The calcium transporter Pmc1 provides Ca2+ tolerance and influences the progression of murine cryptococcal infection.

The Ca(2+)-calcineurin signaling pathway in the human fungal pathogen Cryptococcus neoformans is essential for adaptation to the host environment during infection. Calcium transporters regulate cytosolic calcium concentrations, providing Ca(2+) loading into storage organelles. The three calcium transporters that have been characterized in C. neoformans, Cch1, Eca1 and Vcx1, are required for fungal virulence, supporting a role for calcium-mediated signaling in cryptococcal pathogenesis. In the present study, we report the functional characterization of the putative vacuolar calcium ATPase Pmc1 in C. neoformans. Our results demonstrate that Pmc1 provides tolerance to high Ca(2+) concentrations. The double knockout of C. neoformans PMC1 and VCX1 genes impaired the intracellular calcium transport, resulting in a significant increase in cytosolic calcium levels. Furthermore, Pmc1 was essential for both the progression of pulmonary infection and brain colonization in mice, emphasizing the crucial role of calcium signaling and transport for cryptococcal pathogenesis.

Genetic diversity and lack of artemisinin selection signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion.

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca(2+)-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average d(N)/d(S) ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.

Genome-wide analysis of plant-type II Ca(2+)ATPases gene family from rice and Arabidopsis: potential role in abiotic stresses.

The Plant Ca(2+)ATPases are members of the P-type ATPase superfamily and play essential roles in pollen tube growth, vegetative development, inflorescence architecture, stomatal opening or closing as well as transport of Ca(2+), Mn(2+) and Zn(2+). Their role in abiotic stress adaptation by activation of different signaling pathways is emerging. In Arabidopsis, the P-type Ca(2+)ATPases can be classified in two distinct groups: type IIA (ECA) and type IIB (ACA). The availability of rice genome sequence allowed performing a genome-wide search for P-type Ca(2+)ATPases proteins, and the comparison of the identified proteins with their homologs in Arabidopsis model plant. In the present study, we identified the P-type II Ca(2+)ATPases from rice by analyzing their phylogenetic relationship, multiple alignment, cis-regulatory elements, protein domains, motifs and homology percentage. The phylogenetic analysis revealed that rice type IIA Ca(2+)ATPases clustered with Arabidopsis type IIA Ca(2+)ATPases and showed high sequence similarity within the group, whereas rice type IIB Ca(2+)ATPases presented variable sequence similarities with Arabidopsis type IIB members. The protein homology modeling, identification of putative transmembrane domains and conserved motifs of rice P-type II Ca(2+)ATPases provided information on their functions and structural architecture. The analysis of P-type II Ca(2+)ATPases promoter regions in rice showed multiple stress-induced cis-acting elements. The expression profile analysis indicated vital roles of P-type II Ca(2+)ATPases in stress signaling, plant development and abiotic stress responses. The comprehensive analysis and expression profiling provided a critical platform for functional characterization of P-type II Ca(2+)ATPase genes that could be applied in engineering crop plants with modified calcium signaling and homeostatic pathways.

Ontogeny of plasma membrane Ca2+ ATPase isoforms in the neural retina of the postnatal rat.

Calcium ion (Ca(2+)) signaling has been widely implicated in developmental events in the retina, but little is known about the specific mechanisms utilized by developing neurons to decrease intracellular Ca(2+). Using immunocytochemistry, we determined the expression profiles of all known isoforms of a key Ca(2+) transporter, the plasma membrane Ca(2+) ATPase (PMCA), in the rat retina. During the first postnatal week, the four PMCA isoforms were expressed in patterns that differed from their expression in the adult retina. At birth, PMCA1 was found in the ventricular zone and nascent cell processes in the distal retina as well as in ganglion and amacrine cells. After the first postnatal week, PMCA1 became restricted to photoreceptors and cone bipolar cells. By P10 (by postnatal day 10), most inner retinal PMCA consisted of PMCA2 and PMCA3. Prominent PMCA4 expression appeared after the first postnatal week and was confined primarily to the ON sublamina of the inner plexiform layer (IPL). The four PMCA isoforms could play distinct functional roles in the development of the mammalian retina even before synaptic circuits are established. Their expression patterns are consistent with the hypothesis that inner and outer retinal neurons have different Ca(2+) handling needs.

