Dynamic regulation of [Ca2+]i by plasma membrane Ca(2+)-ATPase and Na+/Ca2+ exchange during capacitative Ca2+ entry in bovine vascular endothelial cells.

The dynamic regulation of Ca2+ extrusion by the plasma membrane Ca(2+)-ATPase (PMCA) and Na+/Ca2+ exchange (NCX) was investigated in single cultured calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorimetry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The quantitative analysis of the recovery from an increase of [Ca2+]i elicited by activation of capacitative Ca2+ entry (CCE) served to characterize kinetic parameters of these Ca2+ extrusion systems in the intact cell. In CPAE cells the PMCA is activated in a Ca(2+)- and time-dependent manner. Full activation of the pump occurs only after [Ca2+]i has been elevated for at least 1 min which results in an increase of the affinity of the pump for Ca2+ and an increase in the apparent maximal extrusion rate (Vmax). Application of calmodulin antagonists W-7 and calmidazolium chloride (compound R 24571) revealed that calmodulin is a major regulator of PMCA activity in vivo. Sequential and simultaneous inhibition of PMCA and NCX suggested that both contribute to Ca2+ extrusion in a non-additive fashion. The activity of one system is dynamically adjusted to compensate for changes in the extrusion rate by the alternative transporter. It was concluded that in vascular endothelial cells, the PMCA functions as a calmodulin-regulated, high-affinity Ca2+ removal system. The contribution by the low-affinity NCX to Ca2+ clearance became apparent at [Ca2+]i > approximately 150 nM under conditions of submaximal activation of the PMCA.     

Kinetics of inhibition of the plasma membrane calcium pump by vanadate in intact human red cells.

The lack of specific inhibitors of the plasma membrane Ca2+ pump (PMCA) has made vanadate (VO3-), a non-specific inhibitor, an invaluable tool in the study of PMCA function. However, three important properties of vanadate as an inhibitor of the PMCA in intact cells, namely its speed of action in different experimental conditions, the reversibility of its inhibitory effects at different doses, and its dose-response, had never been characterized, despite extensive use. We report here the speed, reversibility and dose-response of PMCA inhibition by vanadate in intact human red cells. Near maximal inhibitory concentrations (1mM) in the red cell suspension blocked almost instantly the uphill Ca2+ extrusion by the PMCA, regardless of the intracellular Ca2+ concentration, cation composition of the external media, membrane potential or volume-stability of the cell. PMCA inhibition by vanadate, at concentrations of 10mM and 1mM, was not reversed by washing, resuspending, and incubating the cells for up to 2h in vanadate-free media. Vanadate inhibited PMCA-mediated Ca2+ efflux in intact red cells with a K1/2 of approximately 3 microM, a value similar to that described for the Ca2+-ATPase in isolated red cell membranes.     

Cytochemical localization and quantification of plasma membrane Ca2+-ATPase activity in mollusc digestive gland cells

A cytochemical method allowing the localization and quantification of plasma membrane Ca2+-ATPase (PMCA) in frozen sections obtained from digestive gland cells of Mytilus galloprovincialis, Tapes tapes and Chamelea gallina, is presented. The method utilizes lead as a trapping agent of PO4(2-) ions released by Ca2+-ATPase activity. The amount of lead sulphide precipitate proportionally related to PMCA activity was quantified by a light microscopy digital imaging analysis system. The optimal assay conditions of Ca2+-ATPase activity evaluated at pH 7.4 were: 200 microM free Ca2+, 200 mM KCl, 2 mM ATP, and under such analysis conditions the enzyme showed a linear trend up to 60 min (at 20 degrees C). The PMCA activity was substrate specific: ADP was utilized only at a low rate (24% with respect to an equimolar ATP concentration), while glucose-6-phosphate and beta-glycerophosphate were poorly hydrolyzed. The enzyme activity was strongly inhibited by sodium ortho-vanadate. Our detection of a Ca2-ATPase activity at nanomolar concentrations of free Ca2+ suggests that we have identified a plasma membrane Ca2-ATPase involved in Ca2+ homeostasis. The Ca2+-ATPase was found to be localized in the basal part of the plasma membrane in the digestive gland cells of Mytilus galloprovincialis and Tapes tapes, but in the apical plasma membrane of Chamelea gallina. The possible implications of the different cellular distributions of PMCA activity is discussed.     

