Inhibition of mucin secretion via glucocorticoid-induced regulation of calcium-related proteins in mouse lung.

Calcium is important for physiological functioning in many tissues and is essential in mucus secretion and muscle contraction. Intracellular concentrations of calcium are regulated by calcium-related proteins, such as transient receptor potential cation channel subfamily V member 4 (TRPV 4), TRPV6, Calbindin-D9k (CaBP-9k), sodium-calcium exchanger (NCX1), and plasma membrane Ca2+ ATPase 1 (PMCA1). In this study, the relationship between secretion of pulmonary mucus and calcium regulation was investigated. To confirm the effect of steroid hormones, immature mice were injected with estrogen (E2) or progesterone (P4), and mature mice were injected with dexamethasone (DEX). Subsequently, the location and expression of TRPV4, TRPV6, CaBP-9k, NCX1, and PMCA1 in lung tissue were examined. Periodic acid-Schiff staining was performed to investigate functional aspects of the protein expression. There were no significant differences in calcium-related gene expression in E2- and P4-treated mice, but TRPV4, NCX1, and PMCA1 were increased in DEX-treated mice and were recovered by RU486 treatment. DEX induces the expression of calcium-related proteins through the glucocorticoid receptor-mediated pathway and may involve decreased mucin secretion in the bronchiole. TRPV4, TRPV6, CaBP-9k, NCX1, and PMCA1 were specifically expressed in Clara and alveolar type 2 cells of mouse lung. CC10, a marker of Clara cells, was decreased by DEX. In addition, mucin secretion, which is a functional aspect of this cell, was also decreased by DEX treatment. Control of calcium-related gene expression may affect the control of mucus secretion in the lung. Such a control mechanism can form the basis of studies into diseases such as inflammation attributable to mucus secretion abnormalities, coughing, and respiratory disorders and distress.

Enhancement of intrarenal plasma membrane calcium pump isoform 1 expression in chronic angiotensin II-infused mice.

Plasma membrane calcium pump isoform 1 (PMCA1) is encoded by ATPase plasma membrane Ca2+transporting 1 (ATP2B1), the most likely candidate gene responsible for hypertension. Although PMCA1 is highly expressed in the kidney, little is known about regulation of its renal expression in various pathological conditions in vivo. Our study was designed to elucidate regulation of renal PMCA1 expression in mice. We employed three mouse models for kidney disease. These were the unilateral ureteral obstruction (UUO), the remnant kidney using 5/6 nephrectomy, and chronic angiotensin II administration models. Mice were assessed for systolic blood pressure and renal injury in accordance with the damage induced in the specific model. Kidney PMCA1 mRNA levels were measured in all mice. The UUO model showed renal fibrosis but no changes in blood pressure or renal PMCA1 mRNA expression. Similarly, the 5/6 nephrectomy model exhibited declined renal function without changes in blood pressure or renal PMCA1 mRNA expression. In contrast, chronic angiotensin II administration increased albuminuria and blood pressure as well as significantly increasing renal PMCA1 mRNA and protein expression. These results suggest that renal PMCA1 has a role as one of the molecules involved in angiotensin II-induced hypertension and kidney injury.

Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene.

Plasma membrane Ca2+-ATPases are key calcium exporter proteins in most tissues, and PMCA4b is the main calcium transporter in the human red blood cells (RBCs). In order to assess the expression level of PMCA4b, we have developed a flow cytometry and specific antibody binding method to quantitatively detect this protein in the erythrocyte membrane. Interestingly, we found several healthy volunteers showing significantly reduced expression of RBC-PMCA4b. Western blot analysis of isolated RBC membranes confirmed this observation, and indicated that there are no compensatory alterations in other PMCA isoforms. In addition, reduced PMCA4b levels correlated with a lower calcium extrusion capacity in these erythrocytes. When exploring the potential genetic background of the reduced PMCA4b levels, we found no missense mutations in the ATP2B4 coding regions, while a formerly unrecognized minor haplotype in the predicted second promoter region closely correlated with lower erythrocyte PMCA4b protein levels. In recent GWA studies, SNPs in this ATP2B4 haplotype have been linked to reduced mean corpuscular hemoglobin concentrations (MCHC), and to protection against malaria infection. Our data suggest that an altered regulation of gene expression is responsible for the reduced RBC-PMCA4b levels that is probably linked to the development of human disease-related phenotypes.

Clinical and Steroidogenic Characteristics of Aldosterone-Producing Adenomas With ATPase or CACNA1D Gene Mutations.

