NCX Activity Research Articles

Activation of the cardiac Na+–Ca2+ exchanger by sorcin via the interaction of the respective Ca2+-binding domains

Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca2+ binding. The sarcolemmal Na+/Ca2+ exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca2+ binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca2+ due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3μM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G>wt sorcin>Sorcin Calcium Binding Domain (SCBD)>W99G>E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50µM Ca2+, the interaction with CBD1 followed the order W105G>SCBD>wt sorcin>W99G>E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca2+-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.

Inhibition of sodium–calcium exchange by KB-R7943: Dodecylamine and sphingosine in transfected Chinese hamster ovary cells

Inhibitors of Na +/Ca 2+ exchange (NCX) such as KB-R7943 and SEA0400 are thought to act by promoting an inactive state of the exchanger induced by elevated cytosolic Na + concentrations (Na +-dependent inactivation). This conclusion is based mainly on experiments in excised patches from frog oocytes expressing NCX and has not been fully tested in intact cells. Here we characterize the inhibitory effects of KB-R7943 and other amphiphilic cations on NCX activity in transfected Chinese hamster ovary (CHO) cells expressing the cardiac isoform of the Na +/Ca 2+ exchanger (NCX1.1). Cytosolic Na + concentrations were varied using the channel-forming ionophore gramicidin. KB-R7943 (10 μM) inhibited NCX activity at high (140 mM) but not at low (10 mM) cytosolic Na + concentrations. Cells expressing NCX mutants that were either more (F223E) or less (K229Q) susceptible than the wild-type to Na +-dependent inactivation were either hypersensitive or resistant, respectively, to KB-R7943 inhibition. We recently reported that Na-dependent inactivation in transfected CHO cells could be partially but transiently reversed by increasing the external Ca 2+ concentration (Chernysh et al., 2008). The inhibitory effects of KB-R7943 were likewise partially reversed by elevated Ca 2+. Other amphiphilic cations such as dodecylamine (20 μM) and sphingosine (10 μM) closely mimicked the effects of KB-R7943 on NCX activity. Our results support the hypothesis that KB-R7943 inhibits NCX activity in intact cells by promoting Na +-dependent inactivation and suggest that this is a general mode of inhibition by amphiphilic cations.

Knockout of Na+/Ca2+ exchanger in smooth muscle attenuates vasoconstriction and L-type Ca2+ channel current and lowers blood pressure

Mice with smooth muscle (SM)-specific knockout of Na(+)/Ca(2+) exchanger type-1 (NCX1(SM-/-)) and the NCX inhibitor, SEA0400, were used to study the physiological role of NCX1 in mouse mesenteric arteries. NCX1 protein expression was greatly reduced in arteries from NCX1(SM-/-) mice generated with Cre recombinase. Mean blood pressure (BP) was 6-10 mmHg lower in NCX1(SM-/-) mice than in wild-type (WT) controls. Vasoconstriction was studied in isolated, pressurized mesenteric small arteries from WT and NCX1(SM-/-) mice and in heterozygotes with a global null mutation (NCX1(Fx/-)). Reduced NCX1 activity was manifested by a marked attenuation of responses to low extracellular Na(+) concentration, nanomolar ouabain, and SEA0400. Myogenic tone (MT, 70 mmHg) was reduced by approximately 15% in NCX1(SM-/-) arteries and, to a similar extent, by SEA0400 in WT arteries. MT was normal in arteries from NCX1(Fx/-) mice, which had normal BP. Vasoconstrictions to phenylephrine and elevated extracellular K(+) concentration were significantly reduced in NCX1(SM-/-) arteries. Because a high extracellular K(+) concentration-induced vasoconstriction involves the activation of L-type voltage-gated Ca(2+) channels (LVGCs), we measured LVGC-mediated currents and Ca(2+) sparklets in isolated mesenteric artery myocytes. Both the currents and the sparklets were significantly reduced in NCX1(SM-/-) (vs. WT or NCX1(Fx/-)) myocytes, but the voltage-dependent inactivation of LVGCs was not augmented. An acute application of SEA0400 in WT myocytes had no effect on LVGC current. The LVGC agonist, Bay K 8644, eliminated the differences in LVGC currents and Ca(2+) sparklets between NCX1(SM-/-) and control myocytes, suggesting that LVGC expression was normal in NCX1(SM-/-) myocytes. Bay K 8644 did not, however, eliminate the difference in myogenic constriction between WT and NCX1(SM-/-) arteries. We conclude that, under physiological conditions, NCX1-mediated Ca(2+) entry contributes significantly to the maintenance of MT. In NCX1(SM-/-) mouse artery myocytes, the reduced Ca(2+) entry via NCX1 may lower cytosolic Ca(2+) concentration and thereby reduce MT and BP. The reduced LVGC activity may be the consequence of a low cytosolic Ca(2+) concentration.

