(Phe27), compromised ATP-induced intracellular Ca²+-signaling. Furthermore, hδOR(Cys27) precursors showed slower dissociation from SERCA2b and hδOR(Cys27) expression caused down-regulation of the homocysteine-inducible endoplasmic reticulum-resident ubiquitin domain-like member 1 protein (HERP). We suggest that aging individuals with at least one hδOR(Cys27) encoding allele might have lowered threshold for Ca²+ dysregulation in neurons expressing hδOR.

Inhibition of Nuclear Factor-κB or Bax Prevents Endoplasmic Reticulum Stress- But Not Nitric Oxide-Mediated Apoptosis in INS-1E Cells

Accumulating evidence suggests that endoplasmic reticulum (ER) stress by mechanisms that include ER Ca(2+) depletion via NO-dependent down-regulation of sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) contributes to beta-cell death in type 1 diabetes. To clarify whether the molecular pathways elicited by NO and ER Ca(2+) depletion differ, we here compare the direct effects of NO, in the form of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), with the effects of SERCA2 inhibitor thapsigargin (TG) on MAPK, nuclear factor kappaB (NFkappaB), Bcl-2 proteins, ER stress, and apoptosis. Exposure of INS-1E cells to TG or SNAP caused caspase-3 cleavage and apoptosis. Both TG and SNAP induced activation of the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). However, other classical ER stress-induced markers such as up-regulation of ER chaperone Bip and alternative splicing of the transcription factor Xbp-1 were exclusively activated by TG. TG exposure caused NFkappaB activation, as assessed by IkappaB degradation and NFkappaB DNA binding. Inhibition of NFkappaB or the Bcl-2 family member Bax pathways protected beta-cells against TG- but not SNAP-induced beta-cell death. These data suggest that NO generation and direct SERCA2 inhibition cause two quantitative and qualitative different forms of ER stress. In contrast to NO, direct ER stress induced by SERCA inhibition causes activation of ER stress signaling pathways and elicit proapoptotic signaling via NFkappaB and Bax.

Regulation of Sarco/Endoplasmic Ca2+-ATPase Expression by Calcium in Human Lens Cells

Maintenance of cellular calcium levels is critical to cell function. Loss of calcium homeostasis might be a contributing factor to the development of cataract in the lens. In lens epithelium, calcium is involved in cell signaling and its precise regulation is vital. In this study, we investigated the regulation of sarco/endoplasmic reticulum Ca2+-ATPase2b (SERCA2b) and SERCA3 isoform expression in cultured epithelial B-3 cells from human lenses. Both mRNA and membrane proteins samples were collected for semi quantitative RT-PCR using GAPDH as a control. Western blot analyses were performed on membrane samples.Thapsigargin, a SERCA isoform inhibitor which causes increased cytosolic levels elicited dose-and time-dependent up-regulation of SERCA3 at both mRNA and protein levels; SERCA2b expression was unaffected. Both EGTA and actinomycin partially inhibited the thapsigargin-induced SERCA3 up-regulation. These results indicate that the up-regulation of SERCA3 by thapsigargin is dependent on a calcium-mediated pathway that is likely to occur at the transcription level.

Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca2+-binding sites.

2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 micro m, respectively, measured at pH 7.2). The Ca2+-ATPase is also more potently inhibited at lower pH (IC50 = 70 micro m for SERCA1A at pH 6). 2-APB decreases the affinity for Ca2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca2+-ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca2+ to their binding sites.

Localization and identification of Ca2+ATPases in highly purified human platelet plasma and intracellular membranes. Evidence that the monoclonal antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in human platelets.

The Ca2+ATPase activities of highly purified human platelet membranes prepared by high-voltage free-flow electrophoresis have been analysed by using [gamma-32P]ATP hydrolysis, recognition by antibodies and phosphoenzyme-complex formation. The Ca2+ATPase activity present in mixed membranes was found to be predominantly associated with intracellular membranes after subfractionation, with only a low level of activity associated with plasma membranes. The intracellular-membrane Ca2+ATPase activity was inhibited totally with thapsigargin (Tg), whereas the plasma-membrane Ca2+ATPase was not significantly affected, suggesting that the latter does not belong to the SERCA (sarco-endoplasmic-reticulum Ca2+ATPase) class. A monoclonal antibody, 5F10, raised to the red-cell membrane Ca2+ATPase [Cheng, Magocsi, Cooper, Penniston and Borke (1993) Cell Physiol. Biochem. 4, 31-43] recognized two bands at 135 and 150 kDa in mixed membranes and plasma membranes, and the corresponding bands in red-blood-cell membranes, confirming the Ca2+ATPase to be of the PMCA (plasma-membrane Ca2+ATPase) type. No recognition of any band was detected in intracellular membranes. Identification of the intracellular-membrane Ca2+ATPase activity was carried out with polyclonal antibodies with known specificity towards SERCA 2b (S.2b) and SERCA 3 (N89), and a monoclonal antibody, PL/IM 430, raised against platelet intracellular membranes. All of these antibodies recognized the 100 kDa Ca2+ATPase in mixed membranes and intracellular membranes, with little or no recognition of the activity in the plasma membranes. In some membrane preparations the antibody PL/IM 430 and antiserum N89 recognized similar degradation products, of 74, 70 and 40 kDa, in the intracellular-membrane fraction. The Ca2+ATPase recognized by PL/IM 430 was immunoprecipitated, and the immunoprecipitated protein was specifically recognized by the antiserum N89, but not by S.2b. Analysis of the phosphoenzyme-complex formation revealed potent phosphorylation of the 100 and 74 kDa peptides, both recognized by PL/IM 430 and N89. These studies report the presence of a PMCA in a purified plasma-membrane fraction from human platelets, and that the antibody PL/IM 430 recognizes the SERCA 3 Ca2+ATPase in intracellular membranes.