Calcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced release of calcium stored in the endoplasmic reticulum (ER). Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes), and therefore these enzymes play an important role in ER calcium homeostasis and in the control of B of cell activation. Because Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis, in this work the effects of the virus on SERCA-type calcium pump expression and calcium accumulation in the endoplasmic reticulum of B cells was investigated.
Two Sarco-Endoplasmic Reticulum Calcium transport ATPase isoforms, the low Ca2+-affinity SERCA3, and the high Ca2+-affinity SERCA2 enzymes are simultaneously expressed in B cells. Latency type III infection of Burkitt's lymphoma cell lines with immortalization-competent virus expressing the full set of latency genes selectively decreased the expression of SERCA3 protein, whereas infection with immortalization-deficient virus that does not express the EBNA2 or LMP-1 viral genes was without effect. Down-modulation of SERCA3 expression could be observed upon LMP-1, but not EBNA2 expression in cells carrying inducible transgenes, and LMP-1 expression was associated with enhanced resting cytosolic calcium levels and increased calcium storage in the endoplasmic reticulum. Similarly to virus-induced B cell immortalisation, SERCA3 expression was also decreased in normal B cells undergoing activation and blastic transformation in germinal centers of lymph node follicles.
The data presented in this work indicate that EBV-induced immortalization leads to the remodelling of ER calcium homeostasis of B cells by LMP-1 that copies a previously unknown normal phenomenon taking place during antigen driven B cell activation. The functional remodelling of ER calcium homeostasis by down-regulation of SERCA3 expression constitutes a previously unknown mechanism involved in EBV-induced B cell immortalisation.
Brain cells expend large amounts of energy sequestering calcium (Ca2+), while loss of Ca2+ compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P), a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum (ER) to sequester Ca2+. This led to the hypothesis that G6P regulates Ca2+ accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA). Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, 45Ca2+ accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi) coupled with Ca2+ accumulation was quantified. Addition of G6P significantly and decreased Ca2+ accumulation in a dose-dependent fashion (1–10 mM). The reduction in Ca2+ accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca2+ accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca2+ uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca2+ dystasis caused by altered G6P regulation of SERCA activity.
glucose-6-phosphate; G6P; calcium; SERCA; microsome; endoplasmic reticulum; thapsigargin; brain
Endoplasmic reticulum calcium homeostasis is involved in several essential cell functions including cell proliferation, protein synthesis, stress responses or secretion. Calcium uptake into the endoplasmic reticulum is performed by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). In order to study endoplasmic reticulum calcium homeostasis in situ in mammary tissue, in this work SERCA3 expression was investigated in normal breast and in its benign and malignant lesions in function of the cell type, degree of malignancy, and histological and molecular parameters of the tumors. Our data indicate, that although normal breast acinar epithelial cells express SERCA3 abundantly, its expression is strongly decreased already in very early non-malignant epithelial lesions such as adenosis, and remains low in lobular carcinomas. Whereas normal duct epithelium expresses significant amounts of SERCA3, its expression is decreased in several benign ductal lesions, as well as in ductal adenocarcinoma. The loss of SERCA3 expression is correlated with Elston-Ellis grade, negative hormone receptor expression or triple negative status in ductal carcinomas. The concordance between decreased SERCA3 expression and several histological, as well as molecular markers of ductal carcinogenesis indicates that endoplasmic reticulum calcium homeostasis is remodeled during tumorigenesis in the breast epithelium.
breast cancer; calcium signaling; endoplasmic reticulum; SERCA; calcium pump; ion transport
We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using 45Ca2+ coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca2+-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a full-length SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene.
Spiny lobster; Cloning; Polymerase chain reaction; Rapid amplification of cDNA ends; Sequencing; Real time PCR
In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca2+ is maintained at low levels by pumping Ca2+ into stores in the endoplasmic reticulum (ER) via the sarco ER Ca2+-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca2+, whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca2+ clearance and exaggerating inositol 1,4,5-trisphosphate–mediated Ca2+ liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid β production. Our results point to a physiological role for the presenilins in Ca2+ signaling via regulation of the SERCA pump.
The cardiac isoform of the sarco/endoplasmic reticulum Ca2+ATPase (SERCA2a) plays a major role in controlling excitation/contraction coupling. In both experimental and clinical heart failure (HF), SERCA2a expression is significantly reduced which leads to abnormal Ca2+ handling and deficient contractility. A large number of studies in isolated cardiac myocytes, in small and large animal models of HF showed that restoring SERCA2a expression by gene transfer corrects the contractile abnormalities and improves energetics and electrical remodeling. Following a long line of investigation, a clinical trial is underway to restore SERCA2a expression in patients with HF using adeno-associated virus type 1.
