Ca Homeostasis Research Articles

Extracellular magnesium and calcium blockers modulate macrophage activity

Magnesium (Mg) possesses anti-inflammatory properties, partly because it antagonizes calcium (Ca) and inhibits L-type Ca channels. Our aim was to determine the effects of different concentrations of extracellular Mg, with or without Ca-channel blockers, in macrophages. A macrophage-like cell line J774.E was cultured in different concentrations of extracellular Mg and exposed to i) the phorbol ester PMA to induce the production of reactive oxygen species ii) lipopolysaccharide to induce the production of pro-inflammatory cytokines, or iii) ovalbumin to study endocytosis. The Ca antagonists verapamil and/or TMB-8 were used to interfere with Ca homeostasis. Different concentrations of extracellular Mg did not impact on endocytosis, while Ca antagonists markedly decreased it. Low extracellular Mg exacerbated, whereas Ca antagonists inhibited, PMA-induced production of free radicals. Ca blockers prevented lipopolysaccharide-induced transcription and release of IL-1β, IL-6 and TNF-α, while extracellular Mg had only a marginal effect. Ca channel inhibitors markedly reduced the activity of J774.E cells, thus underscoring the critical role of Ca in the non-specific immune response, a role which was, in some instances, also modulated by extracellular Mg.

Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders

Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction.

Transcriptional Regulation of X-Box-binding Protein One (XBP1) by Hepatocyte Nuclear Factor 4α (HNF4Α) Is Vital to Beta-cell Function

The transcription factor, X-box-binding protein-1 (XBP1), controls the development and maintenance of the endoplasmic reticulum (ER) in multiple secretory cell lineages. We show here that Hepatocyte Nuclear Factor 4α (HNF4α) directly induces XBP1 expression. Mutations in HNF4α cause Mature-Onset Diabetes of the Young I (MODYI), a subset of diabetes characterized by diminished GSIS. In mouse models, cell lines, and ex vivo islets, using dominant negative and human- disease-allele point mutants or knock-out and knockdown models, we show that disruption of HNF4α caused decreased expression of XBP1 and reduced cellular ER networks. GSIS depends on ER Ca(2+) signaling; we show that diminished XBP1 and/or HNF4α in β-cells led to impaired ER Ca(2+) homeostasis. Restoring XBP1 expression was sufficient to completely rescue GSIS in HNF4α-deficient β-cells. Our findings uncover a transcriptional relationship between HNF4α and Xbp1 with potentially broader implications about MODYI and the importance of transcription factor signaling in the regulation of secretion.

Asenapine modulates nitric oxide release and calcium movements in cardiomyoblasts.

Objective:To examine the effects of asenapine on nitric oxide (NO) release and Ca2+ transients in H9C2 cell line, which were either subjected to peroxidation or not.Materials and Methods:H9C2 were treated with asenapine alone or in presence of intracellular kinase blockers, serotoninergic and dopaminergic antagonists, and voltage Ca2+ channels inhibitors. Experiments were also performed in H9C2 treated with hydrogen peroxide. NO release and intracellular Ca2+ were measured through specific probes.Results:In H9C2, asenapine differently modulated NO release and Ca2+ movements depending on peroxidative condition. The Ca2+ pool mobilized by asenapine mainly originated from the extracellular space and was slightly affected by thapsigargin. Moreover, the effects of asenapine were reduced or prevented by kinases blockers, dopaminergic and serotoninergic receptors inhibitors, and voltage Ca2+ channels blockers.Conclusions:On the basis of our findings, we can conclude that asenapine by interacting with its specific receptors, exerts dual effects on NO release and Ca2+ homeostasis in H9C2; this would be of particular clinical relevance when considering their role in cardiac function modulation.

Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions, regulates Ca2+ dynamics in T cells

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca(2+) sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER-PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER-PM junctions to regulate Ca(2+) signaling. Silencing or genetic manipulation of JP4 decreased ER Ca(2+) content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca(2+)-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate-JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca(2+) homeostasis and mediate SOCE in T cells.

Specific cytoarchitectureal changes in hippocampal subareas in daDREAM mice

BackgroundTranscriptional repressor DREAM (downstream regulatory element antagonist modulator) is a Ca2+-binding protein that regulates Ca2+ homeostasis through gene regulation and protein-protein interactions. It has been shown that a dominant active form (daDREAM) is implicated in learning-related synaptic plasticity such as LTP and LTD in the hippocampus. Neuronal spines are reported to play important roles in plasticity and memory. However, the possible role of DREAM in spine plasticity has not been reported.ResultsHere we show that potentiating DREAM activity, by overexpressing daDREAM, reduced dendritic basal arborization and spine density in CA1 pyramidal neurons and increased spine density in dendrites in dentate gyrus granule cells. These microanatomical changes are accompanied by significant modifications in the expression of specific genes encoding the cytoskeletal proteins Arc, Formin 1 and Gelsolin in daDREAM hippocampus.ConclusionsOur results strongly suggest that DREAM plays an important role in structural plasticity in the hippocampus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0204-8) contains supplementary material, which is available to authorized users.

