Evidence for a role of plasma membrane calcium pumps in neurodegenerative disease: Recent developments.

Plasma membrane Ca2+ ATPases (PMCAs) are a major system for calcium extrusion from all cells. Different PMCA isoforms and splice variants are involved in the precise temporal and spatial handling of Ca2+ signals and the re-establishment of resting Ca2+ levels in the nervous system. Lack or inappropriate expression of specific PMCAs leads to characteristic neuronal phenotypes, which may be reciprocally exacerbated by genetic predisposition through alleles in other genes that modify PMCA interactions, regulation, and function. PMCA dysfunction is often poorly compensated in neurons and may lead to changes in synaptic transmission, altered excitability and, with long-term calcium overload, eventual cell death. Decrease and functional decline of PMCAs are hallmarks of neurodegeneration during aging, and mutations in specific PMCAs are responsible for neuronal dysfunction and accelerated neurodegeneration in many sensory and cognitive diseases.

Cortical cytoskeleton dynamics regulates plasma membrane calcium ATPase isoform-2 (PMCA2) activity.

We have previously shown that purified actin can directly bind to human plasma membrane Ca2+ ATPase 4b (hPMCA4b) and exert a dual modulation on its Ca2+-ATPase activity: F-actin inhibits PMCA while short actin oligomers may contribute to PMCA activation. These studies had to be performed with purified proteins given the nature of the biophysical and biochemical approaches used. To assess whether a functional interaction between the PMCAs and the cortical cytoskeleton is of physiological relevance, we characterized this phenomenon in the context of a living cell by monitoring in real-time the changes in the cytosolic calcium levels ([Ca2+]CYT). In this study, we tested the influence of drugs that change the actin and microtubule polymerization state on the activity and membrane expression of the PMCA transiently expressed in human embryonic kidney (HEK293) cells, which allowed us to observe and quantify these relationships in a live cell, for the first time. We found that disrupting the actin cytoskeleton with cytochalasin D significantly increased PMCA-mediated Ca2+ extrusion (~50-100%) whereas pre-treatment with the F-actin stabilizing agent jasplakinolide caused its full inhibition. When the microtubule network was disrupted by pretreatment of the cells with colchicine, we observed a significant decrease in PMCA activity (~40-60% inhibition) in agreement with the previously reported role of acetylated tubulin on the calcium pump. In none of these cases was there a difference in the level of expression of the pump at the cell surface, thus suggesting that the specific activity of the pump was the regulated parameter. Our results indicate that PMCA activity is profoundly affected by the polymerization state of the cortical cytoskeleton in living cells.

Deletion of the intestinal plasma membrane calcium pump, isoform 1, Atp2b1, in mice is associated with decreased bone mineral density and impaired responsiveness to 1, 25-dihydroxyvitamin D3.

The physiological importance of the intestinal plasma membrane calcium pump, isoform 1, (Pmca1, Atp2b1), in calcium absorption and homeostasis has not been previously demonstrated in vivo. Since global germ-line deletion of the Pmca1 in mice is associated with embryonic lethality, we selectively deleted the Pmca1 in intestinal absorptive cells. Mice with loxP sites flanking exon 2 of the Pmca1 gene (Pmca1(fl/fl)) were crossed with mice expressing Cre recombinase in the intestine under control of the villin promoter to give mice in which the Pmca1 had been deleted in the intestine (Pmca1(EKO) mice). Pmca1(EKO) mice were born at a reduced frequency and were small at the time of birth when compared to wild-type (Wt) littermates. At two months of age, Pmca1(EKO) mice fed a 0.81% calcium, 0.34% phosphorus, normal vitamin D diet had reduced whole body bone mineral density (P < 0.037), and reduced femoral bone mineral density (P < 0.015). There was a trend towards lower serum calcium and higher serum parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) concentrations in Pmca1(EKO) mice compared to Wt mice but the changes were not statistically significant. The urinary phosphorus/creatinine ratio was increased in Pmca1(EKO) mice (P < 0.004). Following the administration of 200 ng of 1α,25(OH)2D3 intraperitoneally to Wt mice, active intestinal calcium transport increased ∼2-fold, whereas Pmca1(EKO) mice administered an equal amount of 1α,25(OH)2D3 failed to show an increase in active calcium transport. Deletion of the Pmca1 in the intestine is associated with reduced growth and bone mineralization, and a failure to up-regulate calcium absorption in response to 1α,25(OH)2D3.

