Loss of the Atp2c1 secretory pathway Ca(2+)-ATPase (SPCA1) in mice causes Golgi stress, apoptosis, and midgestational death in homozygous embryos and squamous cell tumors in adult heterozygotes

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.     

Similar Ca(2+)-signaling properties in keratinocytes and in COS-1 cells overexpressing the secretory-pathway Ca(2+)-ATPase SPCA1

Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.     

The Ca2+/Mn2+ pumps in the Golgi apparatus

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed.     

SPCA1 pumps and Hailey-Hailey disease

Both the endoplasmic reticulum and the Golgi apparatus are agonist-sensitive intracellular Ca2+ stores. The Golgi apparatus has Ca2+-release channels and a Ca2+-uptake mechanism consisting of sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and secretory-pathway Ca2+-ATPases (SPCA). SPCA1 has been shown to transport both Ca2+ and Mn2+ in the Golgi lumen and therefore plays an important role in the cytosolic and intra-Golgi Ca2+ and Mn2+ homeostasis. Human genetic studies have provided new information on the physiological role of SPCA1. Loss of one functional copy of the SPCA1 (ATP2C1) gene causes Hailey-Hailey disease, a skin disorder arising in the adult age with recurrent vesicles and erosions in the flexural areas. Here, we review recent experimental evidence showing that the Golgi apparatus plays a much more important role in intracellular ion homeostasis than previously anticipated.     

Squamous cell tumors in mice heterozygous for a null allele of Atp2a2, encoding the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump

Mutations in the human ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), cause Darier disease, an autosomal dominant skin disease characterized by multiple keratotic papules in the seborrheic regions of the body. Mice with a single functional Atp2a2 allele (the mouse homolog of ATP2A2) were shown previously to have reduced levels of SERCA2 in heart and mildly impaired cardiac contractility and relaxation. Here we show that aged heterozygous mutant (Atp2a2(+/-)) mice develop squamous cell tumors of the forestomach, esophagus, oral mucosa, tongue, and skin. Squamous cell tumors occurred in 13/14 Atp2a2(+/-) mice but were not observed in age- and sex-matched wild-type controls. Hyperkeratinized squamous cell papillomas and carcinomas of the upper digestive tract were the most frequent finding among Atp2a2(+/-) mice, and many animals had multiple tumors. Western blot analyses showed that SERCA2 protein levels were reduced in skin and other affected tissues of heterozygous mice. The development of squamous cell tumors in aged Atp2a2(+/-) mice indicates that SERCA2 haploinsufficiency predisposes murine keratinocytes to neoplasia. These findings provide the first direct demonstration that a perturbation of Ca(2+) homeostasis or signaling can serve as a primary initiating event in cancer.     

Store-independent activation of Orai1 by SPCA2 in mammary tumors

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.     

Functional comparison between secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 isoforms by steady-state and transient kinetic analyses

Steady-state and transient kinetic studies were performed to functionally analyze the overall and partial reactions of the Ca(2+) transport cycle of the human secretory pathway Ca(2+)/Mn(2+)-ATPase 1 (SPCA1) isoforms: SPCA1a, SPCA1b, SPCA1c, and SPCA1d (encoded by ATP2C1, the gene defective in Hailey-Hailey disease) upon heterologous expression in mammalian cells. The expression levels of SPCA1 isoforms were 200-350-fold higher than in control cells except for SPCA1c, whose low expression level appears to be the effect of rapid degradation because of protein misfolding. Relative to SERCA1a, the active SPCA1a, SPCA1b, and SPCA1d enzymes displayed extremely high apparent affinities for cytosolic Ca(2+) in activation of the overall ATPase and phosphorylation activities. The maximal turnover rates of the ATPase activity for SPCA1 isoforms were 4.7-6.4-fold lower than that of SERCA1a (lowest for the shortest SPCA1a isoform). The kinetic analysis traced these differences to a decreased rate of the E(1) approximately P(Ca) to E(2)-P transition. The apparent affinity for inorganic phosphate was reduced in the SPCA1 enzymes. This could be accounted for by an enhanced rate of the E(2)-P hydrolysis, which showed constitutive activation, lacking the SERCA1a-specific dependence on pH and K(+).     

Bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)-methane (bis-phenol) is a potent and selective inhibitor of the secretory pathway Ca(2+) ATPase (SPCA1)

The secretory pathway Ca(2+) ATPase (SPCA) provides the Golgi apparatus with a Ca(2+) supply essential for Ca(2+)-dependent enzymes involved in the post-translational modification of proteins in transit through the secretory pathway. Ca(2+) in the Golgi apparatus is also agonist-releasable and plays a role in hormone-induced Ca(2+) transients. Although the Ca(2+) ATPase inhibitors thapsigargin is more selective for the sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA) than for SPCA, no inhibitor has been characterised that selectively inhibits SPCA. A number of inhibitors were assessed for their selectivity to the human SPCA1d compared to the more ubiquitous human SERCA2b. Each isoform was over-expressed in COS-7 cells and the Ca(2+)-dependent ATPase activity measured in their microsomal membranes. Both bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane(bis-phenol) and 2-aminoethoxydiphenylborate (2-APB) selectively inhibited SPCA1d (with IC(50) values of 0.13μM and 0.18mM, respectively), which were of 62- and 8.3-fold greater potency than the values for hSERCA2b (IC(50) values; 8.1μM and 1.5mM, respectively). Other inhibitors tested such as bis-phenol-A, tetrabromobisphenol-A and trifluoperazine inhibited both Ca(2+) ATPases similarly. Furthermore, bis-phenol was able to mobilize Ca(2+) in cells that had been pre-treated with thapsigargin. Therefore we conclude that given the potency and selectivity of bis-phenol it may prove a valuable tool in further understanding the role of SPCA in cellular processes.     

Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases

The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca(2+) ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Targeted disruption of the ATP2A1 gene encoding the sarco(endo)plasmic reticulum Ca2+ ATPase isoform 1 (SERCA1) impairs diaphragm function and is lethal in neonatal mice

Mutations in the ATP2A1 gene, encoding isoform 1 of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1), are one cause of Brody disease, characterized in humans by exercise-induced contraction of fast twitch (type II) skeletal muscle fibers. In an attempt to create a model for Brody disease, the mouse ATP2A1 gene was targeted to generate a SERCA1-null mutant mouse line. In contrast to humans, term SERCA1-null mice had progressive cyanosis and gasping respiration and succumbed from respiratory failure shortly after birth. The percentage of affected homozygote SERCA1(-/-) mice was consistent with predicted Mendelian inheritance. A survey of multiple organs from 10-, 15-, and 18-day embryos revealed no morphological abnormalities, but analysis of the lungs in term mice revealed diffuse congestion and epithelial hypercellularity and studies of the diaphragm muscle revealed prominent hypercontracted regions in scattered fibers and increased fiber size variability. The V(max) of Ca(2+) transport activity in mutant diaphragm and skeletal muscle was reduced by 80% compared with wild-type muscle, and the contractile response to electrical stimulation under physiological conditions was reduced dramatically in mutant diaphragm muscle. No compensatory responses were detected in analysis of mRNAs encoding other Ca(2+) handling proteins or of protein levels. Expression of ATP2A1 is largely restricted to type II fibers, which predominate in normal mouse diaphragm. The absence of SERCA1 in type II fibers, and the absence of compensatory increases in other Ca(2+) handling proteins, coupled with the marked increase in contractile function required of the diaphragm muscle to support postnatal respiration, can account for respiratory failure in term SERCA1-null mice.     

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.     

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.     

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.     

