Expression Of Plasma Membrane Calcium ATPase Research Articles

Neuroplastin expression is essential for hearing and hair cell PMCA expression

Hearing deficits impact on the communication with the external world and severely compromise perception of the surrounding. Deafness can be caused by particular mutations in the neuroplastin (Nptn) gene, which encodes a transmembrane recognition molecule of the immunoglobulin (Ig) superfamily and plasma membrane Calcium ATPase (PMCA) accessory subunit. This study investigates whether the complete absence of neuroplastin or the loss of neuroplastin in the adult after normal development lead to hearing impairment in mice analyzed by behavioral, electrophysiological, and in vivo imaging measurements. Auditory brainstem recordings from adult neuroplastin-deficient mice (Nptn−/−) show that these mice are deaf. With age, hair cells and spiral ganglion cells degenerate in Nptn−/− mice. Adult Nptn−/− mice fail to behaviorally respond to white noise and show reduced baseline blood flow in the auditory cortex (AC) as revealed by single-photon emission computed tomography (SPECT). In adult Nptn−/− mice, tone-evoked cortical activity was not detectable within the primary auditory field (A1) of the AC, although we observed non-persistent tone-like evoked activities in electrophysiological recordings of some young Nptn−/− mice. Conditional ablation of neuroplastin in Nptnlox/loxEmx1Cre mice reveals that behavioral responses to simple tones or white noise do not require neuroplastin expression by central glutamatergic neurons. Loss of neuroplastin from hair cells in adult NptnΔlox/loxPrCreERT mice after normal development is correlated with increased hearing thresholds and only high prepulse intensities result in effective prepulse inhibition (PPI) of the startle response. Furthermore, we show that neuroplastin is required for the expression of PMCA 2 in outer hair cells. This suggests that altered Ca2+ homeostasis underlies the observed hearing impairments and leads to hair cell degeneration. Our results underline the importance of neuroplastin for the development and the maintenance of the auditory system.

Correction: Calcium electroporation for treatment of sarcoma in preclinical studies

[This corrects the article DOI: 10.18632/oncotarget.24352.].

Central and Peripheral Nervous System Progenitors Derived from Human Pluripotent Stem Cells Reveal a Unique Temporal and Cell-Type Specific Expression of PMCAs.

The P-type ATPases family consists of ion and lipid transporters. Their unique diversity in function and expression is critical for normal development. In this study we investigated human pluripotent stem cells (hPSC) and different neural progenitor states to characterize the expression of the plasma membrane calcium ATPases (PMCAs) during human neural development and in mature mesencephalic dopaminergic (mesDA) neurons. Our RNA sequencing data identified a dynamic change in ATPase expression correlating with the differentiation time of the neural progenitors, which was independent of the neuronal progenitor type. Expression of ATP2B1 and ATP2B4 were the most abundantly expressed, in accordance with their main role in Ca2+ regulation and we observed all of the PMCAs to have a subcellular punctate localization. Interestingly in hPSCs ATP2B1 and ATP2B3 were highly expressed in a cell cycle specific manner and ATP2B2 and ATP2B4 were highly expressed in a hPSC sub-population. In neural rosettes a strong apical PMCA expression was identified in the luminal region. Lastly, we confirmed all PMCAs to be expressed in mesDA neurons, however at varying levels. Our results reveal that PMCA expression dynamically changes during stem cell differentiation and highlights the diverging needs of cell populations to regulate and properly integrate Ca2+ changes, which can ultimately correspond to changes in specific stem cell transcription states.

Calcium electroporation for treatment of sarcoma in preclinical studies.

Calcium electroporation (CaEP) describes the use of electric pulses (electroporation) to transiently permeabilize cells to allow supraphysiological doses of calcium to enter the cytosol. Calcium electroporation has successfully been investigated for treatment of cutaneous metastases in a clinical study. This preclinical study explores the possible use of calcium electroporation for treatment of sarcoma.A normal murine muscle cell line (C2C12), and a human rhabdomyosarcoma cell line (RD) were used in the undifferentiated and differentiated state. Electroporation was performed using 8 pulses of 100 μs at 600–1000 V/cm; with calcium (0, 0.5, 1, and 5 mM). Viability was examined by MTS assay, intracellular calcium levels were measured, and expression of plasma membrane calcium ATPase (PMCA) was investigated using western blotting. Calcium/sodium exchanger (NCX1), ryanodine receptor (RyR1) expression and cytoskeleton structure (zyxin/actin) were assessed by immunofluorescence. CaEP efficiency on RD tumors was tested in vivo in immuno-deficient mice.CaEP was significantly more efficient in RD than in normal cells. Intracellular Ca2+ levels after CaEP increased significantly in RD, whereas a lower increase was seen in normal cells. CaEP caused decreased expression of PMCA and NCX1 in malignant cells and RyR1 in both cell lines whereas normal cells exhibited increased expression of NCX1 after CaEP. Calcium electroporation also affected cytoskeleton structure in malignant cells.This study showed that calcium electroporation is tolerated significantly better in normal muscle cells than sarcoma cells and as an inexpensive and simple cancer treatment this could potentially be used in connection with sarcoma surgery for local treatment.

