Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells

Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.     

Allosteric inhibitors of plasma membrane Ca pumps: Invention and applications of caloxins

Plasma membrane Ca2+pumps(PMCA)play a major role in Ca2+homeostasis and signaling by extruding cellular Ca2+with high affinity.PMCA isoforms are encoded by four genes which are expressed differentially in various cell types in normal and disease states.Therefore, PMCA isoform selective inhibitors would aid in delineating their role in physiology and pathophysiology.We are testing the hypothesis that extracellular domains of PMCA can be used as allosteric targets to obtain a novel class of PMCA-specific inhibitors termed caloxins. This review presents the concepts behind the invention of caloxins and our progress in this area.A section is also devoted to the applications of caloxins in literature. We anticipate that isoform-selective caloxins will aid in understanding PMCA physiology in health and disease. With strategies to develop therapeutics from bioactive peptides,caloxins may become clinically useful in car diovascular diseases,neurological disorders,retinopathy,cancer and contraception.     

Partitioning of the plasma membrane Ca2+-ATPase into lipid rafts in primary neurons: effects of cholesterol depletion

Spatial and temporal alterations in intracellular calcium [Ca(2+)](i) play a pivotal role in a wide array of neuronal functions. Disruption in Ca(2+) homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca(2+)-ATPase (PMCA) is a high affinity Ca(2+) transporter that plays a crucial role in the termination of [Ca(2+)](i) signals and in the maintenance of low [Ca(2+)](i) essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or 'lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca(2+) signaling in the central nervous system.     

Delayed activation of the plasma membrane calcium pump by a sudden increase in Ca2+: fast pumps reside in fast cells

There are four genes encoding isoforms of the plasma membrane Ca(2+) pump (PMCA). PMCA variability is increased by the presence of two splicing sites. Functional differences between the variants of PMCA have been described, but little is known about the adaptive advantages of this great diversity of pumps. In this paper we studied how the different isoforms respond to a sudden increase in Ca(2+) concentration. We found that different PMCAs are activated by Ca(2+) at different rates, PMCA 3f and 2a being the fastest, and 4b the slowest. The rate of activation by Ca(2+) depends both on the rate of calmodulin binding and the magnitude of the activation by calmodulin. We found that 2a is located in heart and the stereocilia of inner ear hair cells, 3f in skeletal muscle and 4b was identified in Jurkat cells. Both cardiac and skeletal muscle, and stereocilia recover very rapidly after a cytoplasmic Ca(2+)peak, while in Jurkat cells the recovery takes up to a minute. In stereocilia, 2a is the only method for export of Ca(2+), making the analysis of them unusually straightforward. This indicates that these rates of PMCA activation by Ca(2+) are correlated with the speed of Ca(2+) concentration decay after a Ca2 spike in the cells in which these variants of PMCA are expressed. The results suggest that the type of PMCA expressed will correspond with the speed of Ca(2+) signals in the cell.     

Emanuel Strehler's work on calcium pumps and calcium signaling

Cells are equipped with mechanisms to control tightly the influx, efflux and resting level of free calcium (Ca 2+ ). Inappropriate Ca 2+ signaling and abnormal Ca 2+ levels are involved in many clinical disorders including heart disease, Alzheimer’s disease and stroke. Ca 2+ also plays a major role in cell growth, differentiation and motility; disturbances in these processes underlie cell transformation and the progression of cancer. Accordingly, research in the Strehler laboratory is focused on a better understanding of the molecular "toolkit" needed to ensure proper Ca 2+ homeostasis in the cell, as well as on the mechanisms of localized Ca 2+ signaling. A longterm focus has been on the plasma membrane calcium pumps (PMCAs), which are linked to multiple disorders including hearing loss, neurodegeneration, and heart disease. Our work over the past 20 years or more has revealed a surprising complexity of PMCA isoforms with different functional characteristics, regulation, and cellular localization. Emerging evidence shows how specific PMCAs contribute not only to setting basal intracellular Ca 2+ levels, but also to local Ca 2+ signaling and vectorial Ca 2+ transport. A second major research arearevolves around the calcium sensor protein calmodulin and an enigmatic calmodulin-like protein (CALML3) that is linked to epithelial differentiation. One of the cellular targets of CALML3 is the unconventional motor protein myosin-10, which raises new questions about the role of CALML3 and myosin-10 in cell adhesion and migration in normal cell differentiation and cancer.     