KB-R7943 inhibits high glucose-induced endothelial ICAM-1 expression and monocyte-endothelial adhesion

Hyperglycemia is the major cause of diabetic angiopathy. The aim of our study was to evaluate the impact of KB-R7943, an inhibitor of Na +/Ca 2+ exchanger (NCX) on cell growth and function of human “diabetic” endothelial cells (EC). Intercellular adhesion molecule-1 (ICAM-1) expression and NCX activity were determined after EC were exposed to high glucose in the absence and presence of KB-R7943. Coincubation of EC with high glucose for 24 h resulted in a significant increase of monocyte-endothelial cell adhesion and the expression of ICAM-1. These effects were abolished by KB-R7943 and KB-R7943 significantly decreased the activation of NCX induced by high glucose. These findings suggested that KB-R7943 may play a role in inhibiting expression of adhesion molecules by inhibiting the reverse activation of NCX.

Impact of Loss of T-Tubules on Myocardial Contractile Force and Kinetics

Action potential driven Ca2+ currents via the transverse tubular membrane synchronously trigger Ca2+release from the SR close to the myofibril rapidly activating contraction. Loss of T-tubules has been reported in disease including heart failure, but the effect of detubulation on muscle mechanics has never been investigated.We dissected thin (50-200μm) right ventricular and left atrial trabeculae from adult rats and recorded force and sarcomere length. To achieve acute detubulation, we exposed trabeculae to formamide 1.5M for 20 minutes. Return to normo-osmotic solution increases cell-volume suddenly and disconnects T-tubules from the sarcolemma as was directly verified using di-8-anepps staining and confocal microscopy.In seven ventricular trabeculae, detubulation prolonged the twitch, i.e. time to peak force (by 31±9%) and time to 50% relaxation (by 46±14%) at 1Hz. Detubulation reduced the inotropic effect of increased stimulus-rate (by 29±7%). Maximal twitch force after post-rest potentiation was unchanged. Detubulation increased the fraction of Ca2+ recirculating to the SR (by 17 ± 5%) measured by the decay of potentiation, suggesting an increased SERCA vs. NCX activity. None of these effects was seen in four formamide-treated atrial trabeculae, which constitutively lack T-tubules in rodents.T-tubular disruption from the membrane implies loss of 80% L-Type ICa and 60% INCX. Mathematical modeling shows that in myocytes with EC-Coupling via T tubules and SR alone the loss of the aforementioned currents is not sufficient to explain the differences between control and detubulated trabeculae. Such differences can be predicted assuming that EC-coupling is maintained by a fast Ca2+ rise near the sarcolemma but now followed by, Ca2+-diffusion mediated, propagated Ca2+ induced SR-Ca2+ release toward the core. Enhanced Ca2+_wave spread and recruitment of all myofibril layers can contribute to maintain maximal contractile force in the absence of T-tubules.

Distribution of Voltage Gated Sodium Channels in Rabbit Cardiomyocytes During Development

We and others have postulated that in neonatal cardiomyocytes sodium currents may influence Ca2+ influx through reverse-mode NCX activity by inducing strong membrane depolarization and/or local sodium accumulation profoundly modify contractility. To study the role of voltage-gated sodium channels (NaV1.X) and reverse mode NCX activity through development, the expression and distribution of NaV1.X was determined using Western blot analysis, immunocytochemistry, confocal microscopy and image analysis in cardiomyocytes isolated from 3 and 56 days old rabbits. Immunoblot analysis revealed a robust expression of the skeletal muscle isoform (Nav1.4) in neonatal cardiomyocytes which decreased 6-fold by 56 days (p<0.01). The neuronal isotype Nav1.1 was found to have comparatively low levels of expression throughout development and followed a similar pattern as that of Nav1.4. Nav1.1 levels decreased by 9 fold in the 56 day cardiomyocyte (p<0.01). The cardiac isoform (Nav1.5) expression was also robust in the neonatal and adult cardiomyocytes but the protein levels did not vary significantly throughout development by western blot analysis. The distribution of Nav1.4 and Nav1.5 was punctate in nature on the cell periphery in both 3 and 56 day cardiomyocytes. Both Nav1.4 and Nav1.5 co-localized with NCX in 3 and 56 day cardiomyocytes. In neonatal cardiomyocytes, Nav1.4-NCX and Nav1.5-NCX colocalization relationship remained the same, while in the adult, Nav1.5-NCX colocalization decreased by 50% due to the increase in the separation distances between Nav1.5 and NCX in the adult. Taken together, our results suggest that in the neonate heart, Nav1.4 may dictate the role of NCX in regulating Ca2+ influx during contraction.

