Plasma membrane calcium pump activity in rat pancreatic islets

The aim of this study was to quantify the glucose modulation of the plasma membrane calcium pump (PMCA) function in rat pancreatic islets. Ca²⁺-ATPase activity and levels of phosphorylated PMCA intermediates both transiently declined to a minimum in response to stimulation by glucose. Strictly dependent on Ca²⁺ concentration, this inhibitory effect was fully expressed at physiological concentrations of the cation (less than 0.5 μM), then progressively diminished at higher concentrations. These results, together with those previously reported on the effects of insulin secretagogues and blockers on the activity, expression and cellular distribution of the PMCA, support the concept that the PMCA plays a key role in the regulation of Ca²⁺ signaling and insulin secretion in pancreatic islets.     

Calmodulin effects on steroids‐regulated plasma membrane calcium pump activity

It is now generally accepted that non‐genomic steroids action precedes their genomic effects by modulation of intracellular signaling pathways within seconds after application. Ca²⁺ is a very potent and ubiquitous ion in all cells, and its concentration is precisely regulated. The most sensitive on Ca²⁺ increase is ATP‐consuming plasma membrane calcium pump (PMCA). The enzyme is coded by four genes, but isoforms diversity was detected in excitable and non‐excitable cells. It is the only ion pump stimulated directly by calmodulin (CaM). We examined the role of PMCA isoforms composition and CaM effect in regulation of Ca²⁺ uptake by estradiol, dehydroepiandrosterone (DHEA), pregnenolone (PREG), and their sulfates in a concentration range from 10^?9 to 10^?6 M, using the membranes from rat cortical synaptosomes, differentiated PC12 cells, and human erythrocytes. In excitable membranes with full set of PMCAs steroids apparently increased Ca²⁺ uptake, although to a variable extent. In most of the cases, CaM decreased transport by 30–40% below controls. Erythrocyte PMCA was regulated by the steroids somewhat differently than excitable cells. CaM strongly increased the potency for Ca²⁺ extrusion in membranes incubated with 17‐β‐estradiol and PREG. Our results indicated that steroids may sufficiently control cytoplasmic calcium concentration within physiological and therapeutic range. The response depended on the cell type, PMCA isoforms expression profile, CaM presence, and the steroids structure. Copyright © 2009 John Wiley & Sons, Ltd.     

Mutations in PMCA2 and hereditary deafness: a molecular analysis of the pump defect

The inner ear converts sound waves into hearing signals through the mechanoelectrical transduction (MET) process. Deflection of the stereocilia bundle of hair cells causes the opening of channels that allow the entry of endolymph K⁺ and Ca²⁺. Ca²⁺ that enters is crucial to the hearing process and is exported to the endolymph by the plasma membrane Ca²⁺ pump (isoform PMCA2w/a): disturbances of the balance between Ca²⁺ penetration and ejection, e.g. by pump mutations, generate deafness. Hearing loss caused by PMCA defects is frequently exacerbated by mutations in cadherin 23, a single pass stereociliar Ca²⁺ binding protein that forms the tip links which permit the deflection of the stereocilia bundle and thus the opening of the MET channels. The PMCA2w/a pump ejects Ca²⁺ to the endolymph even in the absence of the natural activator calmodulin. This satisfies the special Ca²⁺ homeostasis requirements of the stereocilia/endolymph system. Here we have analyzed a mice and a human previously described pump mutant. The human mutant only exacerbated the deafness produced by a cadherin 23 mutation. The murine mutant overexpressed in model cells displayed an evident defect both in the basal activity of the pump and in the long range ejection of Ca²⁺, the human mutant instead failed to impair the Ca²⁺ ejection by the pump.     

Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca clearance from MCF-7 breast cancer cells

The expression of the plasma membrane Ca²⁺ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca²⁺ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression – either by treatment or overexpression – led to enhanced Ca²⁺ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca²⁺ homeostasis. These results suggest that modulation of Ca²⁺ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.     