This review addresses the following issues regarding HF gene therapy: 1) new insights on calcium regulation by SERCA2a; 2) SERCA2a as a gene therapy target in animal models of HF; 3) advances in the development of viral vectors and gene delivery; 4) clinical trials of HF using SERCA2a
This review focuses on the new advances in SERCA2a- targeted gene therapy made in the last three years.
SERCA2a is an important therapeutic target in various cardiovascular disorders. Ongoing clinical gene therapy trials will provide answers on its safety and applicability.
Sacroplasmic reticulum Ca2+ ATPase; heart failure; gene therapy
Under certain conditions, cardiac myocytes engage in a mode of calcium signaling in which calcium release from the sarcoplasmic reticulum (SR) to myoplasm occurs in self-propagating succession along the length of the cell. This event is called a calcium wave and is fundamentally a diffusion-reaction phenomenon. We present a simple, continuum mathematical model that simulates calcium waves. The framework features calcium diffusion within the SR and myoplasm, and dual modulation of ryanodine receptor (RyR) release channels by myoplasmic and SR calcium. The model is used to illustrate the effect of varying RyR permeability, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activity and calcium ion mobility in myoplasm and SR on wave velocity. The model successfully reproduces calcium waves using experimentally-derived variables. It also supports the proposal for wave propagation driven by the diffusive spread of myoplasmic calcium, and highlights the importance of SR calcium load on wave propagation.
Calcium; waves; Diffusion; Modeling; Myocyte; Heart; Cardiac; Sarcoplasmic Reticulum
Previous studies showed that overexpression of sarco-endoplasmic reticulum calcium ATPase (SERCA2a) in a variety of heart failure (HF) models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors.
A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated virus 1 (rAAV1) mediated gene transfection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein (control) gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular (LV) sample was determined using two-dimensional (2-D) gel electrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits.
The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers (interleukin-6 and tumor necrosis factor-α) and neuro-hormonal factors (brain natriuretic peptide, endothelin-1 and angiotensin II) compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein (alpha-crystallin-related, B6) were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase.
These findings demonstrate that regional intramyocardial injections of rAAV1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory, excessive neuroendocrine factors and the stress-associated myocardial proteins, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of stress-associated reaction.
Heart failure; Sarco-endoplasmic reticulum calcium ATPase; Gene transfer; Stress reaction; Neuro-hormonal factors
We tested whether calcium-induced calcium release (CICR) contributes to synaptic release from rods in mammalian retina. Electron micrographs and immunofluorescent double labeling for the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) and synaptic ribbon protein, ribeye, showed a close association between ER and synaptic ribbons in mouse rod terminals. Stimulating CICR with 10 μM ryanodine evoked Ca2+ increases in rod terminals from mouse retinal slices visualized using confocal microscopy with the Ca2+-sensitive dye, Fluo-4. Ryanodine also stimulated membrane depolarization of individual mouse rods. Inhibiting CICR with a high concentration of ryanodine (100 μM) reduced the ERG b-wave but not a-wave consistent with inhibition of synaptic transmission from rods. Ryanodine (100 μM) also inhibited light-evoked voltage responses of individual rod bipolar cells (RBCs) and presumptive horizontal cells recorded with perforated patch recording techniques. A presynaptic site of action for ryanodine's effects is further indicated by the finding that ryanodine (100 μM) did not alter currents evoked in voltage-clamped RBCs by puffing the mGluR6 antagonist, (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG), onto bipolar cell dendrites in the presence of the mGluR6 agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4). Ryanodine (100 μM) also inhibited glutamatergic outward currents in RBCs evoked by electrical stimulation of rods using electrodes placed in the outer segment layer. Together, these results indicate that, like amphibian retina, CICR contributes to synaptic release from mammalian (mouse) rods. By boosting synaptic release in darkness, CICR may improve the detection of small luminance changes by post-synaptic neurons.
