Ca2+ signaling differentiation during oocyte maturation

Oocyte maturation is an essential cellular differentiation pathway that prepares the egg for activation at fertilization leading to the initiation of embryogenesis. An integral attribute of oocyte maturation is the remodeling of Ca2+ signaling pathways endowing the egg with the capacity to produce a specialized Ca2+ transient at fertilization that is necessary and sufficient for egg activation. Consequently, mechanistic elucidation of Ca2+ signaling differentiation during oocyte maturation is fundamental to our understanding of egg activation, and offers a glimpse into Ca2+ signaling regulation during the cell cycle.     

Modeling Ca2+ signaling differentiation during oocyte maturation

Ca²⁺ is a fundamental intracellular signal that mediates a variety of disparate physiological functions often in the same cell. Ca²⁺ signals span a wide range of spatial and temporal scales, which endow them with the specificity required to induce defined cellular functions. Furthermore, Ca²⁺ signaling is highly plastic as it is modulated dynamically during normal physiological development and under pathological conditions. However, the molecular mechanisms underlying Ca²⁺ signaling differentiation during cellular development remain poorly understood. Oocyte maturation in preparation for fertilization provides an exceptionally well-suited model to elucidate Ca²⁺ signaling regulation during cellular development. This is because a Ca²⁺ signal with specialized spatial and temporal dynamics is universally essential for egg activation at fertilization. Here we use mathematical modeling to define the critical determinants of Ca²⁺ signaling differentiation during oocyte maturation. We show that increasing IP₃ receptor (IP₃R) affinity replicates both elementary and global Ca²⁺ dynamics observed experimentally following oocyte maturation. Furthermore, our model reveals that because of the Ca²⁺ dependency of both SERCA and the IP₃R, increased IP₃R affinity shifts the system's equilibrium to a new steady state of high cytosolic Ca²⁺, which is essential for fertilization. Therefore our model provides unique insights into how relatively small alterations of the basic molecular mechanisms of Ca²⁺ signaling components can lead to dramatic alterations in the spatio-temporal properties of Ca²⁺ dynamics.     

Modeling Ca2+ signaling differentiation during oocyte maturation

Ca2+ is a fundamental intracellular signal that mediates a variety of disparate physiological functions often in the same cell. Ca2+ signals span a wide range of spatial and temporal scales, which endow them with the specificity required to induce defined cellular functions. Furthermore, Ca2+ signaling is highly plastic as it is modulated dynamically during normal physiological development and under pathological conditions. However, the molecular mechanisms underlying Ca2+ signaling differentiation during cellular development remain poorly understood. Oocyte maturation in preparation for fertilization provides an exceptionally well-suited model to elucidate Ca2+ signaling regulation during cellular development. This is because a Ca2+ signal with specialized spatial and temporal dynamics is universally essential for egg activation at fertilization. Here we use mathematical modeling to define the critical determinants of Ca2+ signaling differentiation during oocyte maturation. We show that increasing IP3 receptor (IP3R) affinity replicates both elementary and global Ca2+ dynamics observed experimentally following oocyte maturation. Furthermore, our model reveals that because of the Ca2+ dependency of both SERCA and the IP3R, increased IP3R affinity shifts the system's equilibrium to a new steady state of high cytosolic Ca2+, which is essential for fertilization. Therefore our model provides unique insights into how relatively small alterations of the basic molecular mechanisms of Ca2+ signaling components can lead to dramatic alterations in the spatio-temporal properties of Ca2+ dynamics.     

Chromosomal rearrangements during human epidermal keratinocyte differentiation

Undifferentiated human epidermal keratinocytes are self‐renewing stem cells that can be induced to undergo a program of differentiation by varying the calcium chloride concentration in the culture media. We utilize this model of cell differentiation and a 3D chromosome painting technique to document significant changes in the radial arrangement, morphology, and interchromosomal associations between the gene poor chromosome 18 and the gene rich chromosome 19 territories at discrete stages during keratinocyte differentiation. We suggest that changes observed in chromosomal territorial organization provides an architectural basis for genomic function during cell differentiation and provide further support for a chromosome territory code that contributes to gene expression at the global level. J. Cell. Physiol. J. Cell. Physiol. 221: 139–146, 2009. © 2009 Wiley‐Liss, Inc.     

