Calcium pumps of plasma membrane and cell interior

Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco/endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping intracellular Ca2+ signals with spatial and temporal resolution. The loss or malfunction of specific Ca2+ pump isoforms is associated with defects such as deafness, ataxia or heart failure. Understanding the involvement of different Ca2+ pump isoforms in the pathogenesis of disease allows their identification as therapeutic targets for the development of selective strategies to prevent or combat the progression of these disorders.     

Phenotypes of SERCA and PMCA knockout mice

P-type Ca2+-ATPases of the sarco(endo)plasmic reticulum (SERCAs) and plasma membrane (PMCAs) are responsible for maintaining the Ca2+ gradients across cellular membranes that are required for regulation of Ca2+-mediated signaling and other biological processes. Gene-targeting studies of SERCA isoforms 1, 2, and 3 and PMCA isoforms 1, 2, and 4 have confirmed some of the general functions proposed for these pumps, such as a major role in excitation-contraction coupling for SERCA1 and SERCA2 and housekeeping functions for PMCA1 and SERCA2, but have also revealed some unexpected phenotypes. These include squamous cell cancer and plasticity in the regulation of Ca2+-mediated exocytosis in SERCA2 heterozygous mutant mice, modulation of Ca2+ signaling in SERCA3-deficient mice, deafness and balance disorders in PMCA2 null mice, and male infertility in PMCA4 null mice. These unique phenotypes provide new information about the cellular functions of these pumps, the requirement of their activities for higher order physiological processes, and the pathophysiological consequences of pump dysfunction.     

Plasma membrane calcium pumps in smooth muscle: from fictional molecules to novel inhibitors

Plasma membrane Ca2+ pumps (PMCA pumps) are Ca2+-Mg2+ ATPases that expel Ca2+ from the cytosol to extracellular space and are pivotal to cell survival and function. PMCA pumps are encoded by the genes PMCA1, -2, -3, and -4. Alternative splicing results in a large number of isoforms that differ in their kinetics and activation by calmodulin and protein kinases A and C. Expression by 4 genes and a multifactorial regulation provide redundancy to allow for animal survival despite genetic defects. Heterozygous mice with ablation of any of the PMCA genes survive and only the homozygous mice with PMCA1 ablation are embryolethal. Some PMCA isoforms may also be involved in other cell functions. Biochemical and biophysical studies of PMCA pumps have been limited by their low levels of expression. Delineation of the exact physiological roles of PMCA pumps has been difficult since most cells also express sarco/endoplasmic reticulum Ca2+ pumps and a Na+-Ca2+-exchanger, both of which can lower cytosolic Ca2+. A major limitation in the field has been the lack of specific inhibitors of PMCA pumps. More recently, a class of inhibitors named caloxins have emerged, and these may aid in delineating the roles of PMCA pumps.     

Sarco/endoplasmic reticulum Ca2+ (SERCA)-pumps: link to heart beats and calcium waves.

Mobilization of endoplasmic reticulum Ca2+ is pivotal to the ability of a cell to send or respond to stimuli. Ca(2+)-Mg(2+)-ATPases, termed SERCA pumps, sequester Ca2+ into the sarco/endoplasmic reticulum. There are several SERCA protein isoforms encoded by three genes. This paper summarizes the structure, function, tissue and subcellular distribution, and regulation of various SERCA isoforms. Then it attempts to link divergence in the signal transduction processes of cells to the types and levels of SERCA proteins they express and to how the cells regulate their SERCA pump activity. The paper examines possible linkages between SERCA pumps and receptor-activated Ca2+ entry, SERCA isoform localization and Ca(2+)-waves, and the role of SERCA pumps in nuclear Ca2+ in cell proliferation and apoptosis. Then it uses available information on cardiac function and chronic stimulation of the fast-twitch muscle to answer a series of basic questions on the regulation of SERCA activity and expression and their linkage to signal transduction. Finally, it discusses the possibility that neurons exhibit complex Ca(2+)-waves whose interactions have the potential to explain the operational basis of neural networks. A series of unanswered questions emerge based on this synthesis, including the unsettling issue of whether all the isoforms are needed to achieve the divergence in signal transduction or if there is a degree of redundancy in the system.     