Modulation of the sodium–calcium exchanger in the rat kidney by different sequential stressors

Stress, if exaggerated, modulates a variety of metabolic pathways and results in development of serious health consequences. The cell membrane sodium–calcium exchanger (NCX) is a major calcium extrusion system and is also modulated by stress. It has been shown previously that mRNA, protein levels and activity of the type 1 NCX (NCX1) in the left ventricle of the rat heart are increased by stressors, such as immobilization or hypoxia. In this study we investigated whether exposure to a subsequent different stressor can affect gene expression, protein level and activity of the NCX1 in rat kidney compared to exposure to only one type of stressor. In these experiments, we used immobilization and cold as the model stressors. We found that cold exposure at 4°C for 24 h, when applied after immobilization repeated seven times, completely abolished the immobilization-induced increase in NCX mRNA level and after 7 days cold exposure the increases in NCX1 protein and activity in rat kidney were also abolished. Permanently increased NCX1 expression can result in imbalance of cellular calcium homeostasis and thus contribute to kidney dysfunction. Based on our results, we conclude that exposure to a cold stressor can have a protective effect on the kidney in rats exposed previously to repeated immobilization stress. This might be explained by differential stimulation of sympathetic neural and adrenal medullary responses by these different stressors.

Expression and Regulation of Shark NCX Gene in Transgenic Mouse Heart

Inward currents generated by the mammalian cardiac Na+-Ca2+-exchanger (NCX1.1) have arrhythmogenic potential, especially in the failing heart where expression of NCX is up-regulated. We hypothesize that arrhythmogenesis might be alleviated if NCX were subject to the same cAMP mediated regulation (suppression of Ca2+-influx, but enhancement of Ca2+-efflux on NCX) as found in the native shark ventricle. To test this hypothesis, we created heterozygous transgenic mice that express the shark NCX protein with a myc-tag, under the control of the alpha-myosin heavy chain promoter (α-MHC). The construct was evaluated by expression and functionality prior to production of transgenic lines. The expression of the transgene was confirmed by immunocytochemistry staining using DAPI and myc-FITC antibody on transfected HL-1 cells. Using dual laser confocal microscopy, the pattern of staining was consistent with NCX expression at the protein level. To determine the functionality of the transgene, HL-1 cells were co-transfected with the shark NCX transgene and GFP. GFP positive cells were incubated with Fluo-4 AM and imaged confocally. These cells showed NCX activity in response to withdrawal and readmission of [Na+]o consistent with electrophysiological data of native shark myocytes.Echocardiography and ECG studies on transgenic mice showed no remarkable cardiac phenotype, but analysis of initial voltage-clamp and western blot studies verify robust exchanger currents and expression levels. Our findings show that shark NCX is functional in the transgenic mouse producing no discernable cardiac pathology, but it remains to be determined as yet whether shark NCX can confer anti-arrhythmic properties to the mammalian heart.

Protein aggregation in neurons following OGD: a role for Na+ and Ca2+ ionic dysregulation

In this study, we investigated whether disruption of Na(+) and Ca(2+) homeostasis via activation of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchange (NCX(rev)) affects protein aggregation and degradation following oxygen-glucose deprivation (OGD). Cultured cortical neurons were subjected to 2 h OGD and 1-24 h reoxygenation (REOX). Redistribution of ubiquitin and formation of ubiquitin-conjugated protein aggregates occurred in neurons as early as 2 h REOX. The protein aggregation progressed further by 8 h REOX. There was no significant recovery at 24 h REOX. Moreover, the proteasome activity in neurons was inhibited by 80-90% during 2-8 h REOX and recovered partially at 24 h REOX. Interestingly, pharmacological inhibition or genetic ablation of NKCC1 activity significantly decreased accumulation of ubiquitin-conjugated protein aggregates and improved proteasome activity. A similar protective effect was obtained by blocking NCX(rev) activity. Inhibition of NKCC1 activity also preserved intracellular ATP and Na(+) homeostasis during 0-24 h REOX. In a positive control study, disruption of endoplasmic reticulum Ca(2+) with thapsigargin triggered redistribution of free ubiquitin and protein aggregation. We conclude that overstimulation of NKCC1 and NCX(rev) following OGD/REOX partially contributes to protein aggregation and proteasome dysfunction as a result of ionic dysregulation.

