Plasma Membrane Ca2+-ATPase Isoforms Research Articles

Effects of dietary calcium (Ca2+) and blood feeding on the immunochemical expression of the plasma membrane Ca2+-ATPase (PMCA) in Malpighian tubules of adult female mosquitoes (Aedes aegypti)

Insect Malpighian tubules contribute to Ca2+ homeostasis via Ca2+ storage in intracellular compartments, Ca2+ secretion into the tubule lumen, and Ca2+ reabsorption into the hemolymph. A plasma membrane Ca2+-ATPase (PMCA) is hypothesized to be a Ca2+-transporter involved in renal Ca2+ transport of insects, however few studies have investigated its immunochemical expression in Malpighian tubules. Here we characterized the abundance and localization of PMCA-like immunoreactivity in Malpighian tubules of adult female mosquitoes Aedes aegypti using an antibody against Drosophila melanogaster PMCA. Western blotting revealed expression of a relatively abundant 109 kDa isoform and a relatively sparse 115 kDa isoform. Feeding mosquitoes 10% sucrose with 50 mM CaCl2 for 7 days did not affect PMCA immunoreactivity. However, at 24, 48, and 96 h post-blood feeding (PBF), the relative abundance of the 109 kDa isoform decreased while that of the 115 kDa isoform increased. Immunolabeling of Malpighian tubules revealed PMCA-like immunoreactivity in both principal and stellate cells; principal cell labeling was intracellular, whereas stellate cell labeling was along the basal membrane. Blood feeding enhanced immunolabeling of PMCA in stellate cells but weakened that in principal cells. Moreover, a unique apicolateral pattern of PMCA-like immunolabeling occurred in principal cells of the proximal segment at 24 h PBF, suggesting potential trafficking to septate junctions. Our results suggest PMCA isoforms are differentially expressed and localized in mosquito Malpighian tubules where they contribute to redistributing tubule Ca2+ during blood meal processing.

Analysis of cholesterol-recognition motifs of the plasma membrane Ca2+-ATPase

The plasma membrane Ca2+-ATPase (PMCA) is crucial for the fine tuning of intracellular calcium levels in eukaryotic cells. In this study, we show the presence of CARC sequences in all human and rat PMCA isoforms and we performed further analysis by molecular dynamics simulations. This analysis focuses on PMCA1, containing three CARC motifs, and PMCA4, with four CARC domains. In PMCA1, two CARC motifs reside within transmembrane domains, while the third is situated at the intracellular interface. The simulations depict more stable RMSD values and lower RMSF fluctuations in the presence of cholesterol, emphasizing its potential stabilizing effect. In PMCA4, a distinct dynamic was found. Notably, the total energy differences between simulations with cholesterol and phospholipids are pronounced in PMCA4 compared to PMCA1. RMSD values for PMCA4 indicate a more energetically favorable conformation in the presence of cholesterol, suggesting a robust interaction between CARCs and this lipid in the membranes. Furthermore, RMSF analysis for CARCs in both PMCA isoforms exhibit lower values in the presence of cholesterol compared to POPC alone. The analysis of H-bond occupancy and total energy values strongly suggests the potential interaction of CARCs with cholesterol. Given the crucial role of PMCAs in physiological calcium regulation and their involvement in diverse pathological processes, this study underscores the significance of CARC motifs and their interaction with cholesterol in elucidating PMCA function. These insights into the energetic preferences associated with CARC-cholesterol interactions offer valuable implications for understanding PMCA function in maintaining calcium homeostasis and addressing potential associated pathologies.

The Puzzling Role of Neuron-Specific PMCA Isoforms in the Aging Process.

The aging process is a physiological phenomenon associated with progressive changes in metabolism, genes expression, and cellular resistance to stress. In neurons, one of the hallmarks of senescence is a disturbance of calcium homeostasis that may have far-reaching detrimental consequences on neuronal physiology and function. Among several proteins involved in calcium handling, plasma membrane Ca2+-ATPase (PMCA) is the most sensitive calcium detector controlling calcium homeostasis. PMCA exists in four main isoforms and PMCA2 and PMCA3 are highly expressed in the brain. The overall effects of impaired calcium extrusion due to age-dependent decline of PMCA function seem to accumulate with age, increasing the susceptibility to neurotoxic insults. To analyze the PMCA role in neuronal cells, we have developed stable transfected differentiated PC12 lines with down-regulated PMCA2 or PMCA3 isoforms to mimic age-related changes. The resting Ca2+ increased in both PMCA-deficient lines affecting the expression of several Ca2+-associated proteins, i.e., sarco/endoplasmic Ca2+-ATPase (SERCA), calmodulin, calcineurin, GAP43, CCR5, IP3Rs, and certain types of voltage-gated Ca2+ channels (VGCCs). Functional studies also demonstrated profound changes in intracellular pH regulation and mitochondrial metabolism. Moreover, modification of PMCAs membrane composition triggered some adaptive processes to counterbalance calcium overload, but the reduction of PMCA2 appeared to be more detrimental to the cells than PMCA3.