Do untranslated introns control Ca2+-ATPase isoform dependence on CaM, found in TN and PM?

Transcript splicing characterization of tomato Ca(2+)-ATPase (LCA1 gene) mRNA indicates that two main transcripts are differentiated in the 3(') terminal region. One of them contains a sequence of about 90bp that could correspond to an untranslated intron that displays sequence homology to calmodulin-binding regions. Calmodulin-binding experiments demonstrate that only one of the two isoforms encoded by LCA1 binds to calmodulin. Since the M(w) calculated for this peptide is 3.7kDa, it is suggested that the presence of this intron is accounted for by the difference in the sizes of the two 116- and 120-kDa isoforms, and it determines calmodulin regulation. This represents a new strategy for a single gene to produce two isoforms that are localized differently (TN and PM), and which are either dependent on or independent of the calmodulin, which in turn is either regulated by the presence or by the absence of a 90bp untranslated intron.

Isoform-specific distribution of the plasma membrane Ca2+ ATPase in the rat brain.

Regulation of cytoplasmic calcium is crucial both for proper neuronal function and cell survival. The concentration of Ca2+ in cytoplasm of a neuron at rest is 10,000 times lower than in the extracellular space, pointing to the importance of the transporters that extrude intracellular Ca2+. The family of plasma membrane calcium-dependent ATPases (PMCAs) represent a major component of the Ca2+ regulatory system. However, little information is available on the regional and cellular distribution of these calcium pumps. We used immunohistochemistry to investigate the distribution of each of the four PMCA isoforms (PMCA1-4) in the rat brain. Each isoform exhibited a remarkably precise and distinct pattern of distribution. In many cases, PMCA isoforms in a single brain structure were differentially expressed within different classes of neurons, and within different subcellular compartments. These data show that each isoform is independently organized and suggest that PMCAs may play a more complex role in calcium homeostasis than generally recognized.

Transconformations of the SERCA1 Ca-ATPase: a normal mode study.

The transport of Ca(2+) by Ca-ATPase across the sarcoplasmic reticulum membrane is accompanied by several transconformations of the protein. Relying on the already established functional importance of low-frequency modes in dynamics of proteins, we report here a normal mode analysis of the Ca(2+)-ATPase based on the crystallographic structures of the E1Ca(2) and E2TG forms. The lowest-frequency modes reveal that the N and A(+Nter) domains undergo the largest amplitude movements. The dynamical domain analysis performed with the DomainFinder program suggests that they behave as rigid bodies, unlike the highly flexible P domain. We highlight two types of movements of the transmembrane helices: i), a concerted movement around an axis perpendicular to the membrane which "twists open" the lumenal side of the protein and ii), an individual translational and rotational mobility which is of lower amplitude for the helices hosting the calcium binding sites. Among all modes calculated for E1Ca, only three are enough to describe the transition to E2TG; the associated movements involve almost exclusively the A and N domains, reflecting the closure of the cytoplasmic headpiece and high displacement of the L7-8 lumenal loop. Subsequently, we discuss the potential contribution of the remaining low-frequency normal modes to the transconformations occurring within the overall calcium transport cycle.

P-type ATPase diversity and evolution: the origins of ouabain sensitivity and subunit assembly.