Multiple Ca2+ signaling pathways regulate intracellular Ca2+ activity in human cardiac fibroblasts

Ca²⁺ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca²⁺ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT‐PCR techniques. It was found that spontaneous intracellular Ca²⁺ (Ca) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry. Ca oscillations were abolished by the store‐operated Ca²⁺ (SOC) entry channel blocker La³⁺, the phospholipase C inhibitor U‐73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2‐aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca oscillations. Inhibition of plasma membrane Ca²⁺ pump (PMCA) and Na⁺–Ca²⁺ exchanger (NCX) also suppressed Ca oscillations. In addition, the frequency of Ca oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca²⁺ oscillations. RT‐PCR revealed that mRNAs for IP3R1‐3, SERCA1‐3, Ca_V1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1‐3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca²⁺ pathways, which are not identical to those of the well‐studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca²⁺ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68–75, 2010. © 2009 Wiley‐Liss, Inc.     

E2F modulates keratinocyte squamous differentiation: implications for E2F inhibition in squamous cell carcinoma

E2F regulation is essential for normal cell cycle progression. Therefore, it is not surprising that squamous cell carcinoma cell lines (SCC) overexpress E2F1 and exhibit deregulated E2F activity when compared with normal keratinocytes. Indeed, deliberate E2F1 deregulation has been shown to induce hyperplasia and skin tumor formation. In this study, we report on a dual role for E2F as a mediator of keratinocyte proliferation and modulator of squamous differentiation. Overexpression of E2F isoforms in confluent primary keratinocyte cultures resulted in suppression of differentiation-associated markers. Moreover, we found that the DNA binding domain and the trans-activation domain of E2F1 are important in mediating suppression of differentiation. Use of a dominant/negative form of E2F1 (E2F d/n) found that E2F inhibition alone is sufficient to suppress the activity of proliferation-associated markers but is not capable of inducing differentiation markers. However, if the E2F d/n is expressed in differentiated keratinocytes, differentiation marker activity is further induced, suggesting that E2F may act as a modulator of squamous differentiation. We therefore examined the effects of E2F d/n in a differentiation-insensitive SCC cell line. We found that treatment with the differentiating agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or expression of E2F d/n alone had no effect on differentiation markers. However, a combination of E2F d/n + TPA induced the expression of differentiation markers. Combined, these data indicate that E2F may play a key role in keratinocyte differentiation. These data also illustrate the unique potential of anti-E2F therapies in arresting proliferation and inducing differentiation of SCCs.     

Antagonistic effects of TGFbeta1 and BMP-6 on skin keratinocyte differentiation

Several members of the transforming growth factor beta (TGFbeta) superfamily are expressed in the developing murine epidermis. Among these are TGFbeta1, which is found in the basal layer, and bone morphogenetic protein (BMP)-6, located in the suprabasal layers. Although the role of TGFbeta in cell growth has been studied extensively, little is known about the effects of these factors on keratinocyte differentiation. This study demonstrates that BMP-6 acts to positively regulate the differentiation of primary skin keratinocytes grown in culture. In contrast, TGFbeta1 antagonizes keratinocyte differentiation blocking the upregulation of keratin markers by BMP-6. We show that the effects of BMP-6 on expression of keratin 1 (K1), a marker of differentiation, requires signaling through the Smad pathway. In addition, regulation of K1 levels by BMP-6 is modulated by the SEK signaling pathway. This suggests that regulation of keratinocyte differentiation by BMP-6 involves multiple signaling systems.     

Chelation of intracellular Ca2+ inhibits murine keratinocyte differentiation in vitro

The role of intracellular Ca²⁺ in the regulation of Ca²⁺-induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca²⁺ chelator 1,2-bis(o-aminophenoxy)-ethane-N, N, N′, N′-tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca²⁺ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca²⁺. Intracellular BAPTA loaded by BAPTA/AM (15–30 μM) inhibited the expression of epidermal differentiation-specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation-associated redistribution of E-cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and Ioricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca²⁺, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca²⁺, the concentration of intracellular free Ca²⁺ (Ca_i) in BAPTA-loaded keratinocytes was examined by digital image analysis using the Ca²⁺-sensitive fluorescent probe fura-2, and Ca²⁺ influx was measured by ⁴⁵Ca²⁺ uptake studies. Increase in extracellular Ca²⁺ (Ca_o) in the culture medium of keratinocytes caused a sustained increase in both Ca_i and Ca²⁺ localized to ionomycin-sensitive intracellular stores in keratinocytes. BAPTA lowered basal Ca_i concentration and prevented the Ca_i increase. After 12 hours of BAPTA treatment, the basal level of Ca_i returned to the control value, but the Ca²⁺ localized in intracellular stores was substantially decreased. ⁴⁵Ca²⁺ uptake was initially (within 30 min) increased in BAPTA-loaded cells. However, the total ⁴⁵Ca²⁺ accumulation over 24 hours in BAPTA-loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Ca_i and total cellular Ca²⁺ content in the presence of increased amount of intracellular Ca²⁺ buffer (e.g., BAPTA) by depleting intracellular Ca²⁺ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca²⁺-stores since this is the major change in intracellular Ca²⁺ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E-cadherin on the cell membrane may be more directly associated with Ca_i change. © 1995 Wiley-Liss, Inc.     

Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

The novel murine Ca2+-binding protein,Scarf, is differentially expressed during epidermal differentiation

Calcium (Ca2+) signaling-dependent systems, such as the epidermal differentiation process, must effectively respond to variations in Ca2+ concentration. Members of the Ca2+-binding proteins play a central function in the transduction of Ca2+ signals, exerting their roles through a Ca2+-dependent interaction with their target proteins, spatially and temporally. By performing a suppression subtractive hybridization screen we identified a novel mouse gene, Scarf (skin calmodulin-related factor), which has homology to calmodulin (CaM)-like Ca2+-binding protein genes and is exclusively expressed in differentiating keratinocytes in the epidermis. The Scarf open reading frame encodes a 148-amino acid protein that contains four conserved EF-hand motifs (predicted to be Ca2+-binding domains) and has homology to mouse CaM, human CaM-like protein, hClp, and human CaM-like skin protein, hClsp. The functionality of Scarf EF-hand domains was assayed with a radioactive Ca2+-binding method. By Southern blot and computational genome sequence analysis, a highly related gene, Scarf2, was found 15 kb downstream of Scarf on mouse chromosome 13. The functional Scarf Ca2+-binding domains suggest a role in the regulation of epidermal differentiation through the control of Ca2+-mediated signaling.     

Structures of Anabaena calcium-binding protein CcbP: insights into Ca2+ signaling during heterocyst differentiation

Ca2+-binding proteins play pivotal roles in both eukaryotic and prokaryotic cells. CcbP from cyanobacterium Anabaena sp. strain PCC 7120 is a major Ca2+-binding protein involved in heterocyst differentiation, a process that forms specialized nitrogen-fixing cells. The three-dimensional structures of both Ca2+-free and Ca2+-bound forms of CcbP are essential for elucidating the Ca2+-signaling mechanism. However, CcbP shares low sequence identity with proteins of known structures, and its Ca2+-binding sites remain unknown. Here, we report the solution structures of CcbP in both Ca2+-free and Ca2+-bound forms determined by nuclear magnetic resonance spectroscopy. CcbP adopts an overall new fold and contains two Ca2+-binding sites with distinct Ca2+-binding abilities. Mutation of Asp38 at the stronger Ca2+-binding site of CcbP abolished its ability to regulate heterocyst formation in vivo. Surprisingly, the β-barrel subdomain of CcbP, which does not participate in Ca2+-binding, topologically resembles the Src homology 3 (SH3) domain and might act as a protein-protein interaction module. Our results provide the structural basis of the unique Ca2+ signaling mechanism during heterocyst differentiation.     

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis

Endoplasmic reticulum (ER)–plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca²⁺ levels has been uncovered in recent years, the functional significance of ER‐PM junctions in non‐excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane‐localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α‐melanocyte‐stimulating hormone (αMSH) depletes ER Ca²⁺ stores, thus recruiting STIM1 to ER‐PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation in vivo. STIM1 domain deletion studies reveal the importance of Ser/Pro‐rich C‐terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH‐cAMP‐MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

The free [Ca2+] in endoplasmic/sarcoplasmic reticulum Ca2+ stores regulates excitability of Ca2+ release by stimulating the Ca2+ release channels. Just how the stored Ca2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca2+-activation to luminal facing regulatory sites, whereas another envisages Ca2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca2+ activation for single cardiac ryanodine receptors (RyR2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca2+ sensors in different parts of the molecule; a luminal activation site (L-site, 60 microM affinity), a cytoplasmic activation site (A-site, 0.9 microM affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2 microM affinity). RyR activation by luminal Ca2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca2+ feedthrough. Ca2+ binding to the L-site initiates brief openings (1 ms duration at 1-10 s(-1)) allowing luminal Ca2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR2 mutations on a range of Ca2+-mediated physiological and pathological processes.     