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.     

Gene knockout studies of Ca2+-transporting ATPases.

The biochemical functions of intracellular and plasma membrane Ca2+-transporting ATPases in the control of cytosolic and organellar Ca2+ levels are well established, but the physiological roles of specific isoforms are less well understood. There appear to be three different types of Ca2+ pumps in mammalian tissues: the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), which sequester Ca2+ within the endoplasmic or sarcoplasmic reticulum, the plasma membrane Ca2+-ATPases (PMCAs), which extrude Ca2+ from the cell, and the putative secretory pathway Ca2+-ATPase (SPCA), the function of which is poorly understood. This review describes the results of recent analyses of mouse models with null mutations in the genes encoding SERCA and PMCA isoforms and genetic studies of SERCA and SPCA dysfunction in both humans and model organisms. These studies are yielding important insights regarding the physiological functions of individual Ca2+-transporting ATPases in vivo.     

Collaborative effect of SERCA and PMCA in cytosolic calcium homeostasis in human platelets

Intracellular free Ca2+ concentration ([Ca2+]c) is finely regulated by several mechanisms that either increase or reduce [Ca2+]c. Two different Ca2+ pumps have been described so far as the main mechanisms for Ca2+ removal from the cytosol, either by its sequestration into the stores, mediated by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or by Ca2+ extrusion to the extracellular medium, by the plasma membrane Ca2+-ATPase (PMCA). We have used inhibitors of these pumps to analyze their Ca2+ clearance efficacy in human platelets stimulated by the physiological agonist thrombin. Results demonstrate that, after platelet stimulation with thrombin, activation of SERCA precedes that of PMCA, although the ability of PMCA to remove Ca2+ from the cytosol last longer than that of SERCA. The efficacy of SERCA and PMCA removing Ca2+ from the cytosol is reduced when the concentration of thrombin increases. This phenomenon correlates with the greater increase in [Ca2+]c induced by higher concentrations of thrombin, which further confirms that SERCA and PMCA activities are regulated by [Ca2+]c.     

Plasma membrane-cytoskeleton-endoplasmic reticulum complexes in neurons and astrocytes

The possibility that certain integral plasma membrane (PM) proteins involved in Ca(2+) homeostasis form junctional units with adjacent endoplasmic reticulum (ER) in neurons and glia was explored using immunoprecipitation and immunocytochemistry. Rat brain membranes were solubilized with the mild, non-ionic detergent, IGEPAL CA-630. Na(+)/Ca(2+) exchanger type 1 (NCX1), a key PM Ca(2+) transporter, was immunoprecipitated from the detergent-soluble fraction. Several abundant PM proteins co-immunoprecipitated with NCX1, including the alpha2 and alpha3 isoforms of the Na(+) pump catalytic (alpha) subunit, and the alpha2 subunit of the dihydropyridine receptor. The adaptor protein, ankyrin 2 (Ank 2), and the cytoskeletal proteins, alpha-fodrin and beta-spectrin, also selectively co-immunoprecipitated with NCX1, as did the ER proteins, Ca(2+) pump type 2 (SERCA 2), and inositol-trisphosphate receptor type 1 (IP(3)R-1). In contrast, a number of other abundant PMs, adaptors, and cytoskeletal proteins did not co-immunoprecipitate with NCX1, including the Na(+) pump alpha1 isoform, PM Ca(2+) pump type 1 (PMCA1), beta-fodrin, and Ank 3. In reciprocal experiments, immunoprecipitation with antibodies to the Na(+) pump alpha2 and alpha3 isoforms, but not alpha1, co-immunoprecipitated NCX1; the antibodies to alpha1 did, however, co-immunoprecipitate PMCA1. Antibodies to Ank 2, alpha-fodrin, beta-spectrin and IP(3)R-1 all co-immunoprecipitated NCX1. Immunocytochemistry revealed partial co-localization of beta-spectrin with NCX1, Na(+) pump alpha3, and IP(3)R-1 in neurons and of alpha-fodrin with NCX1 and SERCA2 in astrocytes. The data support the idea that in neurons and glia PM microdomains containing NCX1 and Na(+) pumps with alpha2 or alpha3 subunits form Ca(2+) signaling complexes with underlying ER containing SERCA2 and IP(3)R-1. These PM and ER components appear to be linked through the cytoskeletal spectrin network, to which they are probably tethered by Ank 2.     