Plasma membrane calcium ATPases: From generic Ca(2+) sump pumps to versatile systems for fine-tuning cellular Ca(2.).

The plasma membrane calcium ATPases (PMCAs) are ATP-driven primary ion pumps found in all eukaryotic cells. They are the major high-affinity calcium extrusion system for expulsion of Ca(2+) ions from the cytosol and help restore the low resting levels of intracellular [Ca(2+)] following the temporary elevation of Ca(2+) generated during Ca(2+) signaling. Due to their essential role in the maintenance of cellular Ca(2+) homeostasis they were initially thought to be "sump pumps" for Ca(2+) removal needed by all cells to avoid eventual calcium overload. The discovery of multiple PMCA isoforms and alternatively spliced variants cast doubt on this simplistic assumption, and revealed instead that PMCAs are integral components of highly regulated multi-protein complexes fulfilling specific roles in calcium-dependent signaling originating at the plasma membrane. Biochemical, genetic, and physiological studies in gene-manipulated and mutant animals demonstrate the important role played by specific PMCAs in distinct diseases including those affecting the peripheral and central nervous system, cardiovascular disease, and osteoporosis. Human PMCA gene mutations and allelic variants associated with specific disorders continue to be discovered and underline the crucial role of different PMCAs in particular cells, tissues and organs.

Plasma membrane Ca²âº-ATPases can shape the pattern of Ca²âº transients induced by store-operated Ca²âº entry.

Calcium (Ca(2+)) is a critical cofactor and signaling mediator in cells, and the concentration of cytosolic Ca(2+) is regulated by multiple proteins, including the plasma membrane Ca(2+)-ATPases (adenosine triphosphatases) (PMCAs), which use ATP to transport Ca(2+) out of cells. PMCA isoforms exhibit different kinetic and regulatory properties; thus, the presence and relative abundance of individual isoforms may help shape Ca(2+) transients and cellular responses. We studied the effects of three PMCA isoforms (PMCA4a, PMCA4b, and PMCA2b) on Ca(2+) transients elicited by conditions that trigger store-operated Ca(2+) entry (SOCE) and that blocked Ca(2+) uptake into the endoplasmic reticulum in HeLa cells, human embryonic kidney (HEK) 293 cells, or primary endothelial cell isolated from human umbilical cord veins (HUVECs). The slowly activating PMCA4b isoform produced long-lasting Ca(2+) oscillations in response to SOCE. The fast-activating isoforms PMCA2b and PMCA4a produced different effects. PMCA2b resulted in rapid and highly PMCA abundance-sensitive clearance of SOCE-mediated Ca(2+) transients, whereas PMCA4a reduced cytosolic Ca(2+), resulting in the establishment of a higher than baseline cytosolic Ca(2+) concentration. Mathematical modeling showed that slow activation was critical to the sustained oscillation induced by the "slow" PMCA4b pump. The modeling and experimental results indicated that the distinct properties of PMCA isoforms differentially regulate the pattern of SOCE-mediated Ca(2+) transients, which would thus affect the activation of downstream signaling pathways.

Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm.