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

Hyperactivation of the Human Plasma Membrane Ca2+ Pump PMCA h4xb by Mutation of Glu99 to Lys

The transport of calcium to the extracellular space carried out by plasma membrane Ca²⁺ pumps (PMCAs) is essential for maintaining low Ca²⁺ concentrations in the cytosol of eukaryotic cells. The activity of PMCAs is controlled by autoinhibition. Autoinhibition is relieved by the binding of Ca²⁺-calmodulin to the calmodulin-binding autoinhibitory sequence, which in the human PMCA is located in the C-terminal segment and results in a PMCA of high maximal velocity of transport and high affinity for Ca²⁺. Autoinhibition involves the intramolecular interaction between the autoinhibitory domain and a not well defined region of the molecule near the catalytic site. Here we show that the fusion of GFP to the C terminus of the h4xb PMCA causes partial loss of autoinhibition by specifically increasing the V_max. Mutation of residue Glu⁹⁹ to Lys in the cytosolic portion of the M1 transmembrane helix at the other end of the molecule brought the V_max of the h4xb PMCA to near that of the calmodulin-activated enzyme without increasing the apparent affinity for Ca²⁺. Altogether, the results suggest that the autoinhibitory interaction of the extreme C-terminal segment of the h4 PMCA is disturbed by changes of negatively charged residues of the N-terminal region. This would be consistent with a recently proposed model of an autoinhibited form of the plant ACA8 pump, although some differences are noted.     

Mammary gland involution is associated with rapid down regulation of major mammary Ca-ATPases

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca²⁺-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca²⁺-ATPases and mammary calcium transport is unknown. We found that 24 h after stopping milk production, PMCA2 and secretory pathway Ca²⁺-ATPases 1 and 2 (SPCA1 and 2) expression decreased 80-95%. PMCA4 and Sarco/Endoplasmic Reticulum Ca²⁺-ATPase 2 (SERCA2) expression increased with the loss of PMCA2, SPCA1, and SPCA2 but did not increase until 72-96 h of involution. The rapid loss of these Ca²⁺-ATPases occurs at a time of high mammary tissue calcium. These results suggest that the abrupt loss of Ca²⁺-ATPases, required by the mammary gland to regulate the large amount of calcium associated with milk production, could lead to accumulation of cell calcium, mitochondria Ca²⁺ overload, calcium mediated cell death and thus play a part in early signaling of mammary involution.     

Role of Ca2+,Mg2+-ATPases in Diabetes-Induced Alterations in Calcium Homeostasis in Input Neurons of the Nociceptive System

In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca²⁺ in input neurons of the nociceptive system increases, Ca²⁺ signals are prolonged, while Ca²⁺ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca²⁺,Mg²⁺-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca²⁺,Mg²⁺-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca²⁺,Mg²⁺-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca²⁺ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities.     

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca²⁺-pump (PMCA) in the membrane region upon interaction with Ca²⁺, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [¹²⁵I]TID-PC/16, measuring the shift of conformation E₂ to the auto-inhibited conformation E₁I and to the activated E₁A state, titrating the effect of Ca²⁺ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca²⁺ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca²⁺ transport but does not modify the affinity for Ca²⁺ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E₁I to the activated E₁A conformation and thus modulating the transport of Ca²⁺. This is reflected in the different apparent constants for Ca²⁺ in the absence of CaM (calculated by Ca²⁺-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca²⁺ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca²⁺ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca²⁺ binding and activation.     

质膜Ca2+-ATPase的结构与功能

质膜Ca²⁺-ATPase（PMCA）是P型ATPase家族的一员,在真核细胞中主要负责信号刺激后胞内高浓度Ca²⁺的清除扫尾工作,并对维持静息状态下较低Ca²⁺浓度起着重要的调节作用.PMCA的一级结构已被确定,拓扑学结构显示,它有10个跨膜区和3个胞浆功能区.它的4个编码基因可产生4种亚型(PMCA 1～4),这些亚型在功能与分布上存在差异.PMCA的活性可被钙调蛋白等多种因素调节,这与其结构特征息息相关.近年来,PMCA已被证实与脂筏结构有一定关联,它在信号传导和细胞凋亡中的作用也成为目前科学研究的焦点.本文主要对PMCA的结构、亚型和功能的研究现状进行综述.     