Ca-induced Ca2+; release; ryanodine; outer retina; synaptic transmission
Intracellular calcium controls several crucial cellular events in apicomplexan parasites, including protein secretion, motility, and invasion into and egress from host cells. The plant compound thapsigargin inhibits the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), resulting in elevated calcium and induction of protein secretion in Toxoplasma gondii. Artemisinins are natural products that show potent and selective activity against parasites, making them useful for the treatment of malaria. While the mechanism of action is uncertain, previous studies have suggested that artemisinin may inhibit SERCA, thus disrupting calcium homeostasis. We cloned the single-copy gene encoding SERCA in T. gondii (TgSERCA) and demonstrate that the protein localizes to the endoplasmic reticulum in the parasite. In extracellular parasites, TgSERCA partially relocalized to the apical pole, a highly active site for regulated secretion of micronemes. TgSERCA complemented a calcium ATPase-defective yeast mutant, and this activity was inhibited by either thapsigargin or artemisinin. Treatment of T. gondii with artemisinin triggered calcium-dependent secretion of microneme proteins, similar to the SERCA inhibitor thapsigargin. Artemisinin treatment also altered intracellular calcium in parasites by increasing the periodicity of calcium oscillations and inducing recurrent, strong calcium spikes, as imaged using Fluo-4 labeling. Collectively, these results demonstrate that artemisinin perturbs calcium homeostasis in T. gondii, supporting the idea that Ca2+-ATPases are potential drug targets in parasites.
The sarco/endoplasmic reticulum calcium ATPase (SERCA) is essential for the control of intracellular free Ca2+ levels. Recently, SERCA has been identified as an important effector of nitric oxide (NO) action in vascular cells. NO stimulates the uptake of cytosolic Ca2+ via SERCA by adducting glutathione to the reactive cysteine-674 thiol. Mutation of this single amino acid prevents the stimulation of Ca2+ uptake by NO, as well as its ability to decrease Ca2+ and cell migration. NO function is impaired in a variety of cardiovascular diseases, including diabetes and atherosclerosis, which are associated with irreversible oxidation of SERCA cysteine-674. Targeting the sources of oxidants in vascular diseases to prevent SERCA oxidation and/or increasing the expression of SERCA may impede vascular disease.
Activity elicits capture of dense-core vesicles (DCVs) that transit through resting Drosophila synaptic boutons to produce a rebound in presynaptic neuropeptide content following release. The onset of capture overlaps with an increase in the mobility of DCVs already present in synaptic boutons. Vesicle mobilization requires Ca2+-induced Ca2+ release by presynaptic endoplasmic reticulum (ER) ryanodine receptors (RyRs) that in turn stimulates Ca2+/calmodulin-dependent kinase II (CamKII). Here we show that the same signaling is required for activity-dependent capture of transiting DCVs. Specifically, the CamKII inhibitor KN-93, but not its inactive analog KN-92, eliminated the rebound replacement of neuropeptidergic DCVs in synaptic boutons. Furthermore, pharmacologically or genetically inhibiting neuronal sarco-endoplasmic reticulum calcium ATPase (SERCA) to deplete presynaptic ER Ca2+ stores or directly inhibiting RyRs prevented the capture response. These results show that the presynaptic RyR-CamKII pathway, which triggers mobilization of resident synaptic DCVs to facilitate exocytosis, also mediates activity-dependent capture of transiting DCVs to replenish neuropeptide stores.
activity-dependent capture; CamKII; dense-core vesicle; Drosophila neuropeptide release; synaptic trafficking
In blood vessels, tone is maintained by agonist-induced cytosolic Ca2+ oscillations of quiescent/contractile vascular smooth muscle cells (VSMCs). However, in synthetic/proliferative VSMCs, Gq/phosphoinositide receptor-coupled agonists trigger a steady-state increase in cytosolic Ca2+ followed by a Store Operated Calcium Entry (SOCE) which translates into activation of the proliferation-associated transcription factor NFAT. Here, we report that in human coronary artery smooth muscle cells (hCASMCs), the sarco/endoplasmic reticulum calcium ATPase type 2a (SERCA2a) expressed in the contractile form of the hCASMCs, controls the nature of the agonist-induced Ca2+ transient and the resulting down-stream signaling pathway. Indeed, restoring SERCA2a expression by gene transfer in synthetic hCASMCs 1) increased Ca2+ storage capacity; 2) modified agonist-induced IP3R Ca2+ release from steady-state to oscillatory mode (the frequency of agonist-induced IP3R Ca2+ signal was 11.66 ± 1.40/100 sec in SERCA2a-expressing cells (n=39) vs 1.37 ± 0.20/100 sec in control cell (n=45), p<0.01); 3) suppressed SOCE by preventing interactions between SR calcium sensor STIM1 and pore forming unit ORAI1; 4) inhibited calcium regulated transcription factor NFAT and its down-stream physiological function such as proliferation and migration.