OBJECT: This comparative study clarified the clinical characteristics and in vitro steroidogenic activities of aldosterone-producing adenomas (APAs) harboring ATPase or CACNA1D gene mutations. DESIGN AND PATIENTS: Genetic testing was performed on 159 unilateral APAs. Somatic ATPase and CACNA1D gene mutations were analyzed in 42 APA tissues without KCNJ5 gene mutations. RESULTS: ATP1A1, ATP2B3, and CACNA1D mutations were detected in one, four, and four patients, respectively. Compared with patients without KCNJ5, ATPase, or CACNA1D mutations (wild type), ATPase mutations tended to have more severe hyperaldosteronism and smaller tumors; those with CACNA1D mutations had clinical characteristics and tumor sizes similar to those with wild-type genes. APAs with ATPase mutations were composed mainly of compact eosinophilic tumor cells, whereas CACNA1D mutations resulted in predominantly clear tumor cells. Aldosterone production in APA cells with ATP2B3 mutations were more responsive to dibutyryl cAMP, whereas those with CACNA1D mutations were more responsive to adrenocorticotropic hormone than the wild-type cells. CONCLUSION: APAs with ATPase mutations demonstrated a potentially severe primary aldosteronism phenotype, whereas those with CACNA1D mutations displayed characteristics similar to wild-type APAs. The status of stimulated aldosterone production was also different according to the cell types, suggesting that the regulatory effects of adrenocorticotropic hormone on aldosterone synthesis could possibly vary according to the intracellular signaling involved in hormone production.

Ethnic variability in the expression of hepatic drug transporters: absolute quantification by an optimized targeted quantitative proteomic approach.

Hepatic OATPs 1B1, 1B3 and 2B1, as well as P-gp, play important roles in regulating liver uptake and biliary excretion of drugs. The intrinsic ethnic variability in OATP1B1-mediated hepatic uptake of statins has been proposed to underlie the ethnic variability in plasma exposures of statins between Caucasians and Asians. Using a targeted quantitative proteomic approach, we determined hepatic protein concentrations of OATP1B1, OATP1B3, OATP2B1, P-gp, and PMCA4 (a housekeeping protein) in a panel of human livers (n = 141) and compared protein expression across Caucasian, Asian, African-American, and unidentified donors. Using an optimized protocol that included sodium deoxycholate as a membrane protein solubilizer, the hepatic protein expression levels (mean ± S.D.) of these transporters across all livers were determined to be 15.0 ± 6.0, 16.1 ± 8.1, 4.1 ± 1.3, 0.6 ± 0.2, and 2.4 ± 1.0 fmol/µg of total membrane protein, respectively. The scaling factor was 3.5 mg of total membrane protein in 100 mg of wet liver tissue. OATP1B1 protein expression was significantly associated with the c.388A>G (rs2306283, N130D) single nucleotide polymorphism. When compared across ethnicity, the hepatic expression levels of OATP1B1 and OATP1B3 were unexpectedly higher in Asians relative to Caucasians, suggesting that hepatic OATP expression alone does not explain the increased systemic statin levels in Asians compared with Caucasians. These findings may help improve physiologically based pharmacokinetic modeling to predict statin pharmacokinetic profiles and enable extrapolation of pharmacokinetic data of OATP substrates across ethnic groups.

The plethora of PMCA isoforms: Alternative splicing and differential expression.

In this review the four different genes of the mammalian plasma membrane calcium ATPase (PMCA) and their spliced isoforms are discussed with respect to their tissue distribution, their differences during development and their importance for regulating Ca²âº homeostasis under different conditions. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

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

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

Altered vascular smooth muscle function in the ApoE knockout mouse during the progression of atherosclerosis.

OBJECTIVES: Relaxation of vascular smooth muscle (VSM) requires re-uptake of cytosolic Ca(2+) into the sarcoplasmic reticulum (SR) via the Sarco/Endoplasmic Reticulum Ca(2+) ATPase (SERCA), or extrusion via the Plasma Membrane Ca(2+) ATPase (PMCA) or sodium Ca(2+) exchanger (NCX). Peroxynitrite, a reactive species formed in vascular inflammatory diseases, upregulates SERCA activity to induce relaxation but, chronically, can contribute to atherogenesis and altered vascular function by escalating endoplasmic reticulum stress. Our objectives were to determine if peroxynitrite-induced relaxation and Ca(2+) handling processes within vascular smooth muscle cells were altered as atherosclerosis develops. METHODS: Aortae from control and ApoE(-/-) mice were studied histologically, functionally and for protein expression levels of SERCA and PMCA. Ca(2+) responses were assessed in dissociated aortic smooth muscle cells in the presence and absence of extracellular Ca(2+). RESULTS: Relaxation to peroxynitrite was concentration-dependent and endothelium-independent. The abilities of the SERCA blocker thapsigargin and the PMCA inhibitor carboxyeosin to block this relaxation were altered during fat feeding and plaque progression. SERCA levels were progressively reduced, while PMCA expression was upregulated. In ApoE(-/-) VSM cells, increases in cytosolic Ca(2+) [Ca(2+)]c in response to SERCA blockade were reduced, while SERCA-independent Ca(2+) clearance was faster compared to control. CONCLUSION: As atherosclerosis develops in the ApoE(-/-) mouse, expression and function of Ca(2+) handling proteins are altered. Up-regulation of Ca(2+) removal via PMCA may offer a potential compensatory mechanism to help normalise the dysfunctional relaxation observed during disease progression.