Reduced expression of the Na+/Ca2+ exchanger in adult cardiomyocytes via adenovirally delivered shRNA results in resistance to simulated ischemic injury

The Na(+)/Ca(2+) exchanger (NCX) is proposed to be an important protein in the regulation of Ca(2+) movements in the heart. This Ca(2+) regulatory action is thought to modulate contractile activity in the heart under normal physiological conditions and may contribute to the Ca(2+) overload that occurs during ischemic reperfusion challenge. To evaluate these hypotheses, adult rat cardiomyocytes were exposed to an adenovirus that codes for short hairpin RNA (shRNA) targeting NCX gene expression through RNA interference. An adenovirus transcribing a short RNA with a scrambled nucleotide sequence was compared with the NCX-shRNA nucleotide sequence and used as a control. Freshly isolated rat cardiomyocytes were infected with virus for 48 h before examination. Cardiomyocytes maintained their characteristic morphological appearance during this short time period after isolation. NCX expression was inhibited by up to approximately 60% by the shRNA treatment as determined by Western blot analysis. The depletion in NCX protein was accompanied by a significant depression of NCX activity in shRNA-treated cells. Ca(2+) homeostasis was unaltered in the shRNA-treated cells upon electrical stimulation compared with control cells. However, when cardiomyocytes were exposed to a simulated ischemic solution, NCX-depleted cells were significantly protected from the rise in cytoplasmic Ca(2+) and damage that was detected in control cells during ischemia and reperfusion. Our data support the role for NCX in ischemic injury to the heart and demonstrate the usefulness of altering gene expression with an adenoviral-delivery system of shRNA in adult cardiomyocytes.

NCX1 Expression and Functional Activity Increase in Microglia Invading the Infarct Core

The sodium-calcium exchanger NCX1 represents a key mediator for maintaining [Na(+)](i) and [Ca(2+)](i) in anoxic conditions. To date, no information is available on NCX1 protein expression and activity in microglial cells under ischemic conditions. By means of Western blotting, patch-clamp electrophysiology, single-cell Fura-2 acetoxymethyl-ester microfluorometry, immunohistochemistry, and confocal microscopy, we investigated the regional and temporal changes of NCX1 protein in microglial cells of the peri-infarct and core regions after permanent middle cerebral artery occlusion. The exchanger expression and activity were measured in primary microglia isolated ex vivo from the core region of adult rat brains 7 days after permanent middle cerebral artery occlusion and in cultured microglia under in vitro hypoxia. One day after permanent middle cerebral artery occlusion, NCX1 protein expression was detected in some microglial cells adjacent to the soma of neurons in the infarct core. More interestingly, 3 and 7 days after permanent middle cerebral artery occlusion, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells obtained from the core also displayed increased NCX1 expression as compared with contralateral cells and showed enhanced NCX activity in the reverse mode of operation. Similarly, NCX activity and NCX1 protein expression were significantly enhanced in BV2 microglia exposed to oxygen and glucose deprivation, whereas NCX2 and NCX3 were downregulated. Interestingly, in NCX1-silenced cells, [Ca(2+)](i) increase induced by hypoxia was completely prevented. Conclusion- The upregulation of NCX1 expression and activity observed in microglia after permanent middle cerebral artery occlusion suggests a relevant role of NCX1 in modulating microglia functions in the postischemic brain.

Ca 2+ regulation in the Na + /Ca 2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na(+)/Ca(2+) exchanger (NCX) occurs via its cytoplasmic Ca(2+)-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca(2+)-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca(2+)-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca(2+). Striking differences in the electrostatic potential between the Ca(2+)-bound and -free forms strongly suggest that Ca(2+)-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C(2)-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca(2+)-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca(2+) is influenced by the closely located cassette exons. We further propose that Ca(2+)-binding to CBD2 induces a second electrostatic switch, required to alleviate Na(+)-dependent inactivation of Na(+)/Ca(2+) exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.

Molecular Pharmacology of the Amiloride Analog 3-Amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a Pan Inhibitor of the Na+-Ca2+Exchanger Isoforms NCX1, NCX2, and NCX3 in Stably Transfected Cells

With the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) and on several other membrane currents including voltage- and pH-sensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC(50) values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the alpha(2) repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca(2+) channels, tetrodotoxin-sensitive Na(+) channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.