Calcium in the Golgi apparatus

The secretory-pathway Ca2+-ATPases (SPCAs) represent a recently recognized family of phosphorylation-type ATPases that supply the lumen of the Golgi apparatus with Ca2+ and Mn2+ needed for the normal functioning of this structure. Mutations of the human SPCA1 gene (ATP2C1) cause Hailey-Hailey disease, an autosomal dominant skin disorder in which keratinocytes in the suprabasal layer of the epidermis detach. We will first review the physiology of the SPCAs and then discuss how mutated SPCA1 proteins can lead to an epidermal disorder.     

Kinetic characterization of calcium uptake by the rat liver Golgi apparatus

We carried out a kinetic characterization of the Ca(2+)active transport in the rat liver Golgi Apparatus (GA) membrane. Calcium accumulation by vesicles of a GA enriched fraction was found to be a function of both Ca(2+)and ATP-Mg concentrations, it was inhibited by 2 microm thapsigargin but not stimulated by 3 microm calmodulin. The kinetic parameter values obtained for the GA Ca(2+)pump were: J(max)of 3.96 nmol/mg min, K(m)for Ca(2+)of 0.150 microm and two K(m)'s for ATP of 1.14 microm and 519 microm. These results were almost identical to those obtained for the endoplasmic reticulum (ER) fraction, indicating that the GA Ca(2+)pump is a sarco/endoplasmic reticulum (SERCA) P-type, analogous-if not identical-to that present in the ER.     

ER‐to‐Golgi blockade of nascent desmosomal cadherins in SERCA2‐inhibited keratinocytes: Implications for Darier's disease

Darier's disease (DD) is an autosomal dominantly inherited skin disorder caused by mutations in sarco/endoplasmic reticulum Ca²⁺‐ATPase 2 (SERCA2), a Ca²⁺ pump that transports Ca²⁺ from the cytosol to the endoplasmic reticulum (ER). Loss of desmosomes and keratinocyte cohesion is a characteristic feature of DD. Desmosomal cadherins (DC) are Ca²⁺‐dependent transmembrane adhesion proteins of desmosomes, which are mislocalized in the lesional but not perilesional skin of DD. We show here that inhibition of SERCA2 by 2 distinct inhibitors results in accumulation of DC precursors in keratinocytes, indicating ER‐to‐Golgi transport of nascent DC is blocked. Partial loss of SERCA2 by siRNA has no such effect, implicating that haploinsufficiency is not sufficient to affect nascent DC maturation. However, a synergistic effect is revealed between SERCA2 siRNA and an ineffective dose of SERCA2 inhibitor, and between an agonist of the ER Ca²⁺ release channel and SERCA2 inhibitor. These results suggest that reduction of ER Ca²⁺ below a critical level causes ER retention of nascent DC. Moreover, colocalization of DC with ER calnexin is detected in SERCA2‐inhibited keratinocytes and DD epidermis. Collectively, our data demonstrate that loss of SERCA2 impairs ER‐to‐Golgi transport of nascent DC, which may contribute to DD pathogenesis.     

Molecular physiology of the SERCA and SPCA pumps

Intracellular Ca(2+)-transport ATPases exert a pivotal role in the endoplasmic reticulum and in the compartments of the cellular secretory pathway by maintaining a sufficiently high lumenal Ca(2+) (and Mn(2+)) concentration in these compartments required for an impressive number of vastly different cell functions. At the same time this lumenal Ca(2+) represents a store of releasable activator Ca(2+) controlling an equally impressive number of cytosolic functions. This review mainly focuses on the different Ca(2+)-transport ATPases found in the intracellular compartments of mainly animal non-muscle cells: the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. Although it is not our intention to treat the ATPases of the specialized sarcoplasmic reticulum in depth, we can hardly ignore the SERCA1 pump of fast-twitch skeletal muscle since its structure and function is by far the best understood and it can serve as a guide to understand the other members of the family. In a second part of this review we describe the relatively novel family of secretory pathway Ca(2+)/Mn(2+) ATPases (SPCA), which in eukaryotic cells are primarily found in the Golgi compartment.     

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production

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 Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-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 Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) 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 beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.