Different effects of Hg2+ and Cu2+ on mussel (Mytilus galloprovincialis) plasma membrane Ca2+-ATPase: Hg2+ induction of protein expression

Deregulation of Ca2+ homeostasis can produce serious effects on cell functioning due to an alteration of Ca2+ signaling. The aim of this study was to evaluate variations in plasma membrane Ca2+-ATPase (PMCA) induced in mussels by in vivo exposure to Cu2+ or Hg2+. PMCA activity was assayed using a cytochemical method allowing localization and in situ quantification of Ca2+-ATPase on cryostat tissue sections. The effects of fixed concentrations of Cu2+ (0.6 microM) or Hg2+ (1.3 microM) were evaluated after different times of exposure (1, 4, 6 days), while those of increasing amounts of Cu2+ (0.3, 0.6, 1.3 microM) or of Hg2+ (0.6, 1.3, 2.4 microM) were evaluated after 4 days. Cu2+ produces dose-dependent inhibition of PMCA in the digestive gland, with a minimum at the fourth day of treatment and a recovery at the sixth day. Conversely, Hg2+ induces a significant rise of PMCA activity, with a maximum at the fourth day. Similar results have been found after biochemical assay of PMCA, using plasma membranes obtained from density-gradient separation of gill homogenates. PMCA expression has been assessed by immunoprecipitation and Western immunoblotting on digestive gland homogenates, showing an induction after exposure to Hg2+ but not to Cu2+. In conclusion, Cu2+ does not vary PMCA expression but reduces PMCA activity, indicating PMCA inhibition; conversely, Hg2+ increases PMCA expression more than PMCA activity, suggesting that it also produces PMCA inhibition, but the induction of PMCA expression leads to a net increase in enzyme activity.     

The plasma membrane Ca2+-ATPase protein from red blood cells is not modified in preeclampsia

Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.     

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.     

Reincorporated plasma membrane Ca2+-ATPase can mediate B-Type Ca2+ channels observed in native membrane of human red blood cells

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1-5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in results between RBC and reincorporated PMCA, most probably attributable to the decrease in ATPase activity following the procedure of reincorporation, the present experimental conditions appear to reveal a channel-mode of the PMCA that shares many similarities with the B-type Ca2+ channel.     

Normal Ca2+ extrusion by the Ca2+ pump of intact red blood cells exposed to high glucose concentrations

The ATPase activity of the plasma membrane Ca2+ pump (PMCA) has been reported to be inhibited by exposure of red blood cell (RBC) PMCA preparations to high glucose concentrations. It has been claimed that this effect could have potential pathophysiological relevance in diabetes. To ascertain whether high glucose levels also affect PMCA transport function in intact RBCs, Ca2+ extrusion by the Ca2+-saturated pump [PMCA maximal velocity (V(max))] was measured in human and rat RBCs exposed to high glucose in vivo or in vitro. Preincubation of normal human RBCs in 30-100 mM glucose for up to 6 h had no effect on PMCA V(max). The mean V(max) of RBCs from 15 diabetic subjects of 12.9 +/- 0.7 mmol. 340 g Hb(-1). h(-1) was not significantly different from that of controls (14.3 +/- 0.5 mmol. 340 g Hb(-1). h(-1)). Similarly, the PMCA V(max) of RBCs from 11 streptozotocin-diabetic rats was not affected by plasma glucose levels more than three times normal for 6-8 wk. Thus exposure to high glucose concentrations does not affect the ability of intact RBCs to extrude Ca2+.     

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration.     

Isoform-specific up-regulation of plasma membrane Ca2+ATPase expression during colon and gastric cancer cell differentiation

In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.     