Estrogen deficiency-induced Ca balance impairment is associated with decrease in expression of epithelial Ca transport proteins in aged female rats.

AIMS: The study is designed to determine whether estrogen and vitamin D endocrine systems interact to regulate calcium (Ca) balance as well as changes in mRNA expression of epithelial Ca transport proteins involved in intestinal and renal Ca transport in aging animals in response to ovariectomy and low dietary Ca intake. MAIN METHODS: Eleven-month-old female sham or ovariectomized (OVX) rats were divided into four groups and fed with either a low-Ca (LCD; 0.1% Ca, 0.65% P) or a high-Ca (HCD; 1.2% Ca, 0.65% P) diet for 12weeks. Ca balance and mRNA expression of Ca transport proteins in the intestine and kidney from rats were systematically studied. KEY FINDINGS: OVX rats fed with LCD resulted in a negative Ca balance. LCD suppressed serum Ca in OVX but not sham rats, resulting in an induction of serum PTH and 1,25(OH)2D3 levels. The surge in serum 1,25(OH)2D3 levels in LCD-fed OVX rats was associated with an increase in mRNA expression of intestinal transient receptor potential cation channel (TRPV6) and calbindin D9k (CaBP9k) as well as renal vitamin D receptor (VDR), but such an induction was unable to restore Ca balance in vivo. In contrast, the negative Ca balance was associated with suppression of intestinal plasma membrane Ca pump (PMCA1b) and renal transient receptor potential cation channel (TRPV5), calbindin D28k (CaBP28k) and PMCA1b mRNA expression in aged OVX rats. SIGNIFICANCE: Negative Ca balance in aged female OVX rats is associated with estrogen-dependent and vitamin D-independent downregulation of epithelial Ca transport protein mRNA expression.

Excessive signal transduction of gain-of-function variants of the calcium-sensing receptor (CaSR) are associated with increased ER to cytosol calcium gradient.

In humans, gain-of-function mutations of the calcium-sensing receptor (CASR) gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA) and reducing expression of Plasma Membrane Calcium-ATPase (PMCA). Wild-type CaSR (hCaSR-wt) and its gain-of-function (hCaSR-R990G; hCaSR-N124K) variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate) receptor inputs to cell function.

Impaired intestinal calcium absorption in protein 4.1R-deficient mice due to altered expression of plasma membrane calcium ATPase 1b (PMCA1b).

Protein 4.1R was first identified in the erythrocyte membrane skeleton. It is now known that the protein is expressed in a variety of epithelial cell lines and in the epithelia of many tissues, including the small intestine. However, the physiological function of 4.1R in the epithelial cells of the small intestine has not so far been explored. Here, we show that 4.1R knock-out mice exhibited a significantly impaired small intestinal calcium absorption that resulted in secondary hyperparathyroidism as evidenced by increased serum 1,25-(OH)2-vitamin D3 and parathyroid hormone levels, decreased serum calcium levels, hyperplasia of the parathyroid, and demineralization of the bones. 4.1R is located on the basolateral membrane of enterocytes, where it co-localizes with PMCA1b (plasma membrane calcium ATPase 1b). Expression of PMCA1b in enterocytes was decreased in 4.1(-/-) mice. 4.1R directly associated with PMCA1b, and the association involved the membrane-binding domain of 4.1R and the second intracellular loop and C terminus of PMCA1b. Our findings have enabled us to define a functional role for 4.1R in small intestinal calcium absorption through regulation of membrane expression of PMCA1b.

Characterization of a plasma membrane Ca(2+) ATPase expressed in olfactory receptor neurons of the moth Spodoptera littoralis.