The Plasma Membrane Calcium ATPases in Calcium Signaling Network.

The plasma membrane Ca2+ ATPases (PMCAs) are responsible for the clearance of Ca2+ out of cells after intracellular Ca2+ transients. Cooperating with Na+/Ca2+ exchangers (NCXs) and Ca2+ buffering proteins, PMCAs play an essential role in maintaining the long-term cellular Ca2+ homeostasis. The plasma membrane Ca2+ ATPase was first discovered in red blood cell membrane about 50 years ago, and then other PMCA isoforms and alternatively spliced variants had been identified from different tissues and different developmental stages, revealing a surprising complexity of the PMCA family. In mammals, there are four PMCA isoforms encoded by four distinct genes. Isoform 1 and 4 are found in virtually all tissues, whereas isoform 2 and 3 are primarily expressed in excitable cells such as neurons and myocytes. Perturbation of PMCAs function has been implicated in a variety of diseases and disorders, including hearing loss, ataxia, paraplegia, and infertility. Here, we would like to review the recent progresses in the study of the PMCAs and related disorders, in particular how these pathological conditions help us to gain an in-depth insight into the function of PMCAs and their contribution in the regulation of Ca2+ signaling network.

Evidence for a role of plasma membrane calcium pumps in neurodegenerative disease: Recent developments.

Plasma membrane Ca2+ ATPases (PMCAs) are a major system for calcium extrusion from all cells. Different PMCA isoforms and splice variants are involved in the precise temporal and spatial handling of Ca2+ signals and the re-establishment of resting Ca2+ levels in the nervous system. Lack or inappropriate expression of specific PMCAs leads to characteristic neuronal phenotypes, which may be reciprocally exacerbated by genetic predisposition through alleles in other genes that modify PMCA interactions, regulation, and function. PMCA dysfunction is often poorly compensated in neurons and may lead to changes in synaptic transmission, altered excitability and, with long-term calcium overload, eventual cell death. Decrease and functional decline of PMCAs are hallmarks of neurodegeneration during aging, and mutations in specific PMCAs are responsible for neuronal dysfunction and accelerated neurodegeneration in many sensory and cognitive diseases.

NFAT1 and NFAT3 cooperate with HDAC4 during regulation of alternative splicing of PMCA isoforms in PC12 cells.

BackgroundThe bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants.ResultsIn this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation.ConclusionsWe suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

Plasma membrane calcium ATPases as novel candidates for therapeutic agent development.

Plasma membrane Ca2+ ATPases (PMCAs) are highly regulated transporters responsible for Ca2+ extrusion from all eukaryotic cells. Different PMCA isoforms are implicated in various tasks of Ca2+ regulation including bulk Ca2+ transport and localized Ca2+ signaling in specific membrane microdomains. Accumulating evidence shows that loss, mutation or inappropriate expression of different PMCAs is associated with pathologies ranging from hypertension, low bone density and male infertility to hearing loss and cerebellar ataxia. Compared to Ca2+ influx channels, PMCAs have lagged far behind as targets for drug development, mainly due to the lack of detailed understanding of their structure and specific function. This is rapidly changing thanks to integrated efforts combining biochemical, structural, cellular and physiological studies suggesting that selective modulation of PMCA isoforms may be of therapeutic value in the management of different and complex diseases. Both structurally informed rational design and high-throughput small molecule library screenings are promising strategies that are expected to lead to specific and isoform-selective modulators of PMCA function. This short review will provide an overview of the diverse roles played by PMCA isoforms in different cells and tissues and their emerging involvement in pathophysiological processes, summarize recent progress in obtaining structural information on the PMCAs, and discuss current and future strategies to develop specific PMCA inhibitors and activators for potential therapeutic applications.

