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

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

Mechanisms of chloride uptake in frog olfactory receptor neurons

Odorant stimulation of olfactory receptor neurons (ORNs) leads to the activation of a Ca²⁺ permeable cyclic nucleotide-gated (CNG) channel followed by opening of an excitatory Ca²⁺-activated Cl⁻ channel, which carries about 70% of the odorant-induced receptor current. This requires ORNs to have a [Cl⁻]_i above the electrochemical equilibrium to render this anionic current excitatory. In mammalian ORNs, the Na⁺-K⁺-2Cl⁻ co-transporter 1 (NKCC1) has been characterized as the principal mechanism by which these neurons actively accumulate Cl⁻. To determine if NKCC activity is needed in amphibian olfactory transduction, and to characterize its cellular location, we used the suction pipette technique to record from Rana pipiens ORNs. Application of bumetanide, an NKCC blocker, produced a 50% decrease of the odorant-induced current. Similar effects were observed when [Cl⁻]_i was decreased by bathing ORNs in low Cl⁻ solution. Both manipulations reduced only the Cl⁻ component of the current. Application of bumetanide only to the ORN cell body and not to the cilia decreased the current by again about 50%. The results show that NKCC is required for amphibian olfactory transduction, and suggest that the co-transporter is located basolaterally at the cell body although its presence at the cilia could not be discarded.     

Inositol-trisphosphate-dependent calcium currents precede cation currents in insect olfactory receptor neurons in vitro

Specialized olfactory receptor neurons in insects respond to species-specific sex pheromones with transient rises in inositol trisphosphate and by opening pheromone-dependent cation channels. These channels resemble cation channels which are directly or indirectly Ca²⁺-dependent. But there appear to be no internal Ca²⁺ stores in the outer dendrite where the olfactory transduction cascade is thought to start. Hence, it remains to be determined whether an influx of external Ca²⁺ precedes pheromone-dependent cation currents. Patch clamp measurements in cultured olfactory receptor neurons from Manduca sexta reveal that a transient inward current precedes pheromone-dependent cation currents. A transient inositol trisphosphate-dependent Ca²⁺ current, also preceding cation currents with the characteristics of pheromone-dependent cation currents, shares properties with the transient pheromone-dependent current. These results match the biochemical measurements with the electrophysiological data obtained in insect olfactory receptor neurons.Abbreviations ORNs Olfactory receptor neurons - IP₃ Inositol-1,4,5-trisphosphate - I_t Transient pheromone-dependent current - I_ir Transient IP₃-dependent current     

Homer2 Protein Regulates Plasma Membrane Ca2+-ATPase-mediated Ca2+ Signaling in Mouse Parotid Gland Acinar Cells

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca²⁺-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca²⁺ signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca²⁺ signaling in parotid gland acinar cells using Homer2-deficient (Homer2^−/−) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca²⁺-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca²⁺-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca²⁺ extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca²⁺ clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca²⁺ signaling in parotid acinar cells.     

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 Ca²⁺ signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca²⁺ ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca²⁺. 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 Ca²⁺ ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca²⁺ transients generated by cell stimulation and impairs its Ca²⁺ extrusion function under conditions of low resting cytosolic Ca²⁺ 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 Ca²⁺-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 Ca²⁺ homeostasis and the previous finding that PMCAs act as digenic modulators in Ca²⁺-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype.     

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 Ca²⁺ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]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 Ca²⁺-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca²⁺-ATPase activity was related to an increase in the apparent affinity for Ca²⁺ 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 Ca²⁺ homeostasis.     

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 Ca²⁺ 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 Ca²⁺. 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 Ca²⁺ 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.     

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

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

Aluminum inhibits the plasma membrane and sarcoplasmic reticulum Ca2+-ATPases by different mechanisms

Aluminum (Al³⁺) is involved in the pathophysiology of neurodegenerative disorders. The mechanisms that have been proposed to explain the action of Al³⁺ toxicity are linked to changes in the cellular calcium homeostasis, placing the transporting calcium pumps as potential targets.The aim of this work was to study the molecular inhibitory mechanism of Al³⁺ on Ca²⁺-ATPases such as the plasma membrane and the sarcoplasmic reticulum calcium pumps (PMCA and SERCA, respectively). These P-ATPases transport Ca²⁺ actively from the cytoplasm towards the extracellular medium and to the sarcoplasmic reticulum, respectively. For this purpose, we performed enzymatic measurements of the effect of Al³⁺ on purified preparations of PMCA and SERCA.Our results show that Al³⁺ is an irreversible inhibitor of PMCA and a slowly-reversible inhibitor of SERCA. The binding of Al³⁺ is affected by Ca²⁺ in SERCA, though not in PMCA. Al³⁺ prevents the phosphorylation of SERCA and, conversely, the dephosphorylation of PMCA. The dephosphorylation time courses of the complex formed by PMCA and Al³⁺ (EPAl) in the presence of ADP or ATP show that EPAl is composed mainly by the conformer E₂P.This work shows for the first time a distinct mechanism of Al³⁺ inhibition that involves different intermediates of the reaction cycle of these two Ca²⁺-ATPases.     