This study provides evidence for the first time that oscillatory and steady-state patterns of Ca2+ transients have different effects on calcium-dependent physiological functions in smooth muscle cells.
SERCA; NFAT; proliferation; signal transduction; calcium oscillations; store-operated calcium entry
Sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and SERCA3 pump Ca2+ in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca2+ concentration ([Ca2+]ER) and the role of SERCA3 in the control of insulin secretion and ER stress.
RESEARCH DESIGN AND METHODS
β-Cell [Ca2+]ER of SERCA3+/+ and SERCA3−/− mice was monitored with an adenovirus encoding the low Ca2+-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca2+ concentration ([Ca2+]c) and [Ca2+]ER were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied.
Glucose elicited synchronized [Ca2+]ER and [Ca2+]c oscillations. [Ca2+]ER oscillations were smaller in SERCA3−/− than in SERCA3+/+ β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca2+]ER rise in SERCA3+/+ and SERCA3−/− β-cells. In a Ca2+-free medium, glucose moderately raised [Ca2+]ER from a highly buffered cytosolic Ca2+ pool. Increasing [Ca2+]c with high [K] elicited a [Ca2+]ER rise that was larger but more transient in SERCA3+/+ than SERCA3−/− β-cells because of the activation of a Ca2+ release from the ER in SERCA3+/+ β-cells. Glucose-induced insulin release was larger in SERCA3−/− than SERCA3+/+ islets. SERCA3 ablation did not induce ER stress.
[Ca2+]c and [Ca2+]ER oscillate in phase in response to glucose. Upon [Ca2+]c increase, Ca2+ is taken up by SERCA2b and SERCA3. Strong Ca2+ influx triggers a Ca2+ release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca2+ uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.
Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca2+ imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca2+ oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate– mediated Ca2+ release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca2+ signaling and controlling Ca2+-sensitive chaperone functions in the ER.
phosphorylation; calnexin; ER lectin chaperones; Ca2+ ATPases; Ca2+ signaling
The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The “2-color” SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.
The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca2+ signaling. However, whether the changes in Ca2+ signaling and Ca2+ signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known.
Materials and Methods
In SERCA2+/- mouse parotid gland acinar cells, Ca2+ signaling, expression levels of Ca2+ signaling proteins, and amylase secretion were investigated.
SERCA2+/- mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca2+ ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP3Rs), but the localization and activities of IP3Rs were not altered. In SERCA2+/- mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice.
These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca2+ signaling proteins in the parotid gland acini, however, overall Ca2+ signaling is unchanged.
sarco/endoplasmic reticulum Ca2+-ATPase; Ca2+ signaling proteins; parotid gland acinar cells
The Golgi apparatus (GA) is an intracellular organelle that plays a central role in lipid and protein posttranslational modification and sorting. In addition, the GA has been also shown to be involved in Ca2+ signalling, as: (i) it accumulates Ca2+ within its lumen in an ATP-dependent process catalyzed by two enzymes, the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and the secretory pathway Ca2+ ATPase1 (SPCA1), and (ii) it releases Ca2+ during cell stimulation in response to inositol 1,4,5-trisphosphate (IP3) receptor activation. Therefore, on this aspect, the GA appears to behave similarly to the major intracellular Ca2+ store, the endoplasmic reticulum (ER). By using a new FRET-based Ca2+ probe, specifically targeted to the trans-compartment of the GA, we demonstrate that the organelle is heterogeneous in terms of Ca2+ handling, the trans-Golgi being insensitive to IP3 and capable of accumulating Ca2+ solely through the activity of SPCA1. The SERCA and the IP3 receptor appear to be restricted to the cis- and intermediate GA compartments. Moreover, selective reduction of Ca2+ concentration within the trans-Golgi, obtained by reducing the level of SPCA1 by RNAi, results in major alterations of protein trafficking within the secretory pathway and induces the collapse of the entire GA morphology.
