Phospholipids and calmodulin modulate the inhibition of PMCA activity by tau.

The disruption of Ca2+ signaling in neurons, together with a failure to keep optimal intracellular Ca2+ concentrations, have been proposed as significant factors for neuronal dysfunction in the Ca2+ hypothesis of Alzheimer's disease (AD). Tau is a protein that plays an essential role in axonal transport and can form abnormal structures such as neurofibrillary tangles that constitute one of the hallmarks of AD. We have recently shown that plasma membrane Ca2+-ATPase (PMCA), a key enzyme in the maintenance of optimal cytosolic Ca2+ levels in cells, is inhibited by tau in membrane vesicles. In the present study we show that tau inhibits synaptosomal PMCA purified from pig cerebrum, and reconstituted in phosphatidylserine-containing lipid bilayers, with a Ki value of 1.5±0.2nM tau. Noteworthy, the inhibitory effect of tau is dependent on the charge of the phospholipid used for PMCA reconstitution. In addition, nanomolar concentrations of calmodulin, the major endogenous activator of PMCA, protects against inhibition of the Ca2+-ATPase activity by tau. Our results in a cellular model such as SH-SY5Y human neuroblastoma cells yielded an inhibition of PMCA by nanomolar tau concentrations and protection by calmodulin against this inhibition similar to those obtained with purified synaptosomal PMCA. Functional studies were also performed with native and truncated versions of human cerebral PMCA4b, an isoform that has been showed to be functionally regulated by amyloid peptides, whose aggregates constitutes another hallmark of AD. Kinetic assays point out that tau binds to the C-terminal tail of PMCA, at a site distinct but close to the calmodulin binding domain. In conclusion, PMCA can be seen as a molecular target for tau-induced cytosolic calcium dysregulation in synaptic terminals. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

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.

Acute inhibition of PMCA4, but not global ablation, reduces blood pressure and arterial contractility via a nNOS-dependent mechanism.

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild-type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild-type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl-l-NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.

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.

Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17ß-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities.

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP.

Evidence suggests that the plasma membrane Ca(2+)-ATPase (PMCA), which is critical for maintaining a low intracellular Ca(2+) concentration ([Ca(2+)]i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca(2+)]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca(2+)]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca(2+)]i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.

Hepcidin and 1,25(OH)2D3 effectively restore Ca2+ transport in ß-thalassemic mice: reciprocal phenomenon of Fe2+ and Ca2+ absorption.

Previously, ß-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of ß-globin gene, has been reported to induce osteopenia and impaired whole body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how ß-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or hepcidin, the latter of which was the liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous ß-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermates, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice. This impaired calcium transport could be restored by 7-day 1,25(OH)2D3 treatment. The 1,25(OH)2D3-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, and PMCA1b, as well as vesicular transport inhibitors. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in the Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors MG132 and bortezomib. In conclusion, both 1,25(OH)2D3 and hepcidin could be used to alleviate the ß-thalassemia-associated impairment of calcium absorption. Therefore, our study has shed light on the development of a treatment strategy to rescue calcium dysregulation in ß-thalassemia.

STIM1 is required for attenuation of PMCA-mediated Ca2+ clearance during T-cell activation.

T-cell activation involves a complex signalling cascade uniquely dependent on elevated cytosolic Ca(2+) levels. Further, the spatiotemporal characteristics of this Ca(2+) signal play a critical role in this process via selective activation of transcription factors. In T cells, store-operated Ca(2+) entry (SOCe) is the primary Ca(2+) influx pathway; however, cytosolic Ca(2+) concentration depends upon the balance between Ca(2+) influx and extrusion. The plasma membrane Ca(2+) ATPase (PMCA) has previously been identified as a critical player in Ca(2+) clearance in T cells. Here, we provide data revealing both functional and physical links between the activation of stromal interacting molecule 1 (STIM1) and PMCA-mediated Ca(2+) clearance. Due to the ubiquitous expression of both STIM1 and PMCA, these findings have wide-ranging implications for Ca(2+) signalling in multiple cell types.

Heterozygous inactivation of plasma membrane Ca(2+)-ATPase in mice increases glucose-induced insulin release and beta cell proliferation, mass and viability.

AIMS/HYPOTHESIS: Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca(2+) extrusion--the Na/Ca exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function. METHODS: Biological and morphological methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo. RESULTS: Pmca (isoform 2) heterozygous inactivation increased intracellular Ca(2+) stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed. CONCLUSIONS/INTERPRETATION: The present data indicates that inhibition of Ca(2+) extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca(2+) extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes.

Presynaptic control of glycine transporter 2 (GlyT2) by physical and functional association with plasma membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ exchanger (NCX).