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

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

Olfactory response termination involves Ca2+-ATPase in vertebrate olfactory receptor neuron cilia

In vertebrate olfactory receptor neurons (ORNs), odorant-induced activation of the transduction cascade culminates in production of cyclic AMP, which opens cyclic nucleotide–gated channels in the ciliary membrane enabling Ca²⁺ influx. The ensuing elevation of the intraciliary Ca²⁺ concentration opens Ca²⁺-activated Cl⁻ channels, which mediate an excitatory Cl⁻ efflux from the cilia. In order for the response to terminate, the Cl⁻ channel must close, which requires that the intraciliary Ca²⁺ concentration return to basal levels. Hitherto, the extrusion of Ca²⁺ from the cilia has been thought to depend principally on a Na⁺–Ca²⁺ exchanger.In this study, we show using simultaneous suction pipette recording and Ca²⁺-sensitive dye fluorescence measurements that in fire salamander ORNs, withdrawal of external Na⁺ from the solution bathing the cilia, which incapacitates Na⁺–Ca²⁺exchange, has only a modest effect on the recovery of the electrical response and the accompanying decay of intraciliary Ca²⁺ concentration. In contrast, exposure of the cilia to vanadate or carboxyeosin, a manipulation designed to block Ca²⁺-ATPase, has a substantial effect on response recovery kinetics. Therefore, we conclude that Ca²⁺-ATPase contributes to Ca²⁺ extrusion in ORNs, and that Na⁺–Ca²⁺exchange makes only a modest contribution to Ca²⁺ homeostasis in this species.     

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

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

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-[¹²⁵I]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 Ca²⁺ 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 Ca²⁺ binding domain, we also studied the occlusion of Ca²⁺, 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 Ca²⁺ 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.     

Differences of Ca2+ handling properties in identified central olfactory neurons of the antennal lobe

Information processing in neurons depends on highly localized Ca²⁺ signals. The spatial and temporal dynamics of these signals are determined by a variety of cellular parameters including the calcium influx, calcium buffering and calcium extrusion. Our long-term goal is to better understand how intracellular Ca²⁺ dynamics are controlled and contribute to information processing in defined interneurons of the insect olfactory system. The latter has served as an excellent model to study general mechanisms of olfaction. Using patch-clamp recordings and fast optical imaging in combination with the ‘added buffer approach’, we analyzed the Ca²⁺ handling properties of different identified neuron types in Periplaneta americana's olfactory system. Our focus was on two types of local interneurons (LNs) with significant differences in intrinsic electrophysiological properties: (1) spiking LNs that generate ‘normal’ Na⁺ driven action potentials and (2) non-spiking LNs that do not express voltage-activated Na⁺ channels. We found that the distinct electrophysiological properties from different types of central olfactory interneurons are strongly correlated with their cell specific calcium handling properties: non-spiking LNs, in which Ca²⁺ is the only cation that enters the cell to contribute to membrane depolarization, had the highest endogenous Ca²⁺ binding ratio and Ca²⁺ extrusion rate.     

Regulation of PKC Mediated Signaling by Calcium during Visceral Leishmaniasis

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

Plasma Membrane Ca2+-ATPase Isoforms Composition Regulates Cellular pH Homeostasis in Differentiating PC12 Cells in a Manner Dependent on Cytosolic Ca2+ Elevations

Plasma membrane Ca²⁺-ATPase (PMCA) by extruding Ca²⁺ outside the cell, actively participates in the regulation of intracellular Ca²⁺ concentration. Acting as Ca²⁺/H⁺ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca²⁺ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pH_mito and pH_cyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca²⁺ clearance and partially attenuated cellular acidification during KCl-stimulated Ca²⁺ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca²⁺ overload. Cyclosporin and bongkrekic acid prevented Ψ_m loss suggesting the involvement of Ca²⁺-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.     

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

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

Glycolytic ATP Fuels the Plasma Membrane Calcium Pump Critical for Pancreatic Cancer Cell Survival

Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca²⁺ ([Ca²⁺]_i), as the ATP-dependent plasma membrane Ca²⁺ ATPase (PMCA) is critical for maintaining low [Ca²⁺]_i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca²⁺]_i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca²⁺]_i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.