The sarcoplasmic reticulum Ca(2+)-ATPase, SERCA1a, contains endoplasmic reticulum targeting information.

The fast-twitch skeletal muscle Ca(2+)-ATPase isoenzyme, SERCA1a, is localized in chick skeletal myotubes to both the sarcoplasmic reticulum (SR) and to the nuclear envelope, an extension of the endoplasmic reticulum (ER). The ER labeling remained after cycloheximide treatment, indicating that it did not represent newly synthesized SERCA1a in transit to the SR. Expression of the cDNA encoding SERCA1a in cultured non-muscle cells led to the localization of the enzyme in the ER, as indicated by organelle morphology and the co-localization of SERCA1a with the endogenous ER luminal protein, BiP. Immunopurification analysis showed that SERCA1a was not bound to BiP, nor was any degradation apparent. Thus, the SR Ca(2+)-ATPase appears to contain ER targeting information.     

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca(2+)-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped viruses indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.     

Sarcalumenin plays a critical role in age-related cardiac dysfunction due to decreases in SERCA2a expression and activity

Impaired Ca(2+) reuptake into the sarcoplasmic reticulum (SR) underlies a primary pathogenesis of heart failure in the aging heart. Sarcalumenin (SAR), a Ca(2+)-binding glycoprotein located in the longitudinal SR, regulates Ca(2+) reuptake by interacting with SR Ca(2+)-ATPase (SERCA). Here we found that the expression levels of both SAR and SERCA2 proteins were significantly downregulated in senescent wild-type mice (18-month old) and that downregulation of SAR protein preceded downregulation of SERCA2 protein. The downregulation of SERCA2 protein was greater in senescent SARKO mice than in age-matched senescent wild-type mice, which was at least in part due to progressive degradation of SERCA2 protein in SARKO mice. Senescent SARKO mice exhibited typical findings of heart failure such as increased sympathetic activity, impaired exercise tolerance, and upregulation of biomarkers of cardiac stress. Consequently, cardiac function was progressively decreased in senescent SARKO. We also found that the expression levels of endoplasmic reticulum (ER) stress-related genes such as x-box binding protein 1 (XBP1) were significantly increased in senescent SARKO mice, indicating that senescent SARKO mice exhibited ER stress. Thus we uncovered the important role of SAR in maintaining Ca(2+) transport activity of SERCA2a and cardiac function in the senescent population.     

Calcium homoeostasis modulator 1 (CALHM1) reduces the calcium content of the endoplasmic reticulum (ER) and triggers ER stress

CALHM1 (calcium homoeostasis modulator 1), a membrane protein with similarity to NMDA (N-methyl-D-aspartate) receptor channels that localizes in the plasma membrane and the ER (endoplasmic reticulum) of neurons, has been shown to generate a plasma-membrane Ca(2+) conductance and has been proposed to influence Alzheimer's disease risk. In the present study we have investigated the effects of CALHM1 on intracellular Ca(2+) handling in HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] cells by using targeted aequorins for selective monitorization of Ca(2+) transport by organelles. We find that CALHM1 increases Ca(2+) leak from the ER and, more importantly, reduces ER Ca(2+) uptake by decreasing both the transport capacity and the Ca(2+) affinity of SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase). As a result, the Ca(2+) content of the ER is drastically decreased. This reduction in the Ca(2+) content of the ER triggered the UPR (unfolded protein response) with induction of several ER stress markers, such as CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein], ERdj4, GRP78 (glucose-regulated protein of 78 kDa) and XBP1 (X-box-binding protein 1). Thus CALHM1 might provide a relevant link between Ca(2+) homoeostasis disruption, ER stress and cell damage in the pathogenesis of neurodegenerative diseases.     

Overexpression of calreticulin sensitizes SERCA2a to oxidative stress

Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.     

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.     