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca(2+)-ATPase (PMCA) isoforms 2 and 3, and Na(+)/Ca(2+)-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2-3·NCX complex would help Na(+)/K(+)-ATPase in controlling local Na(+) increases derived from GlyT2 activity after neurotransmitter release.

Magnesium enhances aurintricarboxylic acid's inhibitory action on the plasma membrane Ca2+-ATPase.

Our research aimed to elucidate the mechanism by which aurintricarboxylic acid (ATA) inhibits plasma membrane Ca2+-ATPase (PMCA), a crucial enzyme responsible for calcium transport. Given the pivotal role of PMCA in cellular calcium homeostasis, understanding how it is inhibited by ATA holds significant implications for potentially regulating physiopathological cellular processes in which this pump is involved. Our experimental findings revealed that ATA employs multiple modes of action to inhibit PMCA activity, which are influenced by ATP but also by the presence of calcium and magnesium ions. Specifically, magnesium appears to enhance this inhibitory effect. Our experimental and in-silico results suggest that, unlike those reported in other proteins, ATA complexed with magnesium (ATA·Mg) is the molecule that inhibits PMCA. In summary, our study presents a novel perspective and establishes a solid foundation for future research efforts aimed at the development of new pharmacological molecules both for PMCA and other proteins.

PMCA2 via PSD-95 controls calcium signaling by α7-containing nicotinic acetylcholine receptors on aspiny interneurons.

Local control of calcium concentration within neurons is critical for signaling and regulation of synaptic communication in neural circuits. How local control can be achieved in the absence of physical compartmentalization is poorly understood. Challenging examples are provided by nicotinic acetylcholine receptors that contain α7 nicotinic receptor subunits (α7-nAChRs). These receptors are highly permeable to calcium and are concentrated on aspiny dendrites of interneurons, which lack obvious physical compartments for constraining calcium diffusion. Using functional proteomics on rat brain, we show that α7-nAChRs are associated with plasma membrane calcium-ATPase pump isoform 2 (PMCA2). Analysis of α7-nAChR function in hippocampal interneurons in culture shows that PMCA2 activity limits the duration of calcium elevations produced by the receptors. Unexpectedly, PMCA2 inhibition triggers rapid calcium-dependent loss of α7-nAChR clusters. This extreme regulatory response is mediated by CaMKII, involves proteasome activity, depends on the second intracellular loop of α7-nAChR subunits, and is specific in that it does not alter two other classes of calcium-permeable ionotropic receptors on the same neurons. A critical link is provided by the scaffold protein PSD-95 (postsynaptic density-95), which is associated with α7-nAChRs and constrains their mobility as revealed by single-particle tracking on neurons. The PSD-95 link is required for PMCA2-mediated removal of α7-nAChR clusters. This three-component combination of PMCA2, PSD-95, and α7-nAChR offers a novel mechanism for tight control of calcium dynamics in neurons.

Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes.

Isoform 4 of the plasma membrane calcium/calmodulin dependent ATPase (PMCA4) has recently emerged as an important regulator of several key pathophysiological processes in the heart, such as contractility and hypertrophy. However, direct monitoring of PMCA4 activity and assessment of calcium dynamics in its vicinity in cardiomyocytes are difficult due to the lack of molecular tools. In this study, we developed novel calcium fluorescent indicators by fusing the GCaMP2 calcium sensor to the N-terminus of PMCA4 to generate the PMCA4-GCaMP2 fusion molecule. We also identified a novel specific inhibitor of PMCA4, which might be useful for studying the role of this molecule in cardiomyocytes and other cell types. Using an adenoviral system we successfully expressed PMCA4-GCaMP2 in both neonatal and adult rat cardiomyocytes. This fusion molecule was correctly targeted to the plasma membrane and co-localised with caveolin-3. It could monitor signal oscillations in electrically stimulated cardiomyocytes. The PMCA4-GCaMP2 generated a higher signal amplitude and faster signal decay rate compared to a mutant inactive PMCA4(mut)GCaMP2 fusion protein, in electrically stimulated neonatal and adult rat cardiomyocytes. A small molecule library screen enabled us to identify a novel selective inhibitor for PMCA4, which we found to reduce signal amplitude of PMCA4-GCaMP2 and prolong the time of signal decay (Tau) to a level comparable with the signal generated by PMCA4(mut)GCaMP2. In addition, PMCA4-GCaMP2 but not the mutant form produced an enhanced signal in response to ß-adrenergic stimulation. Together, the PMCA4-GCaMP2 and PMCA4(mut)GCaMP2 demonstrate calcium dynamics in the vicinity of the pump under active or inactive conditions, respectively. In summary, the PMCA4-GCaMP2 together with the novel specific inhibitor provides new means with which to monitor calcium dynamics in the vicinity of a calcium transporter in cardiomyocytes and may become a useful tool to further study the biological functions of PMCA4. In addition, similar approaches could be useful for studying the activity of other calcium transporters during excitation-contraction coupling in the heart.