Internalization of plasma membrane Ca2+-ATPase during Xenopus oocyte maturation

A transient increase in intracellular Ca²⁺ is the universal signal for egg activation at fertilization. Eggs acquire the ability to mount the specialized fertilization-specific Ca²⁺ signal during oocyte maturation. The first Ca²⁺ transient following sperm entry in vertebrate eggs has a slow rising phase followed by a sustained plateau. The molecular determinants of the sustained plateau are poorly understood. We have recently shown that a critical determinant of Ca²⁺ signaling differentiation during oocyte maturation is internalization of the plasma membrane calcium ATPase (PMCA). PMCA internalization is representative of endocytosis of several integral membrane proteins during oocyte maturation, a requisite process for early embryogenesis. Here we investigate the mechanisms regulating PMCA internalization. To track PMCA trafficking in live cells we cloned a full-length cDNA of Xenopus PMCA1, and show that GFP-tagged PMCA traffics in a similar fashion to endogenous PMCA. Functional data show that MPF activation during oocyte maturation is required for full PMCA internalization. Pharmacological and co-localization studies argue that PMCA is internalized through a lipid raft endocytic pathway. Deletion analysis reveal a requirement for the N-terminal cytoplasmic domain for efficient internalization. Together these studies define the mechanistic requirements for PMCA internalization during oocyte maturation.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

Effects of Phenylalanine and its Metabolites on Cytoplasmic Free Calcium in Cortical Neurons

Classic phenylketonuria (PKU) is characterized by brain lesions. However, its underlying neurotoxic mechanisms remain unknown. Based on our previous studies, we hypothesized that calcium might participate in PKU-associated neuropathy. In cultured cortical neurons, cytoplasmic free calcium concentration ([Ca²⁺]_i) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment immediately increased [Ca²⁺]_i, which began to decrease after 3 min. Moreover, [Ca²⁺]_i decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca²⁺]_i efflux. Phe-induced [Ca²⁺]_i decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca²⁺-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca²⁺]_i efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance ⁴⁵Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger (NCX) and Sarco/Endoplasmic reticulum Ca²⁺-ATPase (SERCA), respectively, did not elicit any changes in [Ca²⁺]_i. Specific siRNA against PMCA had an effect similar to vanadate. Since the brain injury induced by phenylalaninemia was thought to be a chronic process, we cultured cortical neurons in the presence of Phe for 2 weeks and measured [Ca²⁺]_i, PMCA activity and ⁴⁵Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and ⁴⁵Ca-uptake capability decreased from day 9, at the same time [Ca²⁺]_i increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca²⁺]_i and subsequent long-term increase in [Ca²⁺]_i.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Regulation of PKC Mediated Signaling by Calcium during Visceral Leishmaniasis

Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca²⁺ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca²⁺ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca²⁺ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca²⁺-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca²⁺. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca²⁺ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca²⁺-ATPases play prominent roles during visceral leishmaniasis.     

The phosphatase activity of the plasma membrane Ca pump. Activation by acidic lipids in the absence of Ca increases the apparent affinity for Mg

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg²⁺ and K⁺. Micromolar concentrations of Ca²⁺ inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca²⁺ concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca²⁺. The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K⁺ but the apparent affinity of the enzyme for Mg²⁺ increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg²⁺. Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca²⁺ free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca²⁺ and the activation improves the interaction of the enzyme with Mg²⁺.     

Inhibition of plasma membrane Ca2+-atpase pump activity in cultured c6 glioma cells by halothane and xenon

We have compared the effect of two inhalational anesthetics, halothane and xenon, on Ca²⁺-ATPase (PMCA) pumping activity in plasma membrane vesicles prepared from cultured rat C6 glioma cells. Halothane, at concentrations ranging from 0.5 to 1.75 vol% (equivalent to 0.5 to 1.6 MAC), significantly inhibited Ca²⁺ uptake (transport) by plasma membrane vesicles in a dose-related fashion. Xenon, at partial pressures ranging from 0.5 to 1.5 atm (equivalent to 0.5 to 1.6 MAC), similarly inhibited PMCA pumping activity. Additive effects on suppression of PMCA pump activity were observed when C6 cell plasma membrane vesicles were exposed to increasing partial pressures of xenon in the presence of halothane (1 vol%). Halothane also inhibited PMCA pumping in cells from two other lines of neural origin, B104 (rat neuroblastoma) and PC12 (rat pheochromocytoma). Studies described in this report support the thesis that PMCA in cells of neural origin is inhibited by quite different inhalational anesthetics at clinically relevant concentrations.