The response of insect olfactory receptor neurons (ORNs) involves an increase in intracellular Ca(2+) concentration, as in vertebrate ORNs. In order to decipher the Ca(2+) clearance mechanisms in insect ORNs, we have investigated the presence of a plasma membrane Ca(2+) ATPase (PMCA) in the peripheral olfactory system of the moth Spodoptera littoralis. From an analysis of a male antennal expressed-sequence-tag database combined with a strategy of 5'/3' rapid amplification of cDNA ends plus the polymerase chain reaction, we have cloned a full-length cDNA encoding a PMCA. In adult males, the PMCA transcript has been found in various tissues, including the antennae in which its presence has been detected in the sensilla trichodea, and in cultured ORNs. The PMCA gene is slightly expressed at the end of the pupal stage, reaches a maximum at emergence and is maintained at a high level during the adult period. Taken together, these results provide, for the first time, molecular evidence for the putative participation of a PMCA in signalling pathways responsible for the establishment and functioning of the insect peripheral olfactory system.

Plasma membrane calcium ATPase expression in human colon multistep carcinogenesis.

The expression of the plasma membrane Ca(2+) ATPase (PMCA) was analyzed in a series of 84 formalin-fixed and paraffin embedded colon samples including normal mucosa (n = 32), adenoma (n = 19), adenocarcinoma (n = 27), and lymph node metastasis (n = 6) using (i) immunohistochemistry, (ii) mRNA in situ hybridization, and (iii) quantitative reverse-transcriptase PCR. A marked reduction of PMCA4 protein was observed in high-grade adenoma, colon cancer as well as lymph node metastasis, pointing to its potential role in the progression of cancer. However, PMCA4 RNA transcripts were unchanged or even increased in colon carcinomas, suggesting posttranscriptional regulation of PMCA4 during carcinogenesis.

Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2.

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.

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.

Adenovirus-delivered microRNA targeting the vitamin D receptor reduces intracellular Ca²âº concentrations by regulating the expression of Ca²âº-transport proteins in renal epithelial cells.

UNLABELLED: What's known on the subject? and What does the study add? Experimental data have shown that VDR overexpression in the duodenum and kidney cortex is a biological characteristic of genetic hypercalciuric stone-forming rats (GHS rat), and a link between idiopathic calcium stone formation and the microstatellite marker D12S339 (near the VDR locus) has been proven in humans. Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin-D28k and PMCA1b in NRK cell lines. VDR knockdown results in a decrease in intracellular Ca²âº concentration in NRK cell lines. The effect of the elevated VDR level in the kidney on hypercalciuria and the underlying mechanisms need to be further addressed. OBJECTIVE: • To determine the effects of vitamin D receptor (VDR) on hypercalciuria and the mechanisms underlying such effects. MATERIALS AND METHODS: • The adenovirus vector-delivered microRNA targeting rat VDR was constructed. We infected the normal rat kidney epithelial cell line NRK (Cellbank, China) with the adenovirus and then collected the cells at 0, 48, 72, 96, 120 h after infection. The mRNA and protein levels of VDR and VDR-dependent epithelial Ca2+ transport proteins were detected using real-time polymerase chain reaction and Western blot assays, respectively. • Fluorescent Ca²âº indicator Fluo-4 NW (Fluo-4 NW calcium assay kit, Molecular Probes, Invitrogen, USA) and laser scanning confocal microscope (Olympus, FV500-IX71, Japan) were used to detect the cytosolic free Ca²âº concentration at different time points after infection. RESULTS: • The mRNA and protein level of VDR, transient receptor potential vanilloid receptor subtype 5 (TRPV5), calbindin-D28k and plasma membrane Ca²âº-ATPase (PMCA1b) in infected NRK cells was significantly lower at 72 and 96 h after infection than that in control cells. • There was no significant difference between the two groups in the mRNA and protein level of TRPV6 and the Na⁺/Ca²âº-exchanger (NCX1). • Furthermore, VDR knockdown results in a decrease in intracellular Ca²âº concentration ([Ca²âº]i) in NRK cell lines. CONCLUSIONS: • Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin-D28k and PMCA1b, but not of TRPV6 or NCX1, in NRK cell lines. VDR knockdown results in a decrease in [Ca²âº]i in NRK cell lines. • The effect of the elevated VDR level in the kidney on hypercalciuria and the mechanisms underlying need to be further addressed.

Switch of PMCA4 splice variants in bovine epididymis results in altered isoform expression during functional sperm maturation.