Dissecting stimulus-specific Ca2+ signals in amyloplasts and chloroplasts of Arabidopsis thaliana cell suspension cultures.

Calcium is used by plants as an intracellular messenger in the detection of and response to a plethora of environmental stimuli and contributes to a fine-tuned internal regulation. Interest in the role of different subcellular compartments in Ca(2+) homeostasis and signalling has been growing in recent years. This work has evaluated the potential participation of non-green plastids and chloroplasts in the plant Ca(2+) signalling network using heterotrophic and autotrophic cell suspension cultures from Arabidopsis thaliana plant lines stably expressing the bioluminescent Ca(2+) reporter aequorin targeted to the plastid stroma. Our results indicate that both amyloplasts and chloroplasts are involved in transient Ca(2+) increases in the plastid stroma induced by several environmental stimuli, suggesting that these two functional types of plastids are endowed with similar mechanisms for handling Ca(2+) A comparison of the Ca(2+) trace kinetics recorded in parallel in the plastid stroma, the surface of the outer membrane of the plastid envelope, and the cytosol indicated that plastids play an essential role in switching off different cytosolic Ca(2+) signals. Interestingly, a transient stromal Ca(2+) signal in response to the light-to-dark transition was observed in chloroplasts, but not amyloplasts. Moreover, significant differences in the amplitude of specific plastidial Ca(2+) changes emerged when the photosynthetic metabolism of chloroplasts was reactivated by light. In summary, our work highlights differences between non-green plastids and chloroplasts in terms of Ca(2+) dynamics in response to environmental stimuli.

Tissue-specific root ion profiling reveals essential roles of the CAX and ACA calcium transport systems in response to hypoxia in Arabidopsis.

Waterlogging is a major abiotic stress that limits the growth of plants. The crucial role of Ca(2+) as a second messenger in response to abiotic and biotic stimuli has been widely recognized in plants. However, the physiological and molecular mechanisms of Ca(2+) distribution within specific cell types in different root zones under hypoxia is poorly understood. In this work, whole-plant physiological and tissue-specific Ca(2+) changes were studied using several ACA (Ca(2+)-ATPase) and CAX (Ca(2+)/proton exchanger) knock-out Arabidopsis mutants subjected to waterlogging treatment. In the wild-type (WT) plants, several days of hypoxia decreased the expression of ACA8, CAX4, and CAX11 by 33% and 50% compared with the control. The hypoxic treatment also resulted in an up to 11-fold tissue-dependent increase in Ca(2+) accumulation in root tissues as revealed by confocal microscopy. The increase was much higher in stelar cells in the mature zone of Arabidopsis mutants with loss of function for ACA8, ACA11, CAX4, and CAX11 In addition, a significantly increased Ca(2+) concentration was found in the cytosol of stelar cells in the mature zone after hypoxic treatment. Three weeks of waterlogging resulted in dramatic loss of shoot biomass in cax11 plants (67% loss in shoot dry weight), while in the WT and other transport mutants this decline was only 14-22%. These results were also consistent with a decline in leaf chlorophyll fluorescence (F v/F m). It is suggested that CAX11 plays a key role in maintaining cytosolic Ca(2+) homeostasis and/or signalling in root cells under hypoxic conditions.

Calcium signalling in malaria parasites.

Ca(2+) is a ubiquitous intracellular messenger in malaria parasites with important functions in asexual blood stages responsible for malaria symptoms, the preceding liver-stage infection and transmission through the mosquito. Intracellular messengers amplify signals by binding to effector molecules that trigger physiological changes. The characterisation of some Ca(2+) effector proteins has begun to provide insights into the vast range of biological processes controlled by Ca(2+) signalling in malaria parasites, including host cell egress and invasion, protein secretion, motility and cell cycle regulation. Despite the importance of Ca(2+) signalling during the life cycle of malaria parasites, little is known about Ca(2+) homeostasis. Recent findings highlighted that upstream of stage-specific Ca(2+) effectors is a conserved interplay between second messengers to control critical intracellular Ca(2+) signals throughout the life cycle. The identification of the molecular mechanisms integrating stage-transcending mechanisms of Ca(2+) homeostasis in a network of stage-specific regulator and effector pathways now represents a major challenge for a meaningful understanding of Ca(2+) signalling in malaria parasites.