PMCA4, a membrane protein, is the major Ca(2+) efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. We have previously shown that the PMCA4b splice variant interacts with CASK (Ca(2+/)CaM-dependent serine kinase) in regulating sperm Ca(2+). More recently we detected that PMCA4a isoform, in addition to its presence in testis, is secreted in the epididymal luminal fluid and transferred to sperm. Here we show that Pmca4 mRNA is expressed in both the 4a and 4b variants in the vagina, uterus, and oviduct. Immunofluorescence reveals that PMCA4a is similarly expressed and is elevated during estrus, appearing in the glandular and luminal epithelia. Western analysis detected PMCA4a in all tissues and in the luminal fluids (LF) of the vagina (VLF), uterus (ULF), and the oviduct (OLF) collected during estrus. It was ~9- and 4-fold higher in OLF than in VLF and ULF, and only marginally present in LF collected at metestrus/diestrus. Fractionation of the LF collected at estrus, via ultracentrifugation, revealed that 100% of the PMCA4a resides in the vesicular fraction of the ULF and OLF. Transmission electron microscopy (TEM) revealed that OLF vesicles have an exosomal orientation (with the cytoplasmic-side inward), a size range of 25-100 nm, with the characteristic CD9 biomarker. Thus, we dubbed these vesicles "oviductosomes", to which PMCA4a was immunolocalized. Incubation of caudal sperm in the combined LF or exosomes resulted in up to a ~3-fold increase of sperm PMCA4a, as detected by flow cytometry, indicating in vitro uptake. Our results are consistent with the increased requirement of Ca(2+) efflux in the oviduct. They show for the first time the presence of oviductal exosomes and highlight their role, along with uterosomes and vaginal exosomes, in post-testicular sperm acquisition of PMCA4a which is essential for hyperactivated motility and fertility.

Proceedings of conference on Mechanism-Based Development of Natural Products for Human Health.

Mechanism-Based Development of Natural Products in Human Health.

Plasma membrane calcium ATPases as novel candidates for therapeutic agent development.

Plasma membrane Ca2+ ATPases (PMCAs) are highly regulated transporters responsible for Ca2+ extrusion from all eukaryotic cells. Different PMCA isoforms are implicated in various tasks of Ca2+ regulation including bulk Ca2+ transport and localized Ca2+ signaling in specific membrane microdomains. Accumulating evidence shows that loss, mutation or inappropriate expression of different PMCAs is associated with pathologies ranging from hypertension, low bone density and male infertility to hearing loss and cerebellar ataxia. Compared to Ca2+ influx channels, PMCAs have lagged far behind as targets for drug development, mainly due to the lack of detailed understanding of their structure and specific function. This is rapidly changing thanks to integrated efforts combining biochemical, structural, cellular and physiological studies suggesting that selective modulation of PMCA isoforms may be of therapeutic value in the management of different and complex diseases. Both structurally informed rational design and high-throughput small molecule library screenings are promising strategies that are expected to lead to specific and isoform-selective modulators of PMCA function. This short review will provide an overview of the diverse roles played by PMCA isoforms in different cells and tissues and their emerging involvement in pathophysiological processes, summarize recent progress in obtaining structural information on the PMCAs, and discuss current and future strategies to develop specific PMCA inhibitors and activators for potential therapeutic applications.

Human Calmodulin-Like Protein CALML3: A Novel Marker for Normal Oral Squamous Mucosa That Is Downregulated in Malignant Transformation.

Oral cancer is often diagnosed only at advanced stages due to a lack of reliable disease markers. The purpose of this study was to determine if the epithelial-specific human calmodulin-like protein (CALML3) could be used as marker for the various phases of oral tumor progression. Immunohistochemical analysis using an affinity-purified CALML3 antibody was performed on biopsy-confirmed oral tissue samples representing these phases. A total of 90 tissue specimens were derived from 52 patients. Each specimen was analyzed in the superficial and basal mucosal cell layers for overall staining and staining of cellular subcompartments. CALML3 was strongly expressed in benign oral mucosal cells with downregulation of expression as squamous cells progress to invasive carcinoma. Based on the Cochran-Armitage test for trend, expression in the nucleus and at the cytoplasmic membrane significantly decreased with increasing disease severity. Chi-square test showed that benign tissue specimens had significantly more expression compared to dysplasia/CIS and invasive specimens. Dysplasia/CIS tissue had significantly more expression than invasive tissue. We conclude that CALML3 is expressed in benign oral mucosal cells with a statistically significant trend in downregulation as tumorigenesis occurs. CALML3 may thus be a sensitive new marker for oral cancer screening.