Calcium transporters and signalling in smooth muscles

Two P-type Ca transporters, the plasma membrane Ca-ATPase (PMCA) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA), play a crucial role in maintaining Ca homeostasis, controlling contractility and contributing to excitably and cell signalling in smooth muscle cells. There is considerable structural homology between the two Ca-ATPases; they both have transmembrane spanning regions, have similar ATP-phosphorylated intermediaries, counter transport protons and are regulated by several second messengers. They both also exist in several isoforms and have many splice variants, which presumably impart some of their tissue specific functions. We describe the relative contribution of PMCA and the Na-Ca exchanger to Ca efflux in relaxation to smooth muscle, including recent data from transgenic mice, which has begun to elucidate the specific contributions of individual isoforms to Ca signalling. We then consider Ca release and uptake into the SR in smooth muscle. Experiments investigating the distribution of SERCA in smooth muscle cells have provided new insights into control of SR luminal Ca, and the effects of SR Ca load on signalling, is discussed. This is followed by a detailed consideration of the interactions between the surface membrane and SR membrane pumps, exchangers and ion channels in smooth muscle, along with their distribution to caveolae and cholesterol-rich membrane domains. Where relevant the importance of these functions to health and disease are noted. We conclude that the dynamic changes in splice variants expressed, constituents of membrane microdomains and environment of the sub-sarcolemmal space, close to the SR, need to be the focus of future research, so that the full importance of Ca transporters to smooth muscle signalling cascades can be better understood.     

Alternative splicing of the first intracellular loop of plasma membrane Ca2+-ATPase isoform 2 alters its membrane targeting

Plasma membrane Ca(2+)-ATPases (PMCAs) are involved in local Ca(2+) signaling and in the spatial control of Ca(2+) extrusion, but how different PMCA isoforms are targeted to specific membrane domains is unknown. In polarized MDCK epithelial cells, a green fluorescent protein-tagged PMCA4b construct was targeted to the basolateral membrane, whereas a green fluorescent protein-tagged PMCA2b construct was localized to both the apical and basolateral domain. The PDZ protein-binding COOH-terminal tail of PMCA2b was not responsible for its apical membrane localization, as a chimeric pump made of an NH(2)-terminal portion from PMCA4 and a COOH-terminal tail from PMCA2b was targeted to the basolateral domain. Deletion of the last six residues of the COOH terminus of either PMCA2b or PMCA4b did not alter their membrane targeting, suggesting that PDZ protein interactions are not essential for proper membrane localization of the pumps. Instead, we found that alternative splicing affecting the first cytosolic loop determined apical membrane targeting of PMCA2. Only the "w" form, which contains a 45-amino acid residue insertion, showed prominent apical membrane localization. By contrast, the x and z splice variants containing insertions of 14 and 0 residues, respectively, localized to the basolateral membrane. The w splice insert was the crucial determinant of apical PMCA2 localization, and this was independent of the splice configuration at the COOH-terminal end of the pump; both PMCA2w/b and PMCA2w/a showed prominent apical targeting, whereas PMCA2x/b, PMCA2z/b, and PMCA2z/a were confined to the basolateral membrane. These data report the first differential effect of alternative splicing within the first cytosolic loop of PMCA2 and help explain the selective enrichment of specific PMCA2 isoforms in specialized membrane compartments such as stereocilia of auditory hair cells.     