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.     

Alternative splicing of the first intracellular loop of plasma membrane Ca2+-ATPase isoform 2 alters its membrane targeting

Plasma membrane Ca(2+)-ATPases (PMCAs) are involved in local Ca(2+) signaling and in the spatial control of Ca(2+) extrusion, but how different PMCA isoforms are targeted to specific membrane domains is unknown. In polarized MDCK epithelial cells, a green fluorescent protein-tagged PMCA4b construct was targeted to the basolateral membrane, whereas a green fluorescent protein-tagged PMCA2b construct was localized to both the apical and basolateral domain. The PDZ protein-binding COOH-terminal tail of PMCA2b was not responsible for its apical membrane localization, as a chimeric pump made of an NH(2)-terminal portion from PMCA4 and a COOH-terminal tail from PMCA2b was targeted to the basolateral domain. Deletion of the last six residues of the COOH terminus of either PMCA2b or PMCA4b did not alter their membrane targeting, suggesting that PDZ protein interactions are not essential for proper membrane localization of the pumps. Instead, we found that alternative splicing affecting the first cytosolic loop determined apical membrane targeting of PMCA2. Only the "w" form, which contains a 45-amino acid residue insertion, showed prominent apical membrane localization. By contrast, the x and z splice variants containing insertions of 14 and 0 residues, respectively, localized to the basolateral membrane. The w splice insert was the crucial determinant of apical PMCA2 localization, and this was independent of the splice configuration at the COOH-terminal end of the pump; both PMCA2w/b and PMCA2w/a showed prominent apical targeting, whereas PMCA2x/b, PMCA2z/b, and PMCA2z/a were confined to the basolateral membrane. These data report the first differential effect of alternative splicing within the first cytosolic loop of PMCA2 and help explain the selective enrichment of specific PMCA2 isoforms in specialized membrane compartments such as stereocilia of auditory hair cells.     

Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins

Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.     

Plasma membrane Ca2+ ATPase isoform 2b interacts preferentially with Na+/H+ exchanger regulatory factor 2 in apical plasma membranes.

Spatial and temporal regulation of Ca(2+) signaling require the assembly of multiprotein complexes linking molecules involved in Ca(2+) influx, sensing, buffering, and extrusion. Recent evidence indicates that plasma membrane Ca(2+) ATPases (PMCAs) participate in the control of local Ca(2+) fluxes, but the mechanism of multiprotein complex formation of specific PMCAs is poorly understood. Using the PMCA2b COOH-terminal tail as bait in a yeast two-hybrid screen, we identified the PSD-95, Dlg, ZO-1 (PDZ) domain-containing Na(+)/H(+) exchanger regulatory factor-2 (NHERF2) as an interacting partner. Protein pull-down and coimmunoprecipitation experiments using recombinant PMCA2b and PMCA4b as well as NHERF1 and NHERF2 showed that the interaction of PMCA2b with NHERF2 was specific and selective. PMCA4b did not interact with either of the NHERFs, and PMCA2b selectively preferred NHERF2 over NHERF1. Green fluorescent protein-tagged PMCA2b was expressed at the apical membrane in Madin-Darby canine kidney epithelial cells, where it colocalized with apically targeted NHERF2. Our study identifies NHERF2 as the first specific PDZ partner for PMCA2b not shared with PMCA4b, and demonstrates that PMCA splice forms differing only minimally in their COOH-terminal residues interact with unique PDZ proteins. NHERFs have been implicated in the targeting, retention and regulation of membrane proteins including the beta(2)-adrenergic receptor, cystic fibrosis transmembrane conductance regulator, and Trp4 Ca(2+) channel, and NHERF2 is now shown to also interact with PMCA2b. This interaction may allow the functional assembly of PMCA2b in a multiprotein Ca(2+) signaling complex, facilitating integrated cross-talk between local Ca(2+) influx and efflux.     