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.

Immunolocalization of the tumor-sensitive calmodulin-like protein CALML3 in normal human skin and hyperproliferative skin disorders.

BACKGROUND AND OBJECTIVE: Calmodulin-like protein CALML3 is an epithelial-specific protein regulated during keratinocyte differentiation in vitro. CALML3 expression is downregulated in breast cancers and transformed cell lines making it an attractive marker for tumor formation. The objective of this study was to survey CALML3 localization in normal epidermis and in hyperproliferative skin diseases including actinic keratosis, squamous and basal cell carcinoma as well as verruca and psoriasis and to compare CALML3 immunoreactivity with the proliferation marker Ki-67. METHODS: Paraffin-embedded tissue sections from normal human skin and hyperproliferative skin disorders were examined by immunohistochemistry and analyzed for localization and expression of CALML3 and Ki-67. RESULTS: CALML3 was strongly expressed in differentiating layers of normal skin, staining the periphery in suprabasal cells and exhibiting nuclear localization in the stratum granulosum. CALML3 nuclear localization was inversely correlated to Ki-67 staining in each disease, indicating that CALML3 nuclear presence is related to terminal cell differentiation and postmitotic state. CONCLUSIONS: Increased CALML3 expression in suprabasal layers is characteristic for differentiating keratinocytes in normal epidermis, and nuclear expression of CALML3 inversely correlates with expression of the proliferation marker Ki-67. This suggests that CALML3 is a useful marker for normal and benign hyperplastic epidermal development, whereas the loss of nuclear CALML3 indicates progression to a proliferative and potentially malignant phenotype.

Plasma membrane Ca2+-ATPase 4 in murine epididymis: secretion of splice variants in the luminal fluid and a role in sperm maturation.

Plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) is the primary Ca(2+) efflux pump in murine sperm, where it regulates motility. In Pmca4 null sperm, motility loss results in infertility. We have shown that murine sperm PMCA4b interacts with Ca(2+)/CaM-dependent serine kinase (CASK) in regulating Ca(2+) homeostasis and motility. However, recent work indicated that the bovine PMCA4a splice variant (missing in testis) is epididymally expressed, along with 4b, and may be transferred to sperm. Here we show, via conventional and in situ RT-PCR, that both the splice variants of Pmca4 mRNA are expressed in murine testis and throughout the epididymis. Immunofluorescence localized PMCA4a to the apical membrane of the epididymal epithelium, and Western analysis not only confirmed its presence but showed for the first time that PMCA4a and PMCA4b are secreted in the epididymal luminal fluid (ELF), from which epididymosomes containing PMCA4a were isolated. Flow cytometry indicated the presence of PMCA4a on mature caudal sperm where it was increased ~5-fold compared to caput sperm (detected by Western blotting) and ~2-fold after incubation in ELF, revealing in vitro uptake and implicating PMCA4a in epididymal sperm maturation. Coimmunoprecipitation using pan-PMCA4 antibodies, revealed that both variants associate with CASK, suggesting their presence in a complex. Because they have different kinetic properties for Ca(2+) transport and different abilities to bind to CASK, our study suggests a mechanism for combining the functional attributes of both PMCA4 variants, leading to heightened efficiency of the pump in the maintenance of Ca(2+) homeostasis, which is crucial for normal motility and male fertility.

Evaluation of quercetin as a potential drug in osteosarcoma treatment.

BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and young adults. Since the introduction of chemotherapy, the 5-year survival rate of patients with non-metastatic osteosarcoma is ~70%. The main problems in osteosarcoma therapy are the occurrence of metastases, severe side-effects and chemoresistance. Antiproliferative and apoptotic effects of quercetin were shown in several types of cancers, including breast cancer and lung carcinoma. MATERIALS AND METHODS: The present study investigates the cytotoxic potential of quercetin, a dietary flavonoid, in a highly metastasizing human osteosarcoma cell line, 143B. RESULTS: We found that quercetin induces growth inhibition, G2/M phase arrest, and apoptosis in the 143B osteosarcoma cell line. We also observed impaired adhesion and migratory potential after the addition of quercetin. CONCLUSION: Since quercetin has already been shown to have low side effects in a clinical phase I trial in advanced cancer patients, this compound may have considerable potential for osteosarcoma treatment.