Impact of Increasing Dietary Calcium Levels on Calcium Excretion and Vitamin D Metabolites in the Blood of Healthy Adult Cats

BackgroundDietary calcium (Ca) concentrations might affect regulatory pathways within the Ca and vitamin D metabolism and consequently excretory mechanisms. Considering large variations in Ca concentrations of feline diets, the physiological impact on Ca homeostasis has not been evaluated to date. In the present study, diets with increasing concentrations of dicalcium phosphate were offered to ten healthy adult cats (Ca/phosphorus (P): 6.23/6.02, 7.77/7.56, 15.0/12.7, 19.0/17.3, 22.2/19.9, 24.3/21.6 g/kg dry matter). Each feeding period was divided into a 10-day adaptation and an 8-day sampling period in order to collect urine and faeces. On the last day of each feeding period, blood samples were taken.ResultsUrinary Ca concentrations remained unaffected, but faecal Ca concentrations increased (P < 0.001) with increasing dietary Ca levels. No effect on whole and intact parathyroid hormone levels, fibroblast growth factor 23 and calcitriol concentrations in the blood of the cats were observed. However, the calcitriol precursors 25(OH)D2 and 25(OH)D3, which are considered the most useful indicators for the vitamin D status, decreased with higher dietary Ca levels (P = 0.013 and P = 0.033). Increasing dietary levels of dicalcium phosphate revealed an acidifying effect on urinary fasting pH (6.02) and postprandial pH (6.01) (P < 0.001), possibly mediated by an increase of urinary phosphorus (P) concentrations (P < 0.001).ConclusionsIn conclusion, calcitriol precursors were linearly affected by increasing dietary Ca concentrations. The increase in faecal Ca excretion indicates that Ca homeostasis of cats is mainly regulated in the intestine and not by the kidneys. Long-term studies should investigate the physiological relevance of the acidifying effect observed when feeding diets high in Ca and P.

Mitochondrial dynamics and cell death in heart failure.

The highly regulated processes of mitochondrial fusion (joining), fission (division) and trafficking, collectively called mitochondrial dynamics, determine cell-type specific morphology, intracellular distribution and activity of these critical organelles. Mitochondria are critical for cardiac function, while their structural and functional abnormalities contribute to several common cardiovascular diseases, including heart failure (HF). The tightly balanced mitochondrial fusion and fission determine number, morphology and activity of these multifunctional organelles. Although the intracellular architecture of mature cardiomyocytes greatly restricts mitochondrial dynamics, this process occurs in the adult human heart. Fusion and fission modulate multiple mitochondrial functions, ranging from energy and reactive oxygen species production to Ca(2+) homeostasis and cell death, allowing the heart to respond properly to body demands. Tightly controlled balance between fusion and fission is of utmost importance in the high energy-demanding cardiomyocytes. A shift toward fission leads to mitochondrial fragmentation, while a shift toward fusion results in the formation of enlarged mitochondria and in the fusion of damaged mitochondria with healthy organelles. Mfn1, Mfn2 and OPA1 constitute the core machinery promoting mitochondrial fusion, whereas Drp1, Fis1, Mff and MiD49/51 are the core components of fission machinery. Growing evidence suggests that fusion/fission factors in adult cardiomyocytes play essential noncanonical roles in cardiac development, Ca(2+) signaling, mitochondrial quality control and cell death. Impairment of this complex circuit causes cardiomyocyte dysfunction and death contributing to heart injury culminating in HF. Pharmacological targeting of components of this intricate network may be a novel therapeutic modality for HF treatment.

Calcium Ions in Inherited Cardiomyopathies.

Inherited cardiomyopathies are a known cause of heart failure, although the pathways and mechanisms leading from mutation to the heart failure phenotype have not been elucidated. There is strong evidence that this transition is mediated, at least in part, by abnormal intracellular Ca(2+) handling, a key ion in ventricular excitation, contraction and relaxation. Studies in human myocytes, animal models and in vitro reconstituted contractile protein complexes have shown consistent correlations between Ca(2+) sensitivity and cardiomyopathy phenotype, irrespective of the causal mutation. In this review we present the available data about the connection between mutations linked to familial hypertrophic (HCM), dilated (DCM) and restrictive (RCM) cardiomyopathy, right ventricular arrhythmogenic cardiomyopathy/dysplasia (ARVC/D) as well as left ventricular non-compaction and the increase or decrease in Ca(2+) sensitivity, together with the results of attempts to reverse the manifestation of heart failure by manipulating Ca(2+) homeostasis.

