Association between polymorphism and haplotype of ATP2B1 gene and skeletal fluorosis in Han population.

To evaluate the association between ATP2B1 gene polymorphisms and skeletal fluorosis, a cross-sectional study was conducted. In China, 962 individuals were recruited, including 342 cases of skeletal fluorosis. Four TP2BA1 polymorphisms (rs2070759, rs12817819, rs17249754, and rs7136259) were analysed. The results suggested that rs17249754 and rs7136259 were associated with skeletal fluorosis. After controlling confounders, the protective effect of GG genotype in rs17249754 was apparent in individuals over 45 years old, female, with urine fluoride concentration below 1.6 mg/L, serum calcium above 2.25 mmol/L or serum phosphorus between 1.1 and 1.3. Heterozygote TC in rs7136259 increased the risk of skeletal fluorosis in subjects who are elderly, female, with urinary fluoride more than 1.6 mg/L, serum calcium more than 2.25 mmol/L and blood phosphorus between 1.1 and 1.3 mmol/L. Four loci were found to be tightly related by linkage disequilibrium analysis, and the frequency of distribution of haplotype GCGT was lower in the skeletal fluorosis group.

Plasma Membrane Calcium ATPase Regulates Stoichiometry of CD4+ T-Cell Compartments.

Immune responses involve mobilization of T cells within naïve and memory compartments. Tightly regulated Ca2+ levels are essential for balanced immune outcomes. How Ca2+ contributes to regulating compartment stoichiometry is unknown. Here, we show that plasma membrane Ca2+ ATPase 4 (PMCA4) is differentially expressed in human CD4+ T compartments yielding distinct store operated Ca2+ entry (SOCE) profiles. Modulation of PMCA4 yielded a more prominent increase of SOCE in memory than in naïve CD4+ T cell. Interestingly, downregulation of PMCA4 reduced the effector compartment fraction and led to accumulation of cells in the naïve compartment. In silico analysis and chromatin immunoprecipitation point towards Ying Yang 1 (YY1) as a transcription factor regulating PMCA4 expression. Analyses of PMCA and YY1 expression patterns following activation and of PMCA promoter activity following downregulation of YY1 highlight repressive role of YY1 on PMCA expression. Our findings show that PMCA4 adapts Ca2+ levels to cellular requirements during effector and quiescent phases and thereby represent a potential target to intervene with the outcome of the immune response.

Reduced secretion of parathyroid hormone and hypocalcemia in systemic heterozygous ATP2B1-null hypertensive mice.

The ATP2B1 gene is associated with hypertension. We previously reported that systemic heterozygous ATP2B1-null (ATP2B1+/-) mice exhibited hypertension due to impaired endothelial nitric oxide synthase (eNOS) activity and decreased nitric oxide (NO) production. The ATP2B1 gene encodes plasma membrane calcium ATPase 1 (PMCA1), which has been thought to regulate only intracellular Ca2+ concentration. However, recently, it has been suggested that ATP2B1 works not only at cellular levels, but also throughout the entire body, including in the calcium metabolism, using small intestine-specific ATP2B1 knockout mice. To clarify the roles of ATP2B1 in the entire body and the effects of ATP2B1 on blood pressure, we examined the alterations of calcium related factors in ATP2B1+/- mice. ATP2B1+/- mice exhibited hypocalcemia. The expression of ATP2B1 in the kidney and small intestine decreased, and hypercalciuria was confirmed in ATP2B1+/- mice. The intact-PTH levels were lower, and bone mineral density was increased in these mice. These results suggest that hypocalcemia is mainly a result of inhibited bone resorption without compensation by PTH secretion in the case of ATP2B1 knockout. Moreover, NO production may be affected by reduced PTH secretion, which may cause the increase in vascular contractility in these mice. The ATP2B1 gene is important for not only intra-cellular calcium regulation but also for calcium homeostasis and blood pressure control.

Mechanism and effect of shijueming (Concha Haliotidis) on serum calcium in spontaneously hypertensive rats.