Reactive oxygen species directly modify sodium–calcium exchanger activity in a splice variant-dependent manner

The sodium–calcium exchanger isoform 1 (NCX1) operating in calcium-efflux mode plays an important role in maintaining calcium homeostasis in the heart. Paradoxically, activity of NCX1 in calcium-influx mode contributes to the pathological intracellular calcium overload during cardiac ischemia–reperfusion injury. Reactive oxygen species (ROS) also contribute to myocardial dysfunction in ischemia–reperfusion and are reported to alter NCX1 activity. However, the molecular mechanism(s) by which ROS modifies NCX1 activity have not been elucidated. Therefore, the effects of the ROS, H2O2, on recombinant NCX1 splice variants were studied using the patch-clamp technique. H2O2 irreversibly increased calcium-influx mode activity in the cardiac NCX1.1 splice variant, without affecting calcium-efflux mode activity. In direct contrast, H2O2 inhibited the calcium-influx mode of the vascular NCX1.3 splice variant indicating that these disparate effects of H2O2 may be dependent on the exon complement of the alternative splicing region. Using NCX1 splice variants with various exon compositions, the mutually exclusive exons A and B were found to bestow the differential effects of H2O2 on NCX1 function. As NCX1 inhibition is a potential therapeutic strategy for ischemia–reperfusion injury, the effects of the NCX1 inhibitor KB-R7943 were examined. KB-R7943 was ~7-fold less potent at inhibiting NCX1 activity after H2O2 modification. In summary, this study provides insights into the molecular regulation of NCX1 by ROS and indicates that ROS may elicit differential effects in various tissues depending on the exon composition of the splice variant expressed. These results also highlight that the potency of NCX1 inhibitors may be impaired under conditions of oxidative stress.

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na(+)/Ca(2+) exchanger (NCX) is a membrane transporter that can switch Na(+) and Ca(2+) in either direction to maintain the homeostasis of intracellular Ca(2+). Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na(+)-free medium induced a significant increase in intracellular calcium concentration ([Ca(2+)](i)) in both cell types. This response was completely abolished by the NCX inhibitor 5-(N-4-chlorobenzyl)-20,40-dimethylbenzamil (CB-DMB). Moreover, inhibition of NCX activity during Ca(2+)-signaling induced by histamine caused a delay in restoring baseline [Ca(2+)](i). Na(+)-free medium induced TNF-alpha expression and release in HLM comparable to that caused by LPS. TNF-alpha release induced by Na(+)-free medium was blocked by CB-DMB and greatly reduced by RNAi-mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca(2+)](i), to generate Ca(2+)-signals, and to induce TNF-alpha production. Therefore, NCX may contribute to regulate Ca(2+) homeostasis and proinflammatory functions in human macrophages and monocytes.

Regulation of calcium transport by the mammalian retinal pigment epithelium

The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that regulates the composition of the subretinal space bathing the photoreceptor outer segments. Calcium concentration in the subretinal space varies with illumination of the retina. In this study we characterized the transepithelial calcium transport by the mammalian RPE. By immunocytochemistry we localized plasma membrane calcium transport proteins PMCA, TRPV5 and NCX to both apical and basolateral RPE membranes in bovine or human native tissues. Calcium transport activity was assessed as transepithelial 45‐Ca flux in voltage‐clamped bovine RPE/choroid Ussing preparations. Apical‐to‐basal and basal‐to‐apical Ca fluxes were measured in both short and open circuit conditions. We found that bovine RPE/choroid catalyzes a net, active calcium transport in the basal‐to‐apical (choroid‐to‐retina) direction. In normal open circuit condition, the positive apical transepithelial potential drives a paracellular calcium flux that roughly balances this active transport. Application of low sodium Ringer solution to the apical surface decreased net basal‐to‐apical flux by almost 90%, suggesting that NCX activity contributes to the active transepithelial calcium transport in bovine RPE.Supported by American Health Assistance Foundation, Macular Degeneration Research

A key role for dense granule secretion in potentiation of the Ca 2+ signal arising from store-operated calcium entry in human platelets

Recent work has demonstrated a role for Na +/Ca 2+ exchange in potentiation of the Ca 2+ entry elicited through the human platelet store-operated channel by controlling a Mn 2+-impermeable Ca 2+ entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na +/Ca 2+ exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn 2+-impermeable Ca 2+ entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca 2+ signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca 2+ signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion.

Calcium Signalling By Sodium Channels In Developing Rabbit Cardiomyocytes

Recent studies have demonstrated that in the neonatal cardiomyocytes, Ca2+ influx through reverse-mode NCX activity is sufficient to induce calcium induced calcium release. This study is undertaken to study the molecular components of excitation-contraction