Mitochondrial Ca(2+) Processing by a Unit of Mitochondrial Ca(2+) Uniporter and Na(+)/Ca(2+) Exchanger Supports the Neuronal Ca(2+) Influx via Activated Glutamate Receptors.

The current study demonstrates that in hippocampal neurons mitochondrial Ca(2+) processing supports Ca(2+) influx via ionotropic glutamate (Glu) receptors. We define mitochondrial Ca(2+) processing as Ca(2+) uptake via mitochondrial Ca(2+) uniporter (MCU) combined with subsequent Ca(2+) release via mitochondrial Na(+)/Ca(2+) exchanger (NCX). Our tool is to measure the Ca(2+) influx rate in primary hippocampal co-cultures, i.e. neurons and astrocytes, by fluorescent digital microscopy, using a Fura-2-quenching method where we add small amounts of Mn(2+) in the superfusion medium. Thus, Ca(2+) influx is measured with Mn(2+) in the bath. Ru360 as inhibitor of mitochondrial Ca(2+) uptake through MCU strongly reduces the rate of Ca(2+) influx in Glu-stimulated primary hippocampal neurons. Similarly, the Ca(2+) influx rate in Glu-stimulated neurons declines after suppression of potential-dependent MCU, when we depolarize mitochondria with rotenone. With inhibition of Ca(2+) release from mitochondria via NCX using CGP-37157 the Ca(2+) influx via N-methyl-D-aspartate (NMDA)- and kainate-sensitive receptors is slowed down. Working jointly as mitochondrial Ca(2+) processing unit, MCU and NCX, apparently sustain the Ca(2+) throughput of activated Glu-sensitive receptors. Our results revise the role frequently attributed to mitochondria in neuronal Ca(2+) homeostasis, where mitochondria function mainly as Ca(2+) buffer, and prevent excessively high cytosolic Ca(2+) concentration increase during neuronal activity. The mechanism to control Ca(2+) influx in neurons, as discovered in this study, highlights mitochondrial Ca(2+) processing as a promising pharmacological target. We discuss this pathway in relation to the endoplasmic reticulum-related mechanisms of Ca(2+) processing.

Role of TRP ion channels in cancer and tumorigenesis.

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

Enzymes Involved in Pyrophosphate and Calcium Metabolism as Targets for Anti-scuticociliate Chemotherapy.

Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca(2+) homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the invitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+-PPases (H+-PPases). CQ and ART inhibited the invitro growth of ciliates with IC50 values of respectively 74±9μM and 80±8μM. CQ inhibits the H+ translocation (with an IC50 of 13.4±0.2μM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+-PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca(2+) induced by ATP, indicating an effect on the Ca(2+) -ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca(2+) metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis.

Ophiopogonin D maintains Ca2+ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/EETs and suppressing ER stress.

CYP2J3 in myocardium metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs), which have diverse biological activities in rat heart. In this study we examined whether CYP2J3 was involved in cardioprotective effects of ophiopogonin D (OPD), a steroidal glycoside isolated from Chinese herb Radix ophiopogonis. Rat cardiomyoblast cell line (H9c2 cells) was tested. Intracellular Ca(2+) concentrations ([Ca(2+)]i) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with qRT-PCR and Western blot analyses. Cell apoptosis was quantified with Hoechst 33258 staining and TUNEL assay. The level of 14,15-DHET, a stable metabolite of 14,15-EET, was assessed with ELISA. Angiotensin II (10(-6) mol/L) significantly decreased the expression of calcium-regulating molecules (SERCA2a, PLB, RyR2 and FKBP12.6), and elevated [Ca(2+)]i in H9c2 cells. Furthermore, angiotensin II markedly increased the expression of ER stress signaling molecules (GRP78, CHOP, p-JNK and cleaved caspase-12) and ER stress-mediated apoptosis. OPD (100, 250 and 500 nmol/L) dose-dependently increased CYP2J3 expression and 14,15-DHET levels in normal H9c2 cells. Pretreatment of H9c2 cells with OPD suppressed angiotensin II-induced abnormalities in Ca(2+) homeostasis, ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous 14,15-EET also prevented angiotensin II-induced abnormalities in Ca(2+) homeostasis, whereas transfection with CYP2J3 siRNA diminished the effects of OPD on Ca(2+) homeostasis. Furthermore, the intracellular Ca(2+) chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells. OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca(2+) homeostasis and ER stress.