Alternative Pathways for Association and Dissociation of the Calmodulin-binding Domain of Plasma Membrane Ca2+-ATPase Isoform 4b (PMCA4b)

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.

Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid–protein interaction

The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.

Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility.

Plasma-membrane calcium pumps [PMCAs (plasma-membrane Ca(2+)-ATPases)] expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. Recent work indicates functional versatility among PMCA isoforms, with specific pumps being essential for cochlear hair cell function, sperm motility, feedback signalling in the heart and pre- and post-synaptic Ca(2+) regulation in neurons. The functional versatility of PMCAs is due to differences in their regulation by CaM (calmodulin), kinases and other signalling proteins, as well as to their differential targeting and retention in defined plasma membrane domains. The basis for this is the structural diversity of PMCAs. In mammals, four genes encode PMCA isoforms 1-4, and each of these has multiple variants generated by alternative RNA splicing. The alternatively spliced regions are intimately involved in the regulatory interactions and differential membrane localization of the pumps. The alternatively spliced C-terminal tail acts as an autoinhibitory domain by interacting with the catalytic core of the pump. The degree of inhibition and the kinetics of interaction with the major activator CaM differ between PMCA variants. This translates into functional differences in how PMCAs handle Ca(2+) signals of different magnitude and frequency. Accumulating evidence thus demonstrates how structural diversity provides functional versatility in the PMCAs.

Plasma-membrane calcium pumps and hereditary deafness

In mammals, four different genes encode four PMCA (plasma-membrane Ca(2+)-ATPase) isoforms. PMCA1 and 4 are expressed ubiquitously, and PMCA2 and 3 are expressed predominantly in the central nervous system. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of so many isoforms is not clear, but evidently it must be related to the cell-specific demands of Ca(2+) homoeostasis. Recent studies suggest that the alternatively spliced regions in PMCA are responsible for specific targeting to plasma membrane domains, and proteins that bind specifically to the pumps could contribute to further regulation of Ca(2+) control. In addition, the combination of proteins obtained by alternative splicing occurring at two different sites could be responsible for different functional characteristics of the pumps.

Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells

BackgroundFreshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca2+ uptake in gill MR cells, Ca2+ passively enters the cytosol via the epithelium Ca2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data.ResultsUsing a strategy of BLAST from the zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were identified. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady.ConclusionThe present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.

Isoform-specific up-regulation of plasma membrane Ca 2+ATPase expression during colon and gastric cancer cell differentiation

In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca 2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na +/K +–ATPase or that of the sarco/endoplasmic reticulum Ca 2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca 2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.

Plasma Membrane Ca2+ ATPases as Dynamic Regulators of Cellular Calcium Handling

Plasma membrane Ca2+ ATPases (PMCAs) are essential components of the cellular toolkit to regulate and fine-tune cytosolic Ca2+ concentrations. Historically, the PMCAs have been assigned a housekeeping role in the maintenance of intracellular Ca2+ homeostasis. More recent work has revealed a perplexing multitude of PMCA isoforms and alternative splice variants, raising questions about their specific role in Ca2+ handling under conditions of varying Ca2+ loads. Studies on the kinetics of individual isoforms, combined with expression and localization studies suggest that PMCAs are optimized to function in Ca2+ regulation according to tissue- and cell-specific demands. Different PMCA isoforms help control slow, tonic Ca2+ signals in some cells and rapid, efficient Ca2+ extrusion in others. Localized Ca2+ handling requires targeting of the pumps to specialized cellular locales, such as the apical membrane of cochlear hair cells or the basolateral membrane of kidney epithelial cells. Recent studies suggest that alternatively spliced regions in the PMCAs are responsible for their unique targeting, membrane localization, and signaling cross-talk. The regulated deployment and retrieval of PMCAs from specific membranes provide a dynamic system for a cell to respond to changing needs of Ca2+ regulation.