Ca(2+) and Ca(2+)-dependent signals are essential for sperm maturation and fertilization. In mouse sperm the plasma membrane Ca(2+)-ATPase (PMCA) isoform 4 plays a crucial role in Ca(2+) transport. The two major splice variants of PMCA4 are PMCA4a and PMCA4b. PMCA4a differs from PMCA4b in the mechanism of calmodulin binding and activation. PMCA4a shows a much higher basal activity and is more effective than PMCA4b in returning Ca(2+) to resting levels. Knock-out mice carrying a PMCA4-null mutation are infertile because their sperm cannot achieve a hyperactivated state of motility. As sperm reach functional maturity during their transit through the epididymis, the expression of PMCA4a and 4b was assessed in bull testis and epididymis. Quantitative PCR revealed that PMCA4b is the major splice variant in testis, caput, and corpus epididymidis. In contrast, PMCA4a is the major splice variant in cauda epididymidis, whereas sperm are transcriptionally silent. Immunohistochemical staining using a new antibody against bovine PMCA4a located the PMCA4a to the apical membrane of the epithelium of cauda epididymidis, whereas testis, caput, and corpus epididymidis were negative. Western blotting of testis, epididymis, and sperm isolated from caput and cauda epididymidis showed a much higher level of PMCA4a in cauda epididymidis and sperm from cauda epididymidis compared with testis membranes and sperm from caput epididymidis. These findings suggest that PMCA4a is transferred to bovine sperm membranes in cauda epididymidis. This isoform switch may facilitate a higher calcium turnover in sperm necessary to traverse the female genital tract.

atp2b1a regulates Ca(2+) export during differentiation and regeneration of mechanosensory hair cells in zebrafish.

The molecular mechanisms of development of mechanosensory hair cells have been tackled successfully due to in vivo studies in the zebrafish lateral line. The enhancer trap (ET) transgenic line, SqET4 was instrumental in these studies even despite a lack of a link of its GFP expression pattern to a particular gene(s). We mapped the Tol2 transposon insertion of the SqET4 transgenics onto Chr. 4 next to a gene encoding Atp2b1a (Pmca1) - one of the four PMCAs acting to export Ca(2+) from a cell. atp2b1a expression recapitulates that of GFP during the development of mechanoreceptors of the inner ear and lateral line. atp2b1a expression correlates with the regeneration of these cells. Thus, SqET4 represents the Tg:atp2b1a-GFP line, which links Ca(2+) metabolism and the differentiation of mechanoreceptors. The morpholino-mediated knockdown of atp2b1a blocks Ca(2+) export and affects the division of hair cell progenitors, resulting in their accumulation. Under the control of a master gene of hair cells, Atoh1a, Atp2b1a functions during progenitor cell proliferation and hair cell differentiation. Given the similarity between the phenotypes of atp2b1a morphants and embryos treated with the pan-PMCA inhibitor 5(6)-carboxyeosin, Atp2b1a emerges as member of the Atp2b family responsible for Ca(2+) export during the development of hair cells.

Plasma membrane Ca2+-ATPase overexpression depletes both mitochondrial and endoplasmic reticulum Ca2+ stores and triggers apoptosis in insulin-secreting BRIN-BD11 cells.

Ca(2+) may trigger apoptosis in ß-cells. Hence, the control of intracellular Ca(2+) may represent a potential approach to prevent ß-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca(2+)-ATPase (PMCA) overexpression on Ca(2+)-regulated apoptosis in clonal ß-cells. Clonal ß-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca(2+)] using a combination of aequorins with different Ca(2+) affinities and on the ER and mitochondrial pathways of apoptosis. ß-cell stimulation generated microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. Overexpression of PMCA decreased [Ca(2+)] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing ß-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal ß-cell stimulation generates microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. PMCA overexpression depletes intracellular [Ca(2+)] stores and, despite a decrease in mitochondrial [Ca(2+)], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca(2+) homeostasis that could decrease ß-cell apoptosis in diabetes.

Reduced expression of the "fast" calcium transporter PMCA2a during homeostatic plasticity.

In a model of homeostatic plasticity, hippocampal slice culture CA3 pyramidal neurons responded to excitatory synapse inactivity by enhancing glutamate release through an increased number of miniature excitatory post-synaptic currents, mEPSCs and excitatory pre-synaptic terminals. Also accompanying these changes was a specific reduction in the expression of a "fast" calcium transporter, the plasma membrane calcium ATPase, PMCA2a. This transporter normally influences glutamate release from excitatory terminals where it helps control calcium levels. The reduction in PMCA2a expression occurred within 2 days of synapse inactivity; it was specific and reversible in young and mature hippocampal slice cultures and required removal of NMDA receptor mediated activity. Furthermore, the enhanced mEPSCs in the model were resistant to pharmacological inhibition of PMCA transporter activity. Reduced expression of PMCA2a during homeostatic plasticity therefore provides a mechanism to remodel pre-synaptic Ca2+ dynamics as a flexible way to alter glutamate release.