Molecular aspects of the diversity of P-type ATPases are explored in this review. From the substrate specificities among different ATPase molecules, the existence of isoforms within a single class of pump becomes evident and it is now recognized as a universal phenomenon. From the phylogenetic analyses using a vast collection of the deduced amino acid sequences for the P-type ATPase subunits, it also becomes evident that the divergence of substrate-specificity occurred early in the evolution and has been conserved ever since. Further extensive analyses identify a set of novel isoforms that retain an ancestral characteristic of the Na+/K+-(H+/K+-)ATPases in invertebrates.

Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps.

Calcium pumps of the plasma membrane (also known as plasma membrane Ca(2+)-ATPases or PMCAs) are responsible for the expulsion of Ca(2+) from the cytosol of all eukaryotic cells. Together with Na(+)/Ca(2+) exchangers, they are the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular Ca(2+) concentration. Like the Ca(2+) pumps of the sarco/endoplasmic reticulum (SERCAs), which pump Ca(2+) from the cytosol into the endoplasmic reticulum, the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing affects two major locations in the plasma membrane Ca(2+) pump protein: the first intracellular loop and the COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and temporal control of Ca(2+) signaling. The identification of mice carrying PMCA mutations that lead to diseases such as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA "knockout" mice further support the concept of specific, nonredundant roles for each Ca(2+) pump isoform in cellular Ca(2+) regulation.

Expression and functional characterization of a Plasmodium falciparum Ca2+-ATPase (PfATP4) belonging to a subclass unique to apicomplexan organisms.

We have obtained a full-length P type ATPase sequence (PfATP4) encoded by Plasmodium falciparum and expressed PfATP4 in Xenopus laevis oocytes to study its function. Comparison of the hitherto incomplete open reading frame with other Ca(2+)-ATPase sequences reveals that PfATP4 differs significantly from previously defined categories. The Ca(2+)-dependent ATPase activity of PfATP4 is stimulated by a much broader range of [Ca(2+)](free) (3.2-320 micrometer) than are an avian SERCA1 pump or rabbit SERCA 1a (maximal activity < 10 micrometer). The activity of PfATP4 is resistant to inhibition by ouabain (200 micrometer) or thapsigargin (0.8 micrometer) but is inhibited by vanadate (1 mM) or cyclopiazonic acid (1 microM). We used a quantitative polymerase chain reaction to assay expression of mRNA encoding PfATP4 relative to that for beta-tubulin in synchronized asexual stages and found variable expression throughout the life cycle with a maximal 5-fold increase in meronts compared with ring stages. This analysis suggests that PfATP4 defines a novel subclass of Ca(2+)-ATPases unique to apicomplexan organisms and therefore offers potential as a drug target.

Two additional type IIA Ca(2+)-ATPases are expressed in Arabidopsis thaliana: evidence that type IIA sub-groups exist.

High affinity Ca(2+)-ATPases play a central role in calcium homeostasis by catalysing the active efflux of calcium from the cytoplasm. This study reports the identification of two additional type IIA (SERCA-type) Ca(2+)-ATPases from Arabidopsis (AtECA2 and AtECA3), and describes the detailed sequence analysis of these genes in comparison with AtECA1 and other plant and animal Ca(2+)-ATPases. Southern analysis suggests that each of these genes is present as a single copy and also that there may be a small family of moderately related genes that encode type IIA Ca(2+)-ATPases in Arabidopsis. Evidence is also provided from RT-PCR that these genes are expressed in Arabidopsis. Hydropathy analysis predicts that the topology of the Arabidopsis type IIA proteins is similar to the animal SERCA proteins. Sequence and phylogenetic analyses suggest that the type IIA Ca(2+)-ATPases can be further divided into sub-groups.

The plasma membrane calcium pump, its role and regulation: new complexities and possibilities.

Significant progress has been achieved in elucidating the role of the plasma membrane Ca2(+)-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2(+)-ATPase controls resting levels of [Ca2+]CYT has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2(+)-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors.

Purkinje neurons express the SERCA3 isoform of the organellar type Ca(2+)-transport ATPase.