Plasma membrane calcium pumps in smooth muscle: from fictional molecules to novel inhibitors

Plasma membrane Ca2+ pumps (PMCA pumps) are Ca2+-Mg2+ ATPases that expel Ca2+ from the cytosol to extracellular space and are pivotal to cell survival and function. PMCA pumps are encoded by the genes PMCA1, -2, -3, and -4. Alternative splicing results in a large number of isoforms that differ in their kinetics and activation by calmodulin and protein kinases A and C. Expression by 4 genes and a multifactorial regulation provide redundancy to allow for animal survival despite genetic defects. Heterozygous mice with ablation of any of the PMCA genes survive and only the homozygous mice with PMCA1 ablation are embryolethal. Some PMCA isoforms may also be involved in other cell functions. Biochemical and biophysical studies of PMCA pumps have been limited by their low levels of expression. Delineation of the exact physiological roles of PMCA pumps has been difficult since most cells also express sarco/endoplasmic reticulum Ca2+ pumps and a Na+-Ca2+-exchanger, both of which can lower cytosolic Ca2+. A major limitation in the field has been the lack of specific inhibitors of PMCA pumps. More recently, a class of inhibitors named caloxins have emerged, and these may aid in delineating the roles of PMCA pumps.     

Effects of reactive oxygen species on brain synaptic plasma membrane Ca(2+)-ATPase.

The regulation of free intracellular calcium [Ca2+]i is altered in neurons from the aged brain, possibly due to reductions in the activity of Ca2+ transporters. The plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in Ca2+ homeostasis, and its kinetic properties change in aged rat brain. These changes could be due to oxidative modification of PMCA as a result of age-related chronic oxidative stresses. The present studies were undertaken to determine the sensitivity of the neuronal PMCA to in vitro exposure of synaptic plasma membranes (SPMs) to reactive oxygen species (ROS). We examined the effects of three oxidants including peroxyl radicals generated by azo-initiators, 2,2'-Azobis 2-amidinopropane dihydrochloride (AAPH) and 4,4'-Azobis 14-cyanovaleric acid (ACVA), hydrogen peroxide (H2O2), and peroxynitrite (ONOO-). Synaptic plasma membranes briefly exposed to these oxidants were analyzed for functional and structural alterations in PMCA. Although all three oxidants led to significant loss of PMCA activity, the effect of ONOO- was the most potent, followed by peroxyl radicals and H2O2. Kinetic analysis of PMCA activity after oxidant treatment showed decreases in Vmax without significant changes in K(act). Immunoblots revealed oxidant-induced cross-linking of PMCA molecules that were partially reversed under reducing conditions and completely reversed with addition of urea. The PMCA appears to be very sensitive to inhibition by ROS and hence may be a target of oxidative stress in the aging brain. Reduction in its activity may contribute to age-related alterations in neuronal [Ca2+]i regulation.     

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.     

Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes

We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.     

Expression of multiple plasma membrane Ca(2+)-ATPases in rat pancreatic islet cells

When stimulated by glucose, the pancreatic beta-cell displays large oscillations of intracellular free Ca2+ concentration ([Ca2+]i). To control [Ca2+]i, the beta-cell must be equipped with potent mechanisms for Ca2+ extrusion. We studied the expression of the plasma membrane Ca(2+)-ATPases (PMCA) in three insulin secreting preparations (a pure beta-cell preparation, RINm5F cells and pancreatic islet cells), using reverse-transcribed PCR, RNase protection assay and Western blotting. The four main isoforms, PMCA1, PMCA2, PMCA3 and PMCA4 were expressed in the three preparations. Six alternative splice mRNA variants, characterized at splice sites A, B and C were detected in the three preparations (rPMCA1xb, 2yb, 2wb, 3za, 3zc, 4xb), plus two additional variants in pancreatic islet cells (PMCA4za, 1xkb). The latter variant corresponded to a novel variant of rat PMCA1 gene lacking the exon coding for the 10th transmembrane segment, at splice site B. At the mRNA and protein level, five variants predominated (1xb, 2wb, 3za, 3zc, 4xb), whilst one additional isoform (4za), predominated at the protein level only. This provides the first evidence for the presence of PMCA2 and PMCA3 isoforms at the protein level in non-neuronal tissue. Hence, the pancreatic beta-cell is equipped with multiple PMCA isoforms with possible differential regulation, providing a full range of PMCAs for [Ca2+]i regulation.     