The isoform- and location-dependence of the functioning of the plasma membrane calcium pump

The plasma membrane is a specialised multi-component structure with inter- and intracellular signalling functions. Ca2+ plays a crucial role in cellular physiology, and an ATP-driven plasma membrane calcium pump (PMCA) plays the greatest role in the maintenance of a low free Ca2+ concentration in the cytoplasm. The enzyme is coded by four separate genes (PMCA 1-4), and, due to alternative splicing, more than 20 variants can exist. PMCA 1 and 4 isoforms are present in almost all tissues, whereas PMCA 2 and 3 are found in more specialised cell types. The variants differ primarily in their regulatory regions, thus the modulation of calcium pump activity strongly depends on the isoform and the membrane composition. The unique function of PMCA isoforms was confirmed using the practical experimental models - a rat pheochromocytoma cell line, a human neuroblastoma cell line, or, more recently, knockout mice. In addition, based on the finding that PMCA could interact with several specific signaling proteins, it was concluded that its location in defined sites of the cell membrane could be a prerequisite for efficient intercellular communication.     

Calcium fluxes in human trophoblast (BeWo) cells: calcium channels,calcium-ATPase,and sodium-calcium exchanger expression

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are poorly understood. In the current study, we have investigated the characteristics of Ca(2+) fluxes in relation with cell Ca(2+) homeostasis in the human placental trophoblast cell line BeWo. Time-courses of Ca(2+) uptake by BeWo cells displayed rapid initial entry (initial velocity (V(i)) of 3.42 +/- 0.35 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid declined of cell-associated (45)Ca(2+) with a V(i) of efflux (Ve(i)) of 3.30 +/- 0.08 nmol/mg protein/min. Further identification of membrane gates for Ca(2+) entry in BeWo cells was carried out. Expression of Ca(2+) transporter/channel CaT1 and L-type alpha(1S) subunit was showed by RT-PCR. However, mRNA for CaT2 channel and L-type alpha(1C) and alpha(1D) subunits were not revealed. Membrane systems responsible for intracellular Ca(2+) extrusion from BeWo cells were also investigated. Plasma membrane Ca(2+)-ATPases (PMCA) and Na/Ca exchangers (NCX) were detected by Western blot in BeWo cells. Expression of specific isoforms of PMCA and NCX was further investigated by RT-PCR. Messenger RNAs of four isoforms of PMCA (PMCA 1-4) were detected. The presence of messenger RNAs of two NCX isoforms (NCX1 and NCX3) was observed. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. Inorganic ions such as cadmium and manganese did not modify the Ca(2+) fluxes, however, barium increased cell-associated (45)Ca(2+) by, in part, by reducing radiolabel exit.     

RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.     

Plasma membrane calcium ATPases and cancer

The plasma membrane calcium ATPases (PMCAs) are vital regulators of basal Ca(2+) and shape the nature of intracellular free Ca(2+) transients after cellular stimuli and are thus regulators of a plethora of cellular processes. Studies spanning many years have identified that at least some cancers are associated with a remodeling of PMCA isoform expression. This alteration in Ca(2+) efflux capacity may have a variety of consequences including reduced sensitivity to apoptosis and increases in the responsiveness of cancer cells to proliferative stimuli. In this review we provide an overview of studies focused on PMCAs in the context of cancer. We discuss how the remodeling of PMCA expression could provide a survival and/or growth advantage to cancer cells, as well as the potential of pharmacological agents that target specific PMCA isoforms to be novel therapies for the treatment of cancer.     