Cardiomyocyte-specific disruption of Serca2 in adult mice causes sarco(endo)plasmic reticulum stress and apoptosis

Reduced sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA2) contributes to the impaired cardiomyocyte Ca(2+) homeostasis observed in heart failure. We hypothesized that a reduction in SERCA2 also elicits myocardial ER/SR stress responses, including unfolded protein responses (UPR) and cardiomyocyte apoptosis, which may additionally contribute to the pathophysiology of this condition. Left ventricular myocardium from mice with cardiomyocyte-specific tamoxifen-inducible disruption of Serca2 (SERCA2 KO) was compared with aged-matched controls. In SERCA2 KO hearts, SERCA2 protein levels were markedly reduced to 2% of control values at 7 weeks following tamoxifen treatment. Serca2 disruption caused increased abundance of the ER stress-associated proteins CRT, GRP78, PERK, and eIF2α and increased phosphorylation of PERK and eIF2α, indicating UPR induction. Pro-apoptotic signaling was also activated in SERCA2 KO, as the abundance of CHOP, caspase 12, and Bax was increased. Indeed, TUNEL staining revealed an increased fraction of cardiomyocytes undergoing apoptosis in SERCA2 KO. ER-Tracker staining additionally revealed altered ER structure. These findings indicate that reduction in SERCA2 protein abundance is associated with marked ER/SR stress in cardiomyocytes, which induces UPR, apoptosis, and ER/SR structural alterations. This suggests that reduced SERCA2 abundance or function may contribute to the phenotype of heart failure also through induction of ER/SR stress responses.     

Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins

This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (-/-) mice as well as dramatic alterations in intracellular localization of key components of the Ca(2+) homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sarcoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (-/-) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (-/-) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (-/-) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca(2+) homeostasis compartment of the ER.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Cytosolic phosphorylation of calnexin controls intracellular Ca(2+) oscillations via an interaction with SERCA2b

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 Ca(2+) imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca(2+) 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 Ca(2+) 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 Ca(2+) signaling and controlling Ca(2+)-sensitive chaperone functions in the ER.     

Overexpression of calsequestrin in L6 myoblasts: formation of endoplasmic reticulum subdomains and their evolution into discrete vacuoles where aggregates of the protein are specifically accumulated.

Calsequestrin (CSQ), the major low-affinity Ca(2+)-binding glycoprotein of striated muscle fibers, is concentrated to yield aggregates that occupy the lumen of the terminal cisternae of the sarcoplasmic reticulum (SR). When infected or transfected into L6 myoblast, the protein is also concentrated, however, in dense vacuoles apparently separate from the endoplasmic reticulum (ER). CSQ-rich cells appear otherwise normal; in particular, neither other proteins involved in Ca2+ homeostasis nor ER chaperones are increased. The CSQ dense vacuoles are shown herein to be specialized ER subdomains as demonstrated by 1) the endoglycosidase H sensitivity of their CSQ and 2) two markers, calreticulin and calnexin (but not others, protein disulfide isomerase and BiP), intermixed with the vacuole content. Their formation is shown to start with the aggregation of CSQ at discrete sites of the ER lumen. When cells were transfected with both CSQ and calreticulin, only the first gave rise to vacuoles; the second remained diffusely distributed within the ER lumen. The possibility that CSQ aggregation is an artifact of overexpression appears unlikely because 1) within dense vacuoles CSQ molecules are not disulfide cross-linked, 2) their turnover is relatively slow (t = 12 h), and 3) segregated CSQ is bound to large amounts of Ca2+. Transfection of a tagged CSQ into cells already overexpressing the protein revealed the continuous import of the newly synthesized protein into preassembled vacuoles. The tendency to aggregation appears, therefore, as a property contributing to the segregation of CSQ within the ER lumen and to its accumulation within specialized subdomains. The study of L6 cells expressing CSQ-rich vacuoles might thus ultimately help to unravel mechanisms by which the complexity of the sarcoplasmic reticulum is established in muscle fibers.     

Insulin receptor substrate 1 regulation of sarco-endoplasmic reticulum calcium ATPase 3 in insulin-secreting beta-cells