MAP kinases regulate unfertilized egg apoptosis and fertilization suppresses death via Ca2+ signaling

The default fate for eggs from many species is death by apoptosis and thus, successful fertilization depends upon suppression of the maternal death program. Little is known about the molecular triggers which activate this process or how the fertilization signal suppresses the default maternal apoptotic pathway. The MAP kinase (MAPK) family member, ERK, plays a universal and critical role in several stages of oocyte meiotic maturation, and fertilization results in ERK inactivation. In somatic cells, ERK and other MAPK family members, p38 and JNK, provide opposing signals to regulate apoptosis, however, it is not known whether MAPKs play a regulatory role in egg apoptosis, nor whether suppression of apoptosis by fertilization is mediated by MAPK activity. Here we demonstrate that MAPKs are involved in starfish egg apoptosis and we investigate the relationship between the fertilization induced signaling pathway and MAPK activation. ERK is active in post-meiotic eggs just until apoptosis onset and then p38, JNK and a third kinase are activated, and remain active through execution. Sequential activation of ERK and p38 is necessary for apoptosis, and newly synthesized proteins are required both upstream of ERK and downstream of p38 for activation of the full apoptotic program. Fertilization causes a dramatic rise in intracellular Ca2+, and we report that Ca2+ provides a necessary and sufficient pro-survival signal. The Ca2+ pathway following fertilization of both young and aged eggs causes ERK to be rapidly inactivated, but fertilization cannot rescue aged eggs from death, indicating that ERK inactivation is not sufficient to suppress apoptosis.     

Highly Ca2+-selective TRPM channels regulate IP3-dependent oscillatory Ca2+ signaling in the C. elegans intestine

Posterior body wall muscle contraction (pBoc) in the nematode Caenorhabditis elegans occurs rhythmically every 45-50 s and mediates defecation. pBoc is controlled by inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in the intestine. The intestinal epithelium can be studied by patch clamp electrophysiology, Ca2+ imaging, genome-wide reverse genetic analysis, forward genetics, and molecular biology and thus provides a powerful model to develop an integrated systems level understanding of a nonexcitable cell oscillatory Ca2+ signaling pathway. Intestinal cells express an outwardly rectifying Ca2+ (ORCa) current with biophysical properties resembling those of TRPM channels. Two TRPM homologues, GON-2 and GTL-1, are expressed in the intestine. Using deletion and severe loss-of-function alleles of the gtl-1 and gon-2 genes, we demonstrate here that GON-2 and GTL-1 are both required for maintaining rhythmic pBoc and intestinal Ca2+ oscillations. Loss of GTL-l and GON-2 function inhibits I(ORCa) approximately 70% and approximately 90%, respectively. I(ORCa) is undetectable in gon-2;gtl-1 double mutant cells. These results demonstrate that (a) both gon-2 and gtl-1 are required for ORCa channel function, and (b) GON-2 and GTL-1 can function independently as ion channels, but that their functions in mediating I(ORCa) are interdependent. I(ORCa), I(GON-2), and I(GTL-1) have nearly identical biophysical properties. Importantly, all three channels are at least 60-fold more permeable to Ca2+ than Na+. Epistasis analysis suggests that GON-2 and GTL-1 function in the IP3 signaling pathway to regulate intestinal Ca2+ oscillations. We postulate that GON-2 and GTL-1 form heteromeric ORCa channels that mediate selective Ca2+ influx and function to regulate IP3 receptor activity and possibly to refill ER Ca2+ stores.     

Subpopulation of store-operated Ca2+ channels regulate Ca2+-induced Ca2+ release in non-excitable cells

Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+ -free medium with 0.5 microm carbachol releases approximately 5% of the Ca2+ mobilizable by 1 mm carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 microm cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP(3)Rs because it was reduced by only approximately 30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+ -activated outward current and [Ca2+](i) suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365 or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in cardiac cells Ca2+ influx is mediated by voltage-regulated Ca2+ channels whereas in non-excitable cells Ca2+ influx is mediated by store-operated channels.     

Prostaglandins F2alpha and E content and uptake of Ca2+ in the myometrium during different functional states

It is shown that the amount of prostaglandins F2alpha and E in myometrium of female rabbits and a woman decreases in the process of pregnancy and increases during delivery as compared to the control. The 10(-6)M concentration of prostaglandin F2alpha evokes an intensive Ca2+ uptake by myometrium strips both in normal and in pregnant animals but has no effect on the Mg2+, Ca2+-ATPase activity of sarcolemma vesicles. The Ca2+ uptake by the myometrium strips is not affected by prostaglandin F2alpha in the presence of NaF and N-ethylmaleimide inhibiting the ATP-dependent transport of Ca2+.