Phenotypes of SERCA and PMCA knockout mice

P-type Ca2+-ATPases of the sarco(endo)plasmic reticulum (SERCAs) and plasma membrane (PMCAs) are responsible for maintaining the Ca2+ gradients across cellular membranes that are required for regulation of Ca2+-mediated signaling and other biological processes. Gene-targeting studies of SERCA isoforms 1, 2, and 3 and PMCA isoforms 1, 2, and 4 have confirmed some of the general functions proposed for these pumps, such as a major role in excitation-contraction coupling for SERCA1 and SERCA2 and housekeeping functions for PMCA1 and SERCA2, but have also revealed some unexpected phenotypes. These include squamous cell cancer and plasticity in the regulation of Ca2+-mediated exocytosis in SERCA2 heterozygous mutant mice, modulation of Ca2+ signaling in SERCA3-deficient mice, deafness and balance disorders in PMCA2 null mice, and male infertility in PMCA4 null mice. These unique phenotypes provide new information about the cellular functions of these pumps, the requirement of their activities for higher order physiological processes, and the pathophysiological consequences of pump dysfunction.     

Long-term exposure to imatinib reduced cancer stem cell ability through induction of cell differentiation via activation of MAPK signaling in glioblastoma cells

Cellular calcium homeostasis is controlled predominantly by the plasma membrane calcium pump (PMCA). From four PMCA isoforms, PMCA1 and PMCA4 are ubiquitous, while PMCA2 and PMCA3 are found in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in non-differentiated PC12 cells changed the cell morphology and triggered neuritogenesis. Using the microarrays, real-time PCR and immunodetection, we analyzed the effect of PMCA2 or PMCA3 reduction in PC12 cells on gene expression, with emphasis on calmodulin (CaM), neuromodulin (GAP43) and MAP kinases. In PMCA-suppressed lines total CaM increased, and the calm I and calm II genes appeared to be responsible for this effect. mRNA and protein levels of GAP43 were increased, however, the amount of phosphorylated form was lower than in control cells. Localization of CaM/GAP43 and CaM/pGAP43 differed between control and PMCA-reduced cells. In both PMCA-modified lines, amounts of ERK1/2 increased. While pERK1 decreased, the pERK2 level was similar in all examined lines. PMCA suppression did not change the p38 amount, but the p-p38 diminished. JNK2 protein decreased in both PMCA-reduced cells without changes in pJNK level. Microarray analysis revealed distinct expression patterns of certain genes involved in the regulation of cell cycle, proliferation, migration, differentiation, apoptosis and cell signaling. Suppression of neuron-specific PMCA isoforms affected the phenotype of PC12 cells enabling adaptation to the sustained increase in cytosolic Ca(2+) concentration. This is the first report showing function of PMCA2 and PMCA3 isoforms in the regulation of signaling pathways in PC12 cells.     

Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.     

The plasma membrane calcium pump displays memory of past calcium spikes. Differences between isoforms 2b and 4b

To understand how the plasma membrane Ca(2+) pump (PMCA) behaves under changing Ca(2+) concentrations, it is necessary to obtain information about the Ca(2+) dependence of the rate constants for calmodulin activation (k(act)) and for inactivation by calmodulin removal (k(inact)). Here we studied these constants for isoforms 2b and 4b. We measured the ATPase activity of these isoforms expressed in Sf9 cells. For both PMCA4b and 2b, k(act) increased with Ca(2+) along a sigmoidal curve. At all Ca(2+) concentrations, 2b showed a faster reaction with calmodulin than 4b but a slower off rate. On the basis of the measured rate constants, we simulated mathematically the behavior of these pumps upon repetitive changes in Ca(2+) concentration and also tested these simulations experimentally; PMCA was activated by 500 nm Ca(2+) and then exposed to 50 nm Ca(2+) for 10 to 150 s, and then Ca(2+) was increased again to 500 nm. During the second exposure to 500 nm Ca(2+), the activity reached steady state faster than during the first exposure at 500 nm Ca(2+). This memory effect is longer for PMCA2b than for 4b. In a separate experiment, a calmodulin-binding peptide from myosin light chain kinase, which has no direct interaction with the pump, was added during the second exposure to 500 nm Ca(2+). The peptide inhibited the activity of PMCA2b when the exposure to 50 nm Ca(2+) was 150 s but had little or no effect when this exposure was only 15 s. This suggests that the memory effect is due to calmodulin remaining bound to the enzyme during the period at low Ca(2+). The memory effect observed in PMCA2b and 4b will allow cells expressing either of them to remove Ca(2+) more quickly in subsequent spikes after an initial activating spike.