OBJECTIVE: To observe the impact of Shijueming (Concha Haliotidis) on spontaneously hypertensive rats via blood pressure, serum calcium, vascular smooth muscle membrane L-type calcium channel alpha1 C subunit (CaL-alpha1C), plasma membrane calcium-ATPase (PMCA) mRNA expression, and the L-type calcium channel in vascular smooth muscle cells. METHODS: Twelve-week-old male rats with spontaneous hypertension were divided into three groups: a Shijueming (Concha Haliotidis) group (group 1), a nifedipine group (group 2), and a distilled water group (group 3). All were given a four-week treatment. Blood pressure and dissociative serum calcium were examined before treatment. Blood pressure was taken every week during treatment. Atomic absorption spectrometry was used to examine dissociative serum calcium. Reverse transcription-polymerase chain reaction was used to examine the expression of CaL-alpha1C and PMCA1 mRNA. The patch clamp technique was used to examine the electrophysiological characteristics of the vascular smooth muscle cell calcium channels. RESULTS: After treatment, blood pressure of the Shijueming (Concha Haliotidis) group lowered but not significantly (P > 0.05). Blood pressure of the nifedipine group lowered significantly (P < 0.05). Blood pressure of the distilled water group remained high. The concentration of serum calcium in the Shijueming (Concha Haliotidis) and the distilled water groups lowered (P < 0.05). Expression of CaL-alpha1C mRNA in the nifedipine group decreased compared with the distilled water group (P < 0.01). There was the decreasing trend in the Shijueming (Concha Haliotidis) group, but it was not statistically significant. Shijueming (Concha Haliotidis) also had effects on the expression of PMCA1 mRNA but without statistical significance. However, there was a significant decreasing effect on vascular smooth muscle cell I(Ca)-L flow. CONCLUSION: This study indicated that Shijueming (Concha Haliotidis) could increase serum calcium and decrease blood pressure. It may work by influencing calcium channels, expression of PMCA1 mRNA, and regulating ion calcium channels and calcium-ATPase.

Molecular cloning of a plasma membrane Ca²âº ATPase (PMCA) from Y-organs of the blue crab (Callinectes sapidus), and determination of spatial and temporal patterns of PMCA gene expression.

Existing data indicate that a stage-specific increase in intracellular free Ca(2+) stimulates ecdysteroid production by crustacean molting glands (Y-organs). The concentration of Ca(2+) in cytosol is controlled mainly by proteins intrinsic to the plasma membrane and to the membranes of organelles. Several families of proteins are involved, including Ca(2+) channels, Ca(2+) pumps (ATPases), and Ca(2+) exchangers. The family of Ca(2+) pumps includes plasma membrane calcium ATPases (PMCAs). As a step toward understanding the involvement of calcium signaling in regulation of ecdysteroidogenesis, we used a PCR-based cloning strategy (RT-PCR followed by 3'- and 5'-RACE) to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative PMCA. The 4292 base pair (bp) cDNA includes a 3510 bp open reading frame encoding a 1170-residue protein (Cas-PMCA). The conceptually translated protein has a relative molecular mass of 128.8×10(3) and contains all signature domains of an authentic PMCA, including ten transmembrane domains and a calmodulin binding site. The predicted membrane topography of Cas-PMCA is as expected for an authentic PMCA protein. A phylogenetic analysis of nonredundant amino acid sequences of PMCA proteins from different species showed Cas-PMCA clusters with other arthropod PMCA proteins. An assessment of tissue distribution showed the Cas-PMCA transcript to be broadly distributed in both neural and non-neural tissues. Studies using quantitative real-time PCR revealed stage-specific changes in Cas-PMCA abundance during the molting cycle, with peak expression occurring during premolt stage D2, a pattern consistent with the hypothesis that Cas-PMCA functions to maintain cellular Ca(2+) homeostasis in Y-organs.

In vitro studies on intestinal calcium and phosphate transport in horses.

Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca(2+)) and inorganic phosphate (P(i)) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca(2+) and P(i) transport are limited. The aim of the present study was to characterize the intestinal Ca(2+) and P(i) transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca(2+) transport at both mRNA and protein level. Among the different intestinal segments, the duodenum showed significant and highest active Ca(2+) absorption. The findings from RT-PCR and Western blot analysis suggest that the epithelial Ca(2+) channel TRPV6, the cytosolic calcium binding protein calbindin-D(9K) and the plasma membrane calcium ATPase PMCA may be involved in active transcellular Ca(2+) transport. Regarding the P(i) transport, the results indicate significant active P(i) secretion in the jejunum, but the contributing mechanisms remain unclear. A significant inhibiting effect of ouabain as an antagonist of the basolateral Na(+)/K(+)-ATPase on the serosal-to-mucosal P(i) transport suggests a pivotal role of Na(+) in jejunal P(i) transport in the horse.

Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca²+-ATPase.

Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca²+-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed.

Effects of duodenal redox status on calcium absorption and related genes expression in high-fat diet-fed mice.

OBJECTIVE: This study investigated whether duodenal redox imbalance induced by high-fat diet (HFD) influenced expression of genes involved in transcellular calcium absorption, thus leading to reduced intestinal calcium absorption. METHODS: Male C57BL/6 mice were randomly assigned to one of four groups with eight mice in each group. The control group consumed an ordinary diet (4.9% fat, w/w). The other three groups were fed a HFD (21.2% fat), the HFD plus 0.1% lipoic acid, or the HFD plus an additional 0.9% calcium supplement. After 9 wk, plasma and duodenal oxidative stress biomarkers including malondialdehyde, superoxide dismutase, catalase, total antioxidant capacity, reduced glutathione/oxidized glutathione ratio, and reactive oxygen species were examined. The intestinal calcium absorption state was evaluated through examining the calcium balance, bone mineral density, and calcium metabolism biomarkers. Furthermore, quantitative reverse transcription-polymerase chain reaction was carried out to analyze the changes in expression of transcellular calcium absorption-related genes. RESULTS: The HFD induced marked decreases in intestinal calcium absorption and bone mineral density of the whole body, accompanied by redox imbalance and increased oxidative damage in duodenum; duodenal expression of calbindin-D(9K), plasma membrane calcium ATPase (PMCA(1b)), and sodium-calcium exchanger was significantly down-regulated by 1.9-, 2.7-, and 1.5-fold, respectively. Furthermore, duodenal glutathione and oxidized glutathione (GSH/GSSG) ratios were strongly positively correlated with the apparent calcium absorption rate and the expression of PMCA(1b) and Calbindin-D(9K), whereas reactive oxygen species levels were negatively correlated with them. CONCLUSION: Our results demonstrated that a HFD-induced duodenal oxidation state could significantly down-regulate expression of calbindin-D(9K), PMCA(1b), and sodium-calcium exchanger, thus causing an inhibitory effect on intestinal calcium absorption.

High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae.

The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.

In search of the DFNA11 myosin VIIA low- and mid-frequency auditory genetic modifier.

OBJECTIVES: To evaluate the auditory, vestibular, and retinal characteristics of a large American DFNA11 pedigree with autosomal dominant progressive sensorineural hearing loss that first impacts the low- and mid-frequency auditory range. The pedigree (referred to as the HL2 family) segregates a myosin VIIA (MYO7A) mutation in exon 17 at DNA residue G2164C (MYO7A) that seems to be influenced by a genetic modifier that either rescues or exacerbates the MYO7A alteration. DNA analysis to examine single-nucleotide polymorphisms in 2 candidate modifier genes (ATP2B2 and Wolfram syndrome 1 [WFS1]) is summarized in this report. STUDY DESIGN: Family study. RESULTS: The degree of low- and mid-frequency hearing loss in HL2 family members segregating the MYO7A mutation varies from mild to more severe, with approximately the same number of HL2 family members falling at each end of the severity spectrum. The extent of hearing loss in HL2 individuals can vary between family generations. Differences in the degree of hearing loss in MYO7A HL2 family members may be mirrored by vestibular function in at least 2 of these same individuals. The single-nucleotide polymorphisms examined within ATP2B2 and WFS1 did not segregate with the mild versus more severe auditory phenotype. CONCLUSION: The severity of the auditory and vestibular phenotypes in MYO7A HL2 family members may run in parallel, suggesting a common modifier gene within the inner ear. The putative MYO7A genetic modifier is likely to represent a common polymorphism that is not linked tightly to the MYO7A mutation on the MYO7A allele.

Alternative splice variants of plasma membrane calcium-ATPases in human corneal epithelium.

