Monomeric α-synuclein activates the plasma membrane calcium pump.

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

The Plasma Membrane Calcium ATPases and Their Role as Major New Players in Human Disease.

The Ca2+ extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca2+ homeostasis and intracellular Ca2+ signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease.

Analysis of cholesterol-recognition motifs of the plasma membrane Ca2+-ATPase.

The plasma membrane Ca2+-ATPase (PMCA) is crucial for the fine tuning of intracellular calcium levels in eukaryotic cells. In this study, we show the presence of CARC sequences in all human and rat PMCA isoforms and we performed further analysis by molecular dynamics simulations. This analysis focuses on PMCA1, containing three CARC motifs, and PMCA4, with four CARC domains. In PMCA1, two CARC motifs reside within transmembrane domains, while the third is situated at the intracellular interface. The simulations depict more stable RMSD values and lower RMSF fluctuations in the presence of cholesterol, emphasizing its potential stabilizing effect. In PMCA4, a distinct dynamic was found. Notably, the total energy differences between simulations with cholesterol and phospholipids are pronounced in PMCA4 compared to PMCA1. RMSD values for PMCA4 indicate a more energetically favorable conformation in the presence of cholesterol, suggesting a robust interaction between CARCs and this lipid in the membranes. Furthermore, RMSF analysis for CARCs in both PMCA isoforms exhibit lower values in the presence of cholesterol compared to POPC alone. The analysis of H-bond occupancy and total energy values strongly suggests the potential interaction of CARCs with cholesterol. Given the crucial role of PMCAs in physiological calcium regulation and their involvement in diverse pathological processes, this study underscores the significance of CARC motifs and their interaction with cholesterol in elucidating PMCA function. These insights into the energetic preferences associated with CARC-cholesterol interactions offer valuable implications for understanding PMCA function in maintaining calcium homeostasis and addressing potential associated pathologies.

Conformational changes during the reaction cycle of plasma membrane Ca2+-ATPase in the autoinhibited and activated states.

Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity. Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2·Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

Structure, Function and Regulation of the Plasma Membrane Calcium Pump in Health and Disease.

In this review, I summarize the present knowledge of the structural and functional properties of the mammalian plasma membrane calcium pump (PMCA). It is outlined how the cellular expression of the different spliced isoforms of the four genes are regulated under normal and pathological conditions.

Crosstalk among Calcium ATPases: PMCA, SERCA and SPCA in Mental Diseases.

Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca2+ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca2+, neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and secretory pathway Ca2+-ATPase (SPCA) are considered efficient line of defense against abnormal Ca2+ rises. However, their role is not limited only to Ca2+ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein-calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca2+-ATPases to neuropathology, with a special emphasis on mental diseases.

Characterizations of PMCA2-interacting complex and its role as a calcium oxalate crystal-binding protein.

Three isoforms of plasma membrane Ca2+-ATPase (PMCA) are expressed in the kidney. While PMCA1 and PMCA4 play major role in regulating Ca2+ reabsorption, the role for PMCA2 remains vaguely defined. To define PMCA2 function, PMCA2-interacting complex was characterized by immunoprecipitation followed by nanoLC-ESI-Qq-TripleTOF MS/MS (IP-MS). After subtracting non-specific binders using isotype-controlled IP-MS, 474 proteins were identified as PMCA2-interacting partners. Among these, eight were known and 20 were potential PMCA2-interacting partners based on bioinformatic prediction, whereas other 446 were novel and had not been previously reported/predicted. Quantitative immuno-co-localization assay confirmed the association of PMCA2 with these partners. Gene ontology analysis revealed binding activity as the major molecular function of PMCA2-interacting complex. Functional validation using calcium oxalate monohydrate (COM) crystal-protein binding, crystal-cell adhesion, and crystal internalization assays together with neutralization by anti-PMCA2 antibody compared to isotype-controlled IgG and blank control, revealed a novel role of PMCA2 as a COM crystal-binding protein that was crucial for crystal retention and uptake. In summary, a large number of novel PMCA2-interacting proteins have been defined and a novel function of PMCA2 as a COM crystal-binding protein sheds light onto its involvement, at least in part, in kidney stone pathogenesis.

The serum protein renalase reduces injury in experimental pancreatitis.

Acute pancreatitis is a disease associated with inflammation and tissue damage. One protein that protects against acute injury, including ischemic injury to both the kidney and heart, is renalase, which is secreted into the blood by the kidney and other tissues. However, whether renalase reduces acute injury associated with pancreatitis is unknown. Here, we used both in vitro and in vivo murine models of acute pancreatitis to study renalase's effects on this condition. In isolated pancreatic lobules, pretreatment with recombinant human renalase (rRNLS) blocked zymogen activation caused by cerulein, carbachol, and a bile acid. Renalase also blocked cerulein-induced cell injury and histological changes. In the in vivo cerulein model of pancreatitis, genetic deletion of renalase resulted in more severe disease, and administering rRNLS to cerulein-exposed WT mice after pancreatitis onset was protective. Because pathological increases in acinar cell cytosolic calcium levels are central to the initiation of acute pancreatitis, we also investigated whether rRNLS could function through its binding protein, plasma membrane calcium ATPase 4b (PMCA4b), which excretes calcium from cells. We found that PMCA4b is expressed in both murine and human acinar cells and that a PMCA4b-selective inhibitor worsens pancreatitis-induced injury and blocks the protective effects of rRNLS. These findings suggest that renalase is a protective plasma protein that reduces acinar cell injury through a plasma membrane calcium ATPase. Because exogenous rRNLS reduces the severity of acute pancreatitis, it has potential as a therapeutic agent.