Differential effects of G- and F-actin on the plasma membrane calcium pump activity.

We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641-1644, 2007). The results of this study provided evidence for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca(2+) extrusion through the membrane. Our results provide further evidence of the activation-inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.

Alternative pathways for association and dissociation of the calmodulin-binding domain of plasma membrane Ca(2+)-ATPase isoform 4b (PMCA4b).

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.

Plasma membrane calcium ATPase expression in human colon multistep carcinogenesis.

The expression of the plasma membrane Ca(2+) ATPase (PMCA) was analyzed in a series of 84 formalin-fixed and paraffin embedded colon samples including normal mucosa (n = 32), adenoma (n = 19), adenocarcinoma (n = 27), and lymph node metastasis (n = 6) using (i) immunohistochemistry, (ii) mRNA in situ hybridization, and (iii) quantitative reverse-transcriptase PCR. A marked reduction of PMCA4 protein was observed in high-grade adenoma, colon cancer as well as lymph node metastasis, pointing to its potential role in the progression of cancer. However, PMCA4 RNA transcripts were unchanged or even increased in colon carcinomas, suggesting posttranscriptional regulation of PMCA4 during carcinogenesis.

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.

About and beyond the Henri-Michaelis-Menten rate equation for single-substrate enzyme kinetics.

For more than a century the simple single-substrate enzyme kinetics model and related Henri-Michaelis-Menten (HMM) rate equation have been thoroughly explored in various directions. In the present paper we are concerned with a possible generalization of this rate equation recently proposed by F. Kargi (BBRC 382 (2009) 157-159), which is assumed to be valid both in the case that the total substrate or enzyme is in excess and the quasi-steady-state is achieved. We demonstrate that this generalization is grossly inadequate and propose another generalization based on application of the quasi-steady-state condition and conservation equations for both enzyme and substrate. The standard HMM equation is derived by (a) assuming the quasi-steady-state condition, (b) applying the conservation equation only for the enzyme, and (c) assuming that the substrate concentration at quasi-steady-state can be approximated by the total substrate concentration [S](0). In our formula the rate is already expressed through [S](0), and we only assume that when quasi-steady-state is achieved the amount of product formed is negligible compared to [S](0). Numerical simulations show that our formula is generally more accurate than the HMM formula and also can provide a good approximation when the enzyme is in excess, which is not the case for the HMM formula. We show that the HMM formula can be derived from our expression by further assuming that the total enzyme concentration is negligible compared to [S](0).

Regulation of apical membrane enrichment and retention of plasma membrane Ca ATPase splice variants by the PDZ-domain protein NHERF2.

The localization of plasma membrane calcium ATPase (PMCA) isoforms in specified membrane compartments is crucial for their function in local Ca(2+) handling. PMCA2w/b is present in the apical membrane whereas alternative splice variants PMCA2x/b and 2z/b reside in the basolateral membrane in polarized epithelial cells. Here we found that the apical scaffolding protein NHERF2 greatly enhances the apical concentration of PMCA2w/b by tethering the pump to the underlying actin cytoskeleton. The interaction requires the C-terminal PDZ binding sequence in PMCA2b and results in increased membrane retention and decreased lateral mobility of the pump. In contrast, PMCA2x/b remains exclusively basolateral even when NHERF2 is overexpressed. Our results suggest that the alternatively spliced intracellular loop in PMCA2 imposes dominant membrane targeting information. NHERF2-mediated recruitment may be an effective means for polarized cells to regulate the abundance of PMCA2w/b in the apical membrane to meet an increased demand for local Ca(2+) extrusion.