Golgi apparatus; calcium; FRET; SPCA
The sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) is a key intracellular calcium transporter, which regulates cellular calcium concentration [Ca2+] by transporting Ca2+ ions from the cytosol into the endoplasmic reticulum. SERCA-mediated Ca2+ sequestration controls proper folding of newly synthesized proteins within the ER as well as the timing and spatial patterning of depolarization-evoked Ca2+ responses in the cytoplasm. We studied expression and distribution of all three SERCA isoforms in the mouse retina using isoform-specific antibodies. No immunostaining was observed with the SERCA1 antibody. SERCA2 was expressed in photoreceptor inner segments, amacrine and ganglion cells of the mouse retina. Similar SERCA2 localization was observed in adult rat, macaque and ground squirrel retinas. Analysis of distribution of SERCA2 immunofluorescence in the developing mouse retina revealed prominent SERCA2 signals throughout postnatal development. The antibody raised against the SERCA3 isoform labeled inner segments of photoreceptors and cell bodies in the inner nuclear layer of the mouse retina. The SERCA3 signal was detected in the inner plexiform layer of the early postnatal retina, but moved by P10 to the outer retina where it was concentrated in outer segments of cones. These results indicate that SERCA2 represents the dominant SERCA isoform in the mammalian retina. SERCA3 may contribute to calcium regulation in photoreceptors and bipolar cells.
calcium store; photoreceptor; endoplasmic reticulum; mouse; squirrel; rat
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca2+ accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.
SERCA1; endoplasmic reticulum; calcium; apoptosis; splice variants
Two screening protocols based on recursive partitioning and computational ligand docking methodologies, respectively, were employed for virtual screens of a compound library with 345,000 entries for novel inhibitors of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA), a potential target for cancer chemotherapy. A total of 72 compounds that were predicted to be potential inhibitors of SERCA were tested in bioassays and 17 displayed inhibitory potencies at concentrations below 100 µM. The majority of these inhibitors were composed of two phenyl rings tethered to each other by a short link of one to three atoms. Putative interactions between SERCA and the inhibitors were identified by inspection of docking-predicted poses and some of the structural features required for effective SERCA inhibition were determined by analysis of the classification pattern employed by the recursive partitioning models.
hydroquinones; virtual screening; recursive partitioning; ligand docking; calcium pump; enzyme inhibition; structure-activity relationship
In pulmonary arterial smooth muscle, Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) may induce constriction and dilation in a manner that is not mutually exclusive. We show here that the targeting of different sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and RyR subtypes to discrete SR regions explains this paradox. Western blots identified protein bands for SERCA2a and SERCA2b, whereas immunofluorescence labeling of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA2a and SERCA2b and RyR1, RyR2, and RyR3, respectively. Almost all SERCA2a and RyR3 labeling was restricted to a region within 1.5 μm of the nucleus. In marked contrast, SERCA2b labeling was primarily found within 1.5 μm of the plasma membrane, where labeling for RyR1 was maximal. The majority of labeling for RyR2 lay in between these two regions of the cell. Application of the vasoconstrictor endothelin-1 induced global Ca2+ waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca2+ stores by preincubation of cells with the SERCA inhibitor thapsigargin but remained unaffected after preincubation of cells with a second SERCA antagonist, cyclopiazonic acid. We conclude that functionally segregated SR Ca2+ stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca2+ via SERCA2b, and likely releases Ca2+ via RyR1 to mediate vasodilation. The other is located centrally, receives Ca2+ via SERCA2a, and likely releases Ca2+ via RyR3 and RyR2 to initiate vasoconstriction.
Calcium; Calcium ATPase; Calcium Intracellular Release; Ryanodine; Smooth Muscle
Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na+/K+ transporters, H+/K+ transporters, and plasma membrane Ca2+ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca2+ into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.
calreticulin; calcium ATPases; endoplasmic reticulum; calcium oscillations; glycoprotein folding
This review lays out the emerging evidence for the fundamental role of Ca2+ stores and store-operated channels in the Ca2+ homeostasis of rods and cones. Calcium-induced calcium release (CICR) is a major contributor to steady-state and light-evoked photoreceptor Ca2+ homeostasis in the darkness whereas store-operated Ca2+ channels play a more significant role under sustained illumination conditions. The homeostatic response includes dynamic interactions between the plasma membrane, endoplasmic reticulum (ER), mitochondria and/or outer segment disk organelles which dynamically sequester, accumulate and release Ca2+. Coordinated activation of SERCA transporters, ryanodine receptors (RyR), inositol triphosphate receptors (IP3Rs) and TRPC channels amplifies cytosolic voltage-operated signals but also provides a memory trace of previous exposures to light. Store-operated channels, activated by the STIM1 sensor, prevent pathological decrease in [Ca2+]i mediated by excessive activation of PMCA transporters in saturating light. CICR and SOCE may also modulate the transmission of afferent and efferent signals in the outer retina. Thus, Ca2+ stores provide additional complexity, adaptability, tuneability and speed to photoreceptor signaling.
Calcium store; Light; Photoreceptor; Retina; Ryanodine receptor