A V1143F mutation in the neuronal-enriched isoform 2 of the PMCA pump is linked with ataxia.

The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca2+ oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca2+ homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca2+ signaling in neurons demands the continuous activity of Ca2+ pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane Ca2+ATPases (PMCA pumps) play a key role in the regulation of Ca2+ handling in selected sub-plasma membrane microdomains. Among the four basic PMCA pump isoforms existing in mammals, isoforms 2 and 3 are particularly enriched in the nervous system. In humans, genetic mutations in the PMCA2 gene in association with cadherin 23 mutations have been linked to hearing loss phenotypes, while those occurring in the PMCA3 gene were associated with X-linked congenital cerebellar ataxias. Here we describe a novel missense mutation (V1143F) in the calmodulin binding domain (CaM-BD) of the PMCA2 protein. The mutant pump was present in a patient showing congenital cerebellar ataxia but no overt signs of deafness, in line with the absence of mutations in the cadherin 23 gene. Biochemical and molecular dynamics studies on the mutated PMCA2 have revealed that the V1143F substitution alters the binding of calmodulin to the CaM-BD leading to impaired Ca2+ ejection.

Selectivity of plasma membrane calcium ATPase (PMCA)-mediated extrusion of toxic divalent cations in vitro and in cultured cells.

In the recent years, the toxicity of certain divalent cations has been associated with the alteration of intracellular Ca2+ homeostasis. Among other mechanisms, these cations may affect the functionality of certain Ca2+-binding proteins and/or Ca2+ pumps. The plasma membrane calcium pump (PMCA) maintains Ca2+ homeostasis in eukaryotic cells by mediating the efflux of this cation in a process coupled to ATP hydrolysis. The aim of this work was to investigate both in vitro and in cultured cells if other divalent cations (Sr2+, Ba2+, Co2+, Cd2+, Pb2+ or Be2+) could be transported by PMCA. Current results indicate that both purified and intact cell PMCA transported Sr2+ with kinetic parameters close to those of Ca2+ transport. The transport of Pb2+ and Co2+ by purified PMCA was, respectively, 50 and 75% lower than that of Ca2+, but only Co2+ was extruded by intact cells and to a very low extent. In contrast, purified PMCA-but not intact cell PMCA-transported Ba2+ at low rates and only when activated by limited proteolysis or by phosphatidylserine addition. Finally, purified PMCA did not transport Cd2+ or Be2+, although minor Be2+ transport was measured in intact cells. Moreover, Cd2+ impaired the transport of Ca2+ through various mechanisms, suggesting that PMCA may be a potential target of Cd2+-mediated toxicity. The differential capacity of PMCA to transport these divalent cations may have a key role in their detoxification, limiting their noxious effects on cell homeostasis.

3D-QSAR and Molecular Docking Studies on the TcPMCA1-Mediated Detoxification of Scopoletin and Coumarin Derivatives.

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is an economically important agricultural pest that is difficult to prevent and control. Scopoletin is a botanical coumarin derivative that targets Ca2+-ATPase to exert a strong acaricidal effect on carmine spider mites. In this study, the full-length cDNA sequence of a plasma membrane Ca2+-ATPase 1 gene (TcPMCA1) was cloned. The sequence contains an open reading frame of 3750 bp and encodes a putative protein of 1249 amino acids. The effects of scopoletin on TcPMCA1 expression were investigated. TcPMCA1 was significantly upregulated after it was exposed to 10%, 30%, and 50% of the lethal concentration of scopoletin. Homology modeling, molecular docking, and three-dimensional quantitative structure-activity relationships were then studied to explore the relationship between scopoletin structure and TcPMCA1-inhibiting activity of scopoletin and other 30 coumarin derivatives. Results showed that scopoletin inserts into the binding cavity and interacts with amino acid residues at the binding site of the TcPMCA1 protein through the driving forces of hydrogen bonds. Furthermore, CoMFA (comparative molecular field analysis)- and CoMSIA (comparative molecular similarity index analysis)-derived models showed that the steric and H-bond fields of these compounds exert important influences on the activities of the coumarin compounds.Notably, the C3, C6, and C7 positions in the skeletal structure of the coumarins are the most suitable active sites. This work provides insights into the mechanism underlying the interaction of scopoletin with TcPMCA1. The present results can improve the understanding on plasma membrane Ca2+-ATPase-mediated (PMCA-mediated) detoxification of scopoletin and coumarin derivatives in T. cinnabarinus, as well as provide valuable information for the design of novel PMCA-inhibiting acaricides.

A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations.

The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype.

Modulation of plasma membrane Ca2+-ATPase by neutral phospholipids: effect of the micelle-vesicle transition and the bilayer thickness.