R-Cepia1er as a New Tool to Directly Measure [Ca] in the Sarcoplasmic Reticulum of Ventricular Myocytes

In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR) determines, in part, the amplitude of SR Ca release which, in turn, governs myocardial contraction. Therefore, it is critical to understand the molecular mechanisms that regulate [Ca]SR handling. Until recently, the best tool available to directly measure [Ca]SR was a low affinity Ca indicator (Fluo-5N). However this dye presents several difficulties, including species-limitation (it does not work in small rodents), dye extrusion, and non-specific cellular localization. Recently a new genetically encoded family of Ca indicators has been generated, named calcium-measuring organelle-entrapped protein indicators (CEPIA). Here we tested the red emitting fluorescence Ca indicator targeted to the SR, R-CEPIA1er, as a tool to directly measure [Ca]SR dynamics in ventricular myocytes. Infection of rabbit and rat myocytes with an adenovirus expressing the R-CEPIA1er gene, displayed prominent localization of the indicator in the SR and nuclear envelope. Calibration of R-CEPIA1er in rabbit myocytes resulted in a Kd of 617 microM, suggesting that this sensor is sensitive in the whole physiological range of [Ca]SR. [Ca]SR dynamics measured with R-CEPIA1er were tested in rabbit myocytes and compared to Fluo5-N signal. We found that the time course of the [Ca]SR depletion and fractional [Ca]SR release (FR) induced by an action potential was similar between R-CEPIA1er and Fluo-5N. R-CEPIA1er fluorescence did not decline during experiments, indicating lack of dye extrusion or photobleaching. Furthermore, this Ca sensor allowed us to reliably measure [Ca]SR dynamics in rat myocytes. Measurements of [Ca]SR with R-CEPIA1er can be combined with [Ca]cyt indicators (e.g. Fluo-4) to obtain detailed information regarding Ca homeostasis in cardiac myocytes. Moreover, R-CEPIA1er is an optimal tool that can be used to study [Ca]SR handling in commonly used transgenic animals (such as mice and rats).

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

Impaired Mitochondrial Function due to Familial Alzheimers Disease-Causing Presenilins Mutants via Calcium Disruptions

Mutants in presenilins (PS1 or PS2) is the major cause of familial Alzheimer's disease (FAD). FAD causing PS mutants affect intracellular Ca(2+) homeostasis by enhancing the gating of inositol trisphosphate (IP3) receptor (IP3R) Ca(2+) release channel on the endoplasmic reticulum, leading to exaggerated Ca(2+) release into the cytoplasm. Using experimental IP3R-mediated Ca(2+) release data, in conjunction with a computational model of cell bioenergetics, we explore how the differences in mitochondrial Ca(2+) uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca(2+). We find that as a result of exaggerated cytosolic Ca(2+) in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca(2+) dependent enzymes that stimulate NADH production. This leads to decreased rates in proton pumping due to diminished membrane potential along with less ATP and enhanced ROS production. These results show that through Ca(2+) signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially to cell death.

High-Throughput Screens to Discover Inhibitors of Leaky Ryanodine Receptor Calcium Channels

Using fluorescence lifetime (FLT) detection of fluorescence resonance energy transfer (FRET), we have developed high-throughput screening (HTS) methods to discover compounds that modulate the ryanodine receptor (RyR) Ca2+ (Ca) channel for therapeutic applications. Regulation of cellular Ca homeostasis is critical for skeletal and cardiac muscle function, and RyR is a central player. In cardiomyocytes, high Ca “leak” via the cardiac isoform of RyR (RyR2), and reduced SR Ca uptake, conspire to reduce the SR Ca content and elevate diastolic [Ca]i, both hallmarks of heart failure (HF). RyRs that open inappropriately during diastole contribute to both systolic and diastolic dysfunction and arrhythmias in HF. Therefore, inhibitors of the RyR leaky state could become highly effective drugs. Our HTS methods specifically detect binding of key RyR modulatory proteins (CaM and FKBP12.6) that have been implicated in controlling the pathological RyR2 leak. Thus, under oxidizing conditions that mimic a pathological state, a drug that restores normal CaM and/or FKBP binding may correct the leaky RyR state. Integration of fluorescently labeled FKBP12.6 and CaM and FRET enables translation of these tools into ultrasensitive HTS assays to assess the RyR leaky conformation. We have carried out a pilot screen of the 731-compound NIH Clinical Collections, which yielded several compounds that changed FRET by >3SD (a typical threshold used to select hits in primary HTS). Ongoing studies will show how the HTS structural readout correlates with effects on RyR function.