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.

Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells

Zinc oxide nanoparticle is one of the most important materials with diverse applications. However, it has been reported that zinc oxide nanoparticles are toxic to organisms, and that oxidative stress is often hypothesized to be an important factor in cytotoxicity mediated by zinc oxide nanoparticles. Nevertheless, the mechanism of toxicity of zinc oxide nanoparticles has not been completely understood. In this study, we investigated the cytotoxic effect of zinc oxide nanoparticles and the possible molecular mechanism involved in calcium homeostasis mediated by plasma membrane calcium ATPase in rat retinal ganglion cells. Real-time cell electronic sensing assay showed that zinc oxide nanoparticles could exert cytotoxic effect on rat retinal ganglion cells in a concentration-dependent manner; flow cytometric analysis indicated that zinc oxide nanoparticles could lead to cell damage by inducing the overproduction of reactive oxygen species. Furthermore, zinc oxide nanoparticles could also apparently decrease the expression level and their activity of plasma membrane calcium ATPase, which finally disrupt the intracellular calcium homeostasis and result in cell death. Taken together, zinc oxide nanoparticles could apparently decrease the plasma membrane calcium ATPase expression, inhibit their activity, cause the elevated intracellular calcium ion level and disrupt the intracellular calcium homeostasis. Further, the disrupted calcium homeostasis will trigger mitochondrial dysfunction, generate excessive reactive oxygen species, and finally initiate cell death. Thus, the disrupted calcium homeostasis is involved in the zinc oxide nanoparticle-induced rat retinal ganglion cell death.

Placental development and expression of calcium transporting proteins in the extraembryonic membranes of a placentotrophic lizard

Pseudemoia pagenstecheri is a viviparous Australian scincid lizard in which the maternal-embryonic placental interface is differentiated into structurally distinct regions. The chorioallantoic placenta contains an elliptical-shaped region, the placentome, characterized by hypertrophied uterine and embryonic epithelial cells supported by dense vascular networks. The remainder of the chorioallantoic placenta, the paraplacentome, is also highly vascularized but uterine and chorionic epithelia are thin. An omphaloplacenta with hypertrophied epithelia is located in the abembryonic hemisphere of the egg. There is extensive placental transport of organic and inorganic nutrients, e.g., 85-90% of neonatal calcium is received via placental transfer. Calcium uptake by extraembryonic membranes of squamates correlates with expression of the intracellular calcium binding protein, calbindin-D(28K) , and plasma membrane calcium ATPase (PMCA) is a marker for active calcium transport. We estimated expression of calbindin-D(28K) and PMCA in the chorioallantoic membrane in a developmental series of embryos using immunoblotting and used immunohistochemistry to define the cellular localization of calbindin-D(28K) to test the hypotheses that 1) expression of calcium transporting proteins is coincident with placental transport of calcium and 2) the placenta is functionally specialized for calcium transport in regions of structural differentiation. Calbindin-D(28K) and PMCA were detected at low levels in early stages of development and increased significantly prior to birth, when embryonic calcium uptake peaks. These data support the hypothesis that placental calcium secretion occurs over an extended interval of gestation, with increasing activity as embryonic demand escalates in late development. In addition, calbindin-D(28K) expression is localized in chorionic epithelial cells of the placentome and in the epithelium of the omphalopleure of the omphaloplacenta, which supports the hypothesis that regional structural differentiation in the placenta reflects functional specializations for calcium transport.

Endothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells

Nitric oxide (NO) plays a pivotal role in the regulation of cardiovascular physiology. Endothelial NO is mainly produced by the endothelial nitric oxide synthase (eNOS) enzyme. eNOS enzymatic activity is regulated at several levels, including Ca(2+)/calmodulin binding and the interaction of eNOS with associated proteins. There is emerging evidence indicating a role for the plasma membrane calcium ATPase (PMCA) as a negative regulator of Ca(2+)/calmodulin-dependent signal transduction pathways via its interaction with partner proteins. The aim of our study was to investigate the possibility that the activity of eNOS is regulated through its association with endothelial PMCA. We show here a novel interaction between endogenous eNOS and PMCA in human primary endothelial cells. The interaction domains were located to the region 735-934 of eNOS and the catalytic domain of PMCA. Ectopic expression of PMCA in endothelial cells resulted in an increase in phosphorylation of the residue Thr-495 of endogenous eNOS. However, disruption of the PMCA-eNOS interaction by expression of the PMCA interaction domain significantly reversed the PMCA-mediated effect on eNOS phosphorylation. These results suggest that eNOS activity is negatively regulated via interaction with PMCA. Moreover, NO production by endothelial cells was significantly reduced by ectopic expression of PMCA. Our results show strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways.