The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca(2+) pump (PMCA). We found that Ca(2+)-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca(2+)-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA.

The plasma membrane calcium pump: new ways to look at an old enzyme.

The three-dimensional structure of the PMCA pump has not been solved, but its basic mechanistic properties are known to repeat those of the other Ca(2+) pumps. However, the pump also has unique properties. They concern essentially its numerous regulatory mechanisms, the most important of which is the autoinhibition by its C-terminal tail. Other regulatory mechanisms involve protein kinases and the phospholipids of the membrane in which the pump is embedded. Permanent activation of the pump, e.g. by calmodulin, is physiologically as harmful to cells as its absence. The concept is now emerging that the global control of cell Ca(2+) may not be the main function of the pump; in some cell types, it could even be irrelevant. The main pump role would be the regulation of Ca(2+) in cell microdomains in which the pump co-segregates with partners that modulate the Ca(2+) message and transduce it to important cell functions.

Plasma membrane Ca(2+)-ATPase is a novel target for ketamine action.

Ketamine, a high affinity uncompetitive antagonist of voltage-dependent NMDA receptor, has been used for years as a dissociative anesthetic. Although the drug is considered as safe and well-tolerable, it is now evident that it can exert dose-dependent multidirectional effects acting on different cellular targets and pathways. The latest clinical studies also demonstrated its promising antidepressant action. However, the widespread use of this drug in humans is largely limited by a broad range of cognitive adverse effects that resemble some core symptoms of schizophrenia. In line with the hypothesis of unifying role of calcium in schizophrenia symptomology, we used ketamine-induced rat model of experimental psychosis to study the effect of 5-day ketamine treatment (30 mg/kg, ip) on the activity of plasma membrane Ca(2+)-ATPase. Whereas no change in a total amount of the enzyme in cortical synaptosomal membranes was observed, a decrease by ∼50% in hydrolytic activity, as well as lowered phosphointermediate formation were detected. Moreover, ketamine action appeared to be isoform-independent. The experiments on intact Ca(2+)-ATPase purified from vehicle-treated rat cortex revealed dose-dependent inhibition of enzymatic activity. Furthermore, ketamine decreased, but not eliminated, the stimulation by calmodulin. The inhibitory effect, although much weaker, was also evident for truncated form of calcium pump obtained following digestion by trypsin. Our results indicate that plasma membrane Ca(2+)-ATPase is a novel target for ketamine and putative interaction sites may involve central catalytic loop and calmodulin-binding domain.

Mutation of plasma membrane Ca2+ ATPase isoform 3 in a family with X-linked congenital cerebellar ataxia impairs Ca2+ homeostasis.

Ca(2+) in neurons is vital to processes such as neurotransmission, neurotoxicity, synaptic development, and gene expression. Disruption of Ca(2+) homeostasis occurs in brain aging and in neurodegenerative disorders. Membrane transporters, among them the calmodulin (CaM)-activated plasma membrane Ca(2+) ATPases (PMCAs) that extrude Ca(2+) from the cell, play a key role in neuronal Ca(2+) homeostasis. Using X-exome sequencing we have identified a missense mutation (G1107D) in the CaM-binding domain of isoform 3 of the PMCAs in a family with X-linked congenital cerebellar ataxia. PMCA3 is highly expressed in the cerebellum, particularly in the presynaptic terminals of parallel fibers-Purkinje neurons. To study the effects of the mutation on Ca(2+) extrusion by the pump, model cells (HeLa) were cotransfected with expression plasmids encoding its mutant or wild-type (wt) variants and with the Ca(2+)-sensing probe aequorin. The mutation reduced the ability of the PMCA3 pump to control the cellular homeostasis of Ca(2+). It significantly slowed the return to baseline of the Ca(2+) transient induced by an inositol-trisphosphate (InsP(3))-linked plasma membrane agonist. It also compromised the ability of the pump to oppose the influx of Ca(2+) through the plasma membrane capacitative channels.

Spontaneous Reconstitution of Functional Transmembrane Proteins During Bioorthogonal Phospholipid Membrane Synthesis.

Transmembrane proteins are critical for signaling, transport, and metabolism, yet their reconstitution in synthetic membranes is often challenging. Non-enzymatic and chemoselective methods to generate phospholipid membranes in situ would be powerful tools for the incorporation of membrane proteins. Herein, the spontaneous reconstitution of functional integral membrane proteins during the de novo synthesis of biomimetic phospholipid bilayers is described. The approach takes advantage of bioorthogonal coupling reactions to generate proteoliposomes from micelle-solubilized proteins. This method was successfully used to reconstitute three different transmembrane proteins into synthetic membranes. This is the first example of the use of non-enzymatic chemical synthesis of phospholipids to prepare proteoliposomes.

Conformational changes produced by ATP binding to the plasma membrane calcium pump.

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction.

As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca(2+) with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca(2+)-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca(2+)-ATPase activity was related to an increase in the apparent affinity for Ca(2+) and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca(2+) homeostasis.

A C-terminal di-leucine motif controls plasma membrane expression of PMCA4b.

Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.