Role of Na/Ca Exchange and the Plasma Membrane Ca2+–ATPase in β Cell Function and Death

Recent progresses concerning the Na/Ca exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA) in the pancreatic beta cell are reviewed. The rat beta cell expresses two splice variants of NCX1 and six splice variants of the 4 PMCA isoforms. At the protein level, the most abundant forms are PMCA2 and PMCA3, providing the first evidence for the presence of these two isoforms in a non-neuronal tissue. Overexpression of NCX1 in an insulinoma cell line altered the initial rise in cytosolic-free Ca2+ concentration ([Ca2+]i) induced by membrane depolarization and the return of the [Ca2+]i to the baseline value on membrane repolarization, indicating that NCX contributes to both Ca2+ inflow and outflow in the beta cell. In contrast, overexpression of the PMCA markedly reduced the global rise in Ca2+ induced by membrane depolarization, indicating that the PMCA has a capacity higher than expected to extrude Ca2+. Glucose, the main physiological stimulus of insulin release from the beta cell, has opposite effect on NCX and PMCA transcription, expression and activity, inducing an increase in the case of NCX and a decrease in the case of the PMCA. This indicates that when exposed to glucose, the beta cell switches from a low-efficiency Ca2+ extruding mechanism, the PMCA, to a high-capacity system, the NCX, in order to better face the increase in Ca2+ inflow induced by the sugar. To our knowledge, this is the first demonstration of a reciprocal change in PMCA and NCX1 expression and activity in response to a given stimulus in any tissue.

Role of Na/Ca exchange and the plasma membrane Ca 2+ ‐ATPase in cell function

The Fifth International Conference on Na/Ca Exchange took place between 23 and 27 August 2006 in Brussels, Belgium, and was organized by A. Herchuelz. Image taken from the meeting poster showing hypothetical models for the Na/Ca exchanger (left) and the plasma membrane Ca2+‐ATPase (right), courtesy of M. Hilge. ![][1] Cytosolic Ca2+ regulates several cellular processes and its concentration is, in turn, finely regulated by various channels, pumps and exchangers. The Na/Ca exchanger (NCX) and the plasma membrane Ca2+‐ATPase (PMCA) pump are two concurrent mechanisms for Ca2+ extrusion from the cell (Carafoli, 1994; Blaustein & Lederer, 1999; Philipson & Nicoll, 2000; Strehler & Treiman, 2004; Prasad et al , 2004). PMCA and NCX were cloned 17 and 19 years ago, respectively; four mammalian isoforms have been identified for the former (PMCA1–4) and three have been identified for the latter (NCX1–3; Carafoli, 1994; Philipson & Nicoll, 2000). Na/Ca exchange (NCX) is a unique mechanism that allows Ca2+ extrusion from the cell against its gradient without energy consumption. Indeed, it is the entry of Na+ along its electrochemical gradient that provides the energy for Ca2+ extrusion. In addition, because NCX is electrogenic and voltage sensitive, it can reverse during cellular activation and contribute to Ca2+ entry into the cell (Blaustein & Lederer, 1999). Recently, the crucial role of NCX in cellular Ca2+ homeostasis has been definitively shown by the generation of mice with functional destruction of one Ncx gene. Homozygous Ncx1 ‐deficient mice are not viable, and die between embryonic days 9 and 10, owing to the lack of development of the cardiovascular system (Wakimoto et al , 2000); therefore, NCX is essential for life. PMCA belongs to the P‐type family of transport ATPases, which form a phosphorylated intermediate … [1]: /embed/graphic-1.gif

Plasma membrane Ca 2+-ATPase isoforms in frog crista ampullaris: Identification of PMCA1 and PMCA2 specific splice variants

Ca 2+ ions play a pivotal role in inner ear hair cells as they are involved from the mechano-electrical transduction to the transmitter release. Most of the Ca 2+ that enters into hair cells via mechano-transduction and voltage-gated channels is extruded by the plasma membrane Ca 2+-ATPases (PMCAs) that operate in both apical and basal cellular compartments. Here, we determined the identity and distribution of PMCA isoforms in frog crista ampullaris: we showed that PMCA1, PMCA2 and PMCA3 are expressed, while PMCA4 appears to be negligible. We also identify PMCA1bx, PMCA2av and PMCA2bv as the major splice variants produced from PMCA1 and PMCA2 genes. PMCA2av appears to be the major Ca 2+-pump operating at the apical pole of the cell, even if PMCA1b is also expressed in the stereocilia. PMCA1bx is, instead, the principal PMCA of hair cell basolateral compartment, where it is expressed together with PMCA2 (probably PMCA2bv) and PMCA3. Frog crista ampullaris hair cells lack a Na/Ca exchanger, therefore PMCAs are the only mechanism of Ca 2+ extrusion. The coexpression of specific isozymes in the different cellular compartments responds to the need of a fine regulation of both basal and dynamic Ca 2+ levels at the apical and basal pole of the cell.