Plasma membrane calcium-ATPases (PMCAs) play a critical role in regulating intracellular calcium concentration. Four genes encode PMCA proteins with alternative splicing of transcripts at three sites (A, B and C) serving to increase isoform diversity. Our previous work shows that all four PMCAs are expressed and have specific locations in human corneal epithelium (hCE). The present work examined which splice variants of PMCAs are expressed in hCE. Total RNA was extracted from hCE scraped from cadaver corneas of five different donors (two females and three males, age range 55-76 years). RT-PCR was performed using PMCA isoform-specific primers designed to amplify transcripts that included either splice site A or splice sites B and C. PMCA cDNAs were sequenced or cloned, and then sequenced. There was uniformity in the PMCA1 and PMCA4 expression profile among the five donors. Specifically, every donor expressed PMCA4 transcripts (4x at site A and 4b at site B/C). Every donor also expressed PMCA1 transcripts at sites B/C, specifically PMCA1b and PMCA1kb. In contrast, PMCA2 and PMCA3 expression varied; PCR DNAs were detected in two of five donors. One donor expressed PMCA2a and a novel PMCA2 variant we termed PMCA2((i)). PMCA3a transcript was demonstrated in a different donor. Finally, for all the donors, bands encoding site A transcripts for PMCA4 were obtained but no PCR transcripts were detected at site A for PMCA1, PMCA2 and PMCA3. This investigation showed that hCE expressed multiple splice variants of PMCA isoforms. Furthermore, this study documented the expression of the PMCA1k variant (PMCA1kb) previously only described in intestine and pancreatic beta cells and describes a novel PMCA2((i)) variant. Finally, this study suggests that the molecular configuration of PMCA1, PMCA2 and PMCA3 in the region of splice site A in hCE must be different than in other tissues since the same primers that produced site A transcripts in several other tissues were ineffective in priming PCR in hCE.

Sequence, annotation and developmental expression of the sea urchin Ca(2+) -ATPase family.

Whole genome sequence data permit the study of protein families regulating cellular homeostasis during development. Here we present a study of the sea urchin Ca(2+)-ATPases made possible by the Sea Urchin Genome Sequencing Project. This is of potential interest because adult sea urchins, their gametes and embryos live in the relatively high Ca(2+) concentration of 10 mM. Three Ca(2+)-ATPases regulate Ca(2+) levels in animal cells: plasma membrane Ca(2+)-ATPase (PMCA), sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and secretory pathway Ca(2+)-ATPase (SPCA). The primary structures of Sp-PMCA and Sp-SERCA in the sea urchin, Strongylocentrotus purpuratus (Sp), have been published. Here, we present the primary structure of Sp-SPCA, which is 912 amino acids and has 66% identity and 80% similarity to human SPCA1. Southern blots and genome analysis show that Sp-SPCA is a single copy gene. Each Sp Ca(2+)-ATPase is highly conserved when compared to its human ortholog, indicating that human and sea urchin share structurally similar energy driven Ca(2+) homeostasis mechanisms that have been maintained throughout the course of deuterostome evolution. Annotation using the assembled sea urchin genome reveals that Sp-SPCA, Sp-PMCA and Sp-SERCA have 23, 17 and 24 exons. RT-Q-PCR shows that transcripts of Sp-SPCA are at low levels compared to Sp-PMCA and Sp-SERCA. Gradual increases in Sp-PMCA and Sp-SERCA mRNA begin at the 18 hour hatched blastula stage and peak 4-5-fold higher by 25 h at the mid to late blastulae stage.

Molecular determinants for differential membrane trafficking of PMCA1 and PMCA2 in mammalian hair cells.

The plasma membrane Ca2+-ATPase-2 (PMCA2) is expressed in stereocilia of hair cells of the inner ear, whereas PMCA1 is expressed in the basolateral plasma membrane of hair cells. Both extrude excess Ca2+ from the cytosol. They are predicted to contain ten membrane-spanning segments, two large cytoplasmic loops as well as cytosolic N- and C-termini. Several isoform variants are generated for both PMCA1 and PMCA2 by alternative splicing, affecting their first cytosolic loop (A-site) and their C-terminal tail. To understand how these isoforms are differentially targeted in hair cells, we investigated their targeting regions and expression in hair cells. Our results show that a Leu-Ile motif in 'b'-tail splice variants promotes PMCA1b and PMCA2b basolateral sorting in hair cells. Moreover, apical targeting of PMCA2 depends on the size of the A-site-spliced insert, suggesting that the conformation of the cytoplasmic loop plays a role in apical targeting.