The role of Ca2+ transport across the plasma membrane for cell migration.

Cell migration plays a central role in many physiological and pathophysiological processes. On a cellular level it is based on a highly coordinated restructuring of the cytoskeleton, a continuous cycle of adhesion and de-adhesion as well as on the activity of ion channels and transporters. The cytoplasmic Ca2+ ([Ca2+]i) concentration is an important coordinator of these intracellular processes. Thus, [Ca2+]i must be tightly controlled in migrating cells. This is among other things achieved by the activity of Ca2+ permeable channels, the plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX) in the plasma membrane. Here, we wanted to determine the functional role of these transport proteins in cell migration. We therefore quantified the acute effect of inhibitors of these transport proteins (Gd3+, vanadate, KB-R7943) on migration, [Ca2+]i, and intracellular pH (pHi) of MDCK-F cells. Migration was monitored with computer-assisted time-lapse video microscopy. [Ca2+]i and pHi were measured with the fluorescent indicators fura-2 and BCECF. NCX expression in MDCK-F cells was verified with ion substitution experiments, and expression of PMCA was tested with RT-PCR. All blockers lead to a rapid impairment of cell migration. However, the most prominent effect is elicited by NCX-inhibition with KB-R7943. NCX-blockade leads to an almost complete inhibition of migration which is accompanied by a dose-dependent increase of [Ca2+]i and an intracellular alkalinisation. We show that inhibition of NCX and PMCA strongly affects lamellipodial dynamics of migrating MDCK-F cells. Taken together, our results show that PMCA and in particular NCX are of critical importance for cell migration.     

Role of third extracellular domain of plasma membrane Ca2+-Mg2+-ATPase based on the novel inhibitor caloxin 3A1

The plasma membrane Ca2+ pump (PMCA) is a Ca2+-Mg2+-ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+ ([Ca2+]i). It contains five putative extracellular domains (PEDs). Earlier we had reported that binding to PED2 leads to PMCA inhibition. Mutagenesis of residues in transmembrane domain 6 leads to loss of PMCA activity. PED3 connects transmembrane domains 5 and 6. PED3 is only five amino acid residues long. By screening a phage display library, we obtained a peptide sequence that binds this target. After examining a number of peptides related to this original sequence, we selected one that inhibits the PMCA pump (caloxin 3A1). Caloxin 3A1 inhibits PMCA but not the sarcoplasmic reticulum Ca2+-pump. Caloxin 3A1 did not inhibit formation of the 140 kDa acylphosphate intermediate from ATP or its degradation. Thus, PEDs play a role in the reaction cycle of PMCA even though sites for binding to the substrates Ca2+ and Mg-ATP2-, and the activator calmodulin are all in the cytosolic domains of PMCA. In endothelial cells exposed to low concentration of a Ca2+-ionophore, caloxin 3A1 caused a further increase in [Ca2+]i proving its ability to inhibit PMCA pump extracellularly. Thus, even though PED3 is the shortest PED, it plays key role in the PMCA function.     

The plasma membrane Ca2+ pump isoform 4a differs from isoform 4b in the mechanism of calmodulin binding and activation kinetics: implications for Ca2+ signaling

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., T?r?k, K., and Caride, A. J. (2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.     

(Ca2+ + Mg2+)-ATPase activator: its presence in human red blood cells and rabbit skeletal muscle.

We find that both human red blood cells and rabbit skeletal muscle contain a soluble activator which can stimulate (Ca2+ + Mg2+)-ATPase activity. The activator protein from either source can enhance the (Ca2+ + Mg2+)-ATPase of both the red blood cell membrane and the microsomal fraction from skeletal muscle. The data suggest that they are members of the class of Ca2+-binding modulator proteins. A possible physiological role for the skeletal muscle activator protein in the contractile process is discussed.