Plasma membrane calcium pumps and their emerging roles in cancer

Alterations in calcium signaling and/or the expression of calcium pumps and channels are an increasingly recognized property of some cancer cells. Alterations in the expression of plasma membrane calcium ATPase (PMCA) isoforms have been reported in a variety of cancer types, including those of breast and colon, with some studies of cancer cell line differentiation identifying specific PMCA isoforms, which may be altered in some cancers. Some studies have also begun to assess levels of PMCA isoforms in clinical tumor samples and to address mechanisms of altered PMCA expression in cancers. Both increases and decreases in PMCA expression have been reported in different cancer types and in many cases these alterations are isoform specific. In this review, we provide an overview of studies investigating the expression of PMCA in cancer and discuss how both the overexpression and reduced expression of a PMCA isoform in a cancer cell could bestow a growth advantage, through augmenting responses to proliferative stimuli or reducing sensitivity to apoptosis.

Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways

Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular free calcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulin-dependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.

Antisense-mediated Inhibition of the Plasma Membrane Calcium-ATPase Suppresses Proliferation of MCF-7 Cells

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anti-cancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

Localization of Alkaline Phosphatase and Ca2+-ATPase in the Cat Placenta

We localized alkaline phosphatase and plasma membrane calcium-ATPase (PMCA) in the cat placental syncytiotrophoblast to address their polarized distribution and their potential as markers for specific plasma membrane purification. We used enzyme- (alkaline phosphatase) and immuno- (PMCA) histochemistry and, for alkaline phosphatase, compared data to observations on the human placenta. Alkaline phosphatase activity in the cat was localized to the decidual cell membranes, to within the associated interstitial space and on the subjacent apical (maternal facing) plasma membrane of the syncytiotrophoblast. Occasional maternal capillaries were positive on their basal surface and there was focal staining within the syncytiotrophoblast. This widespread distribution is less specific than in the human placenta where alkaline phosphatase was restricted to the apical and basal plasma syncytiotrophoblast membranes, with much greater density on the apical membrane. Expression of PMCA in the cat was restricted to the basal membrane of the syncytiotrophoblast only. This specific localization of PMCA is identical to the human placenta and all other species in which its placental localization has been studied. We conclude that the plasma membranes of the cat syncytiotrophoblast show a broadly similar functional polarization to the human and that PMCA would prove a useful marker in isolation of the cat syncytiotrophoblast basal plasma membrane.

Decreased PMCA4b expression has no effect on calcium homeostasis in Meg-01 cells

PMCA (plasma membrane calcium ATPase) is an energy-driven membrane transporter that pumps calcium out of the cell cytosol. Stable resting calcium and highly regulated cytosolic calcium fluxes must be maintained for proper cellular function. The primary function of PMCA in calcium homeostasis is to regulate the steady-state calcium concentration while cells are still at rest. We examined the effects of stable production of antisense RNAs targeted to the PMCA subtype 4b (PMCA4b) on cultured human megakaryoblastic (Meg-01) cells. The expression of PMCA-4b in these cells was diminished by approximately 50% as assessed by Western immunoblotting and in vitro ATPase assay. It was also determined that endogenous expression of PMCA1b in these cells was at a level such as that it can not be detected by Western immunoblotting. The rate of calcium efflux catalyzed by PMVA4b was inhibited in cells with decreased PMCA4b expression by approximately 60%. However, there was no difference in extrusion rate when sarco(endo)plasmic reticular ATPases (SERCA) were not functional. The resting levels of intracellular calcium concentrations in these cells were also not distinguishable from those of wild-type cells. These results suggest that a decrease in PMCA expression in Meg-01 cells is compensated to maintain normal intracellular calcium levels.

Plasma membrane calcium ATPase plays a role in reducing Ca(2+)-mediated cytotoxicity in PC12 cells.

In many cell types, cell death induced by a variety of insults is accompanied by an increase in intracellular calcium. The Ca(2+) homeostatic mechanisms affected by such insults, however, have not been fully determined. Recent evidence indicates that kainic acid-induced seizures alter plasma membrane calcium ATPase mRNA expression within vulnerable hippocampal cell populations before the onset of cell death. We examined the effects of altering plasma membrane calcium ATPase expression on cell vulnerability in rat pheochromocytoma 12 cells. Pheochromocytoma 12 cells are vulnerable to Ca(2+) overload induced by the Ca(2+) ionophore A23187. Reverse transcriptase-PCR and Western blot data indicated that plasma membrane calcium ATPase isoform 4b constitutes a major calcium pump isoform in the pheochromocytoma 12 cells. Therefore, permanently transfected pheochromocytoma 12-derived cell lines were established that either over-expressed plasma membrane calcium ATPase isoform 4b, or suppressed the expression of the endogenous plasma membrane calcium ATPase isoform 4. Over-expressing clones were less vulnerable to Ca(2+)-mediated cell death induced by A23187 whereas "antisense" clones were considerably more susceptible. These data indicate that regulation of plasma membrane calcium ATPase expression may be critical to cellular survival when cells are exposed to pathological increases in intracellular calcium.