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

We previously showed that plasma membrane Ca(2+)-ATPase (PMCA) activity accounted for 25-30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca(2+) ([Ca(2+)](i)) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1(+/-), Pmca4(+/-), Pmca4(-/-), and Pmca1(+/-)Pmca4(-/-) mice. There were no differences in basal [Ca(2+)](i) values between bladder preparations. KCl (80 mM) elicited both larger forces (150-190%) and increases in [Ca(2+)](i) (130-180%) in smooth muscle from Pmca1(+/-) and Pmca1(+/-)Pmca4(-/-) bladders than those in WT or Pmca4(-/-). The responses to carbachol (CCh: 10 muM) were also greater in Pmca1(+/-) (120-150%) than in WT bladders. In contrast, the responses in Pmca4(-/-) and Pmca1(+/-)Pmca4(-/-) bladders to CCh were significantly smaller (40-50%) than WT. The rise in half-times of force and [Ca(2+)](i) increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4(-/-) (130-190%) and Pmca1(+/-)Pmca4(-/-) (120-250%) bladders, but not in Pmca1(+/-) bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca(2+) clearance, while PMCA4 is essential for the [Ca(2+)](i) increase and contractile response to the CCh receptor-mediated signal transduction pathway.

Human platelet Ca2+-ATPases: New markers of cell differentiation as illustrated in idiopathic scoliosis

The aetiology of adolescent idiopathic scoliosis (AIS), the most common form of scoliosis, is unclear. Previous studies showed controversial platelet abnormalities including intracellular calcium. Platelet Ca2+ homeostasis is controlled by a multi-Ca2+-ATPase system including SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) and PMCA (plasma membrane Ca2+-ATPase) isoforms. Here, we first investigated the expression of PMCA4b, SERCA3a and SERCA2b isoforms in platelets of 17 patients with AIS. Patients presenting thoracic curves were found to present a higher PMCA4b expression coupled to a lower SERCA3a one in agreement with an abnormality in platelet maturation. Indeed, using PMA-treated MEG 01 cells, an in vitro model of megakaryocytopoiesis, we found an increase in SERCA3a expression, associated to a caspase-3 mediated C terminal proteolysis of PMCA4b. To look whether platelets reflect a basic defect in cell differentiation, we next identified osteoblast Ca2+-ATPases and studied their expressions in AIS. Major expressions of PMCA4b and SERCA2b were found in normal osteoblasts. Comparing platelets and osteoblasts in two additional patients with AIS, we found opposite and concerted regulations of the expressions of PMCA4b and caspase-3 substrate, PARP in both cell types. A systemic defect in cell differentiation involving caspase-3 can be proposed as a novel mechanism in the etiopathogenesis of the most frequent type of AIS.

Plasma membrane calcium ATPase is concentrated in the head of sea urchin spermatozoa

Plasma membrane Ca2+ATPases (PMCAs) export Ca2+ from cells in a highly regulated manner, providing fine-tuning to the maintenance of intracellular Ca2+ concentrations. There are few studies of PMCAs in spermatozoa, which is surprising considering the importance of this enzyme in all cell types. Here we describe the primary structure and localization of the PMCA of sea urchin spermatozoa (suPMCA). The suPMCA is 1,154 amino acids and has 56% identity and 76% similarity to all 4 human PMCA isoforms. The suPMCA shares the features of a typical PMCA, including domains for calmodulin binding, ATP binding, ATPase phosphorylation, and 10 putative transmembrane segments with two large cytoplasmic loops. Southern blots show that suPMCA is a single copy gene. Treatment of live sea urchin sperm with the PMCA inhibitor, 5-(-6)-carboxyeosin, results in elevations of intracellular Ca2+ and loss of flagellar motility. Immunoblotting and immunoflorescence show that suPMCA is concentrated in the sperm head plasma membrane. In previous work, we showed that a plasma membrane K+ dependent Na+/Ca2+ exchanger (suNCKX), which also keeps Ca2+ low in these cells, is concentrated in the sperm flagellum. Thus, the sperm head and flagellum localize different gene products, both functioning to keep intracellular Ca2+ low, while the sperm swims in seawater containing 10 mM Ca2+.