We have previously characterized an insulin receptor substrate 1 (IRS-1)-overexpressing beta-cell line. These beta-cells demonstrated elevated fractional insulin secretion and elevated cytosolic Ca(2+) levels compared with wild-type and vector controls. This effect of IRS-1 may be mediated via an interaction with the sarco-endoplasmic reticulum calcium ATPase (SERCA). Here we demonstrate that IRS-1 and IRS-2 localize to an endoplasmic reticulum (ER)-enriched fraction in beta-cells using subcellular fractionation. We also observe co-localization of both IRS-1 and IRS-2 with ER marker proteins using immunofluorescent confocal microscopy. Furthermore, immuno-electron microscopy studies confirm that IRS-1 and SERCA3b localize to vesicles derived from the ER. In Chinese hamster ovary-T (CHO-T) cells transiently transfected with SERCA3b alone or together with IRS-1, SERCA3b co-immunoprecipitates with IRS-1. This interaction is enhanced with insulin treatment. SERCA3b also co-immunoprecipitates with IRS-1 in wild-type and IRS-1-overexpressing beta-cell lines. Ca(2+) uptake in ER-enriched fractions prepared from wild-type and IRS-1-overexpressing cell lines shows no significant difference, indicating that the previously observed decrease in Ca(2+) uptake by IRS-1-overexpressing cells is not the result of a defect in SERCA. Treatment of wild-type beta-cells with thapsigargin, an inhibitor of SERCA, resulted in an increase in glucose-stimulated fractional insulin secretion similar to that observed in IRS-1-overexpressing cells. The colocalization of IRS proteins and SERCA in the ER of beta-cells increases the likelihood that these proteins can interact with one another. Co-immunoprecipitation of IRS-1 and SERCA in CHO-T cells and beta-cells confirms that these proteins do indeed interact directly. Pharmacological inhibition of SERCA in beta-cells results in enhanced secretion of insulin. Taken together, our data suggest that interaction between IRS proteins and SERCA is an important regulatory step in insulin secretion.     

Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.     

Sarco/endoplasmic reticulum Ca2+ATPase type 3 isoforms (SERCA3b and SERCA3f): distinct roles in cell adhesion and ER stress

Sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs) pump free Ca(2+) from the cytosol into the endoplasmic reticulum. The human SERCA3 family counts six members named SERCA3a to 3f. However, the exact role of these different isoforms in cellular physiology remains undetermined. In this study, we compared some physiological consequences of SERCA3b and SERCA3f overexpression in HEK-293 cells. We observed that overexpression of SERCA3b affected cell adhesion capacity associated with a major disorganization of F-actin and a decrease in focal adhesion. Furthermore, we found that SERCA3f overexpression resulted in an increase in endoplasmic reticulum stress markers (including processing of X-box-binding protein-1 (XBP-1) mRNA and expression of chaperone glucose-regulated protein 78 (GRP78)). This was associated with the activation of caspase cascade and a higher spontaneous cell death. In conclusion, these data point for the first time to distinct physiological roles of SERCA3 isoforms in cell functions.     

Modulation of STIM1 and capacitative Ca2+ entry by the endoplasmic reticulum luminal oxidoreductase ERp57

STIM1 is an endoplasmic reticulum (ER) membrane Ca(2+) sensor responsible for activation of store-operated Ca(2+) influx. We discovered that STIM1 oligomerization and store-operated Ca(2+) entry (SOC) are modulated by the ER oxidoreductase ERp57. ERp57 interacts with the ER luminal domain of STIM1, with this interaction involving two conserved cysteine residues, C(49) and C(56). SOC is accelerated in the absence of ERp57 and inhibited in C(49) and C(56) mutants of STIM1. We show that ERp57, by ER luminal interaction with STIM1, has a modulatory role in capacitative Ca(2+) entry. This is the first demonstration of a protein involved in ER intraluminal regulation of STIM1.     

Effects of PMCA and SERCA pump overexpression on the kinetics of cell Ca(2+) signalling

The dynamic interactions of the main pathways for active Ca(2+) transport have been analysed in living cells by altering the expression of their components. The plasma membrane (PMCA) and the endoplasmic reticulum (ER) (SERCA) Ca(2+) pumps were transiently overexpressed in CHO cells, and the Ca(2+) homeostasis in the subcellular compartments was investigated using specifically targeted chimaeras of the Ca(2+)- sensitive photoprotein aequorin. In resting cells, overexpression of the PMCA and SERCA pumps caused a reduction and an increase in ER [Ca(2+)] levels, respectively, while no significant differences were detected in cytosolic and mitochondrial [Ca(2+)]. Upon stimulation with an inositol 1,4, 5-trisphosphate (IP(3))-generating agonist, the amplitude of the mitochondrial and cytosolic Ca(2+) rises correlated with the ER [Ca(2+)] only up to a threshold value, above which the feedback inhibition of the IP(3) channel by Ca(2+) appeared to be limiting.