Thyroid hormone downregulates the expression and function of sarcoplasmic reticulum-associated CaM kinase II in the rabbit heart

Phosphorylation of sarcoplasmic reticulum (SR) Ca2+-cycling proteins by a membrane-associated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is a well-documented physiological mechanism for regulation of transmembrane Ca2+ fluxes and the cardiomyocyte contraction-relaxation cycle. The present study investigated the effects of L-thyroxine-induced hyperthyroidism on protein expression of SR CaM kinase II and its substrates, endogenous CaM kinase II-mediated SR protein phosphorylation, and SR Ca2+ pump function in the rabbit heart. Membrane vesicles enriched in junctional SR (JSR) or longitudinal SR (LSR) isolated from euthyroid and hyperthyroid rabbit hearts were utilized. Endogenous CaM kinase II-mediated phosphorylation of ryanodine receptor-Ca2+ release channel (RyR-CRC), Ca2+-ATPase, and phospholamban (PLN) was significantly lower (30-70%) in JSR and LSR vesicles from hyperthyroid than from euthyroid rabbit heart. Western immunoblotting analysis revealed significantly higher (approximately 40%) levels of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) in JSR, but not in LSR, from hyperthyroid than from euthyroid rabbit heart. Maximal velocity of Ca2+ uptake was significantly increased in JSR (130%) and LSR (50%) from hyperthyroid compared with euthyroid rabbit hearts. Apparent affinity of the Ca2+-ATPase for Ca2+ did not differ between the two groups. Protein levels of PLN and CaM kinase II were significantly lower (30-40%) in JSR, LSR, and ventricular tissue homogenates from hyperthyroid rabbit heart. These findings demonstrate selective downregulation of expression and function of CaM kinase II in hyperthyroid rabbit heart in the face of upregulated expression and function of SERCA2 predominantly in the JSR compartment.     

The isoform- and location-dependence of the functioning of the plasma membrane calcium pump

The plasma membrane is a specialised multi-component structure with inter- and intracellular signalling functions. Ca2+ plays a crucial role in cellular physiology, and an ATP-driven plasma membrane calcium pump (PMCA) plays the greatest role in the maintenance of a low free Ca2+ concentration in the cytoplasm. The enzyme is coded by four separate genes (PMCA 1-4), and, due to alternative splicing, more than 20 variants can exist. PMCA 1 and 4 isoforms are present in almost all tissues, whereas PMCA 2 and 3 are found in more specialised cell types. The variants differ primarily in their regulatory regions, thus the modulation of calcium pump activity strongly depends on the isoform and the membrane composition. The unique function of PMCA isoforms was confirmed using the practical experimental models - a rat pheochromocytoma cell line, a human neuroblastoma cell line, or, more recently, knockout mice. In addition, based on the finding that PMCA could interact with several specific signaling proteins, it was concluded that its location in defined sites of the cell membrane could be a prerequisite for efficient intercellular communication.     

Expression,purification, and characterization of isoform 1 of the plasma membrane Ca2+ pump: focus on calpain sensitivity

The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms.     

Delayed activation of the plasma membrane calcium pump by a sudden increase in Ca2+: fast pumps reside in fast cells

There are four genes encoding isoforms of the plasma membrane Ca(2+) pump (PMCA). PMCA variability is increased by the presence of two splicing sites. Functional differences between the variants of PMCA have been described, but little is known about the adaptive advantages of this great diversity of pumps. In this paper we studied how the different isoforms respond to a sudden increase in Ca(2+) concentration. We found that different PMCAs are activated by Ca(2+) at different rates, PMCA 3f and 2a being the fastest, and 4b the slowest. The rate of activation by Ca(2+) depends both on the rate of calmodulin binding and the magnitude of the activation by calmodulin. We found that 2a is located in heart and the stereocilia of inner ear hair cells, 3f in skeletal muscle and 4b was identified in Jurkat cells. Both cardiac and skeletal muscle, and stereocilia recover very rapidly after a cytoplasmic Ca(2+)peak, while in Jurkat cells the recovery takes up to a minute. In stereocilia, 2a is the only method for export of Ca(2+), making the analysis of them unusually straightforward. This indicates that these rates of PMCA activation by Ca(2+) are correlated with the speed of Ca(2+) concentration decay after a Ca2 spike in the cells in which these variants of PMCA are expressed. The results suggest that the type of PMCA expressed will correspond with the speed of Ca(2+) signals in the cell.     

A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells

The four basic isoforms of the plasma membrane Ca2+ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII(3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca2+ in the native intracellular environment. The level of [Ca2+] in the endoplasmic reticulum and the height of the [Ca2+] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca2+ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.     

Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells

Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.