Insulin protects pancreatic acinar cells from cytosolic calcium overload and inhibition of plasma membrane calcium pump.

Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca(2+) ([Ca(2+)](i)) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca(2+) overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca(2+)-ATPase (PMCA) provides a final common path for cells to "defend" [Ca(2+)](i) during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca(2+) overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca(2+) overload, even in the face of impaired mitochondrial function.

Calmodulin antagonizes amyloid-ß peptides-mediated inhibition of brain plasma membrane Ca(2+)-ATPase.

The synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in regulating intracellular Ca(2+) concentration in brain. We have recently found that PMCA is the only Ca(2+) pump in brain which is inhibited by amyloid-ß peptide (Aß), a neurotoxic peptide implicated in the pathology of Alzheimer's disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aß. Results from kinetic assays indicate that Aß aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aß could be blocked by pretreating the purified protein with Ca(2+)-calmodulin, the main endogenous activator of PMCA, and the activity of truncated PMCA lacking the calmodulin binding domain was not affected by Aß. Dot-overlay experiments indicated a physical association of Aß with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aß by burying exposed sites on PMCA, making them inaccessible to Aß, and also by direct binding to the peptide. These results suggest a protective role of calmodulin against neuronal Ca(2+) dysregulation by PMCA inhibition induced by Aß.

Presynaptic pH and vesicle fusion in Drosophila larvae neurones.

Both intracellular pH (pHi) and synaptic cleft pH change during neuronal activity yet little is known about how these pH shifts might affect synaptic transmission by influencing vesicle fusion. To address this we imaged pH- and Ca(2+) -sensitive fluorescent indicators (HPTS, Oregon green) in boutons at neuromuscular junctions. Electrical stimulation of motor nerves evoked presynaptic Ca(2+) i rises and pHi falls (∼0.1 pH units) followed by recovery of both Ca(2+) i and pHi. The plasma-membrane calcium ATPase (PMCA) inhibitor, 5(6)-carboxyeosin diacetate, slowed both the calcium recovery and the acidification. To investigate a possible calcium-independent role for the pHi shifts in modulating vesicle fusion we recorded post-synaptic miniature end-plate potential (mEPP) and current (mEPC) frequency in Ca(2+) -free solution. Acidification by propionate superfusion, NH(4)(+) withdrawal, or the inhibition of acid extrusion on the Na(+)/H(+) exchanger (NHE) induced a rise in miniature frequency. Furthermore, the inhibition of acid extrusion enhanced the rise induced by propionate addition and NH(4)(+) removal. In the presence of NH(4)(+), 10 out of 23 cells showed, after a delay, one or more rises in miniature frequency. These findings suggest that Ca(2+) -dependent pHi shifts, caused by the PMCA and regulated by NHE, may stimulate vesicle release. Furthermore, in the presence of membrane permeant buffers, exocytosed acid or its equivalents may enhance release through positive feedback. This hitherto neglected pH signalling, and the potential feedback role of vesicular acid, could explain some important neuronal excitability changes associated with altered pH and its buffering.

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.

Optimisation and validation of a high throughput screening compatible assay to identify inhibitors of the plasma membrane calcium ATPase pump--a novel therapeutic target for contraception and malaria.

PURPOSE: ATPases, which constitute a major category of ion transporters in the human body, have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. We have recently found that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) results in infertility in male mice due to a selective defect in sperm motility. In addition, recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determines the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimise and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF® Transcreener® ADP (TR-FRET) assay to screen a drug library. METHODS AND RESULTS: PMCA4 membrane microsomes were prepared from HEK293 cells overexpressing PMCA4. Western blot quantification revealed nearly nine-fold increased expression of PMCA4 compared to LacZ (control virus)-infected cells. Maximal PMCA4 microsomal activity was achieved in the TR-FRET assay with 15ng/µl microsomal concentration, 30-minute pre-incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1µM free-calcium. Finally a dose-response curve for carboxyeosin (a non-specific PMCA inhibitor) under optimised conditions showed significant PMCA4 inhibition. Upon confirmation that the assay was suitable for high-throughput screening, we have screened the ChemBioNet small molecule library (~21,000 compounds) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1,494 primary hits. CONCLUSIONS: We have optimised the HTRF® Transcreener® ADP assay for high-throughput screening to identify PMCA4 inhibitors. The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non-hormonal contraception or anti-malaria therapy.

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion.

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 µM) and capsaicin (1 µM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 µM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 µM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.