We report the distribution of the sarco(endo)plasmic reticulum Ca2+ ATPase 3 (SERCA3) isoform in the rat brain. Compared to SERCA2 isoform, which is found in all brain regions, SERCA3 is specifically expressed in the Purkinje neurons. This conclusion is based on immunochemical observations using SERCA3- and SERCA2b-specific antibodies, in-situ hybridization using SERCA3-specific oligonucleotide probes and single-cell reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry clearly revealed the expression of SERCA3 in the cell body and in the dentritic processes of the Purkinje neurons. Single-cell ratio RT-PCR showed that Purkinje neurons expressed 3-fold lower levels of SERCA3 mRNA compared to SERCA2 mRNA. SERCA3 expression is very low or absent in the rat cerebrum and brainstem. It is known that the SERCA3 Ca2+ pump has an approximately 5-fold lower affinity for Ca2+ when expressed in COS cells as compared to other SERCA members [15]. If this property is also valid in a neuronal context, the expression of the SERCA3 Ca(2+)-pump isoform could have important functional implications for the regulation of the cytosolic Ca2+ concentration in Purkinje neurons.

Transcript distribution of plasma membrane Ca2+ pump isoforms and splice variants in the human brain.

Plasma membrane calcium pumps (PMCAs) play a major role in the maintenance and fine regulation of the intracellular Ca2+ concentration. Fourteen subregions of the normal human brain were carefully dissected and analyzed by reverse transcriptase-polymerase chain reaction for the distribution of mRNAs corresponding to the four known PMCA genes as well as their alternative splicing products at two previously defined 'hotspots' A and C. All PMCA genes were found to be expressed in every brain subregion; however, consistent differences were found in the distribution of alternative splice options. The four cortical regions and hippocampus were characterized by the relative preference of variants that include an entire exon at site C and lead to the expression of isoforms of the a-type. Inferior olive and olfactory bulb showed a relative preponderance of the b-form 'default' types of alternative splicing at site C, and a decrease or even the lack of 'differentiated' forms such as variants 1a and 1c. At the N-terminal splice site A, the default x-type variants were predominant in all brain regions for PMCA 1, 3, and 4. By contrast, the pattern of PMCA2 variants was the most variable, ranging from the presence of the entire set of 2x, 2w, and 2z forms in inferior olive to the almost exclusive presence of form 2z (excluding all alternatively spliced sequences) in the four cortical regions, caudate, and hippocampus. Regional differences in the PMCA splice type distribution in normal human brain may correlate with different demands on the regulation of the set-point resting Ca2+ levels in these areas. Changes in these patterns may correlate with altered physiological states of the affected regions and/or reflect an (early) sign of Ca2+ dyshomeostasis characteristic of many neurodegenerative diseases.

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.

The plasma membrane calcium pump: functional domains, regulation of the activity, and tissue specificity of isoform expression.

The plasma membrane Ca2+ pump is responsible for the fine regulation of the intracellular Ca2+ level and is thus involved in the control of several cellular processes. The activity of the pump is regulated by a multiplicity of mechanisms, among which are calmodulin, acidic phospholipids, kinase-mediated phosphorylation, or an oligomerization process. The C-terminal part of the molecule interacts with the region of the pump close to the active site, leading to the decrease of the activity in the resting state. Four genes coding for different isoforms of the plasma membrane Ca2+ ATPase are known in humans. Isoform 1 and 4 represent housekeeping isoforms, whereas isoforms 2 and 3 are only present in specialized tissues. The variability of the protein is further increased by alternative RNA splicing at two sites (A, C). Alternative splicing occurs within (splice site C) or near (splice site A) regions coding for regulatory domains of the protein. In all isoforms a corresponding splice form exists at both splice sites. These common splice forms are present in all tissues, whereas isoform unique splice forms are normally only present in specialized tissues. In neuronal tissues all isoforms and almost the complete set of splice forms are found. The transcripts of the different isoforms are distributed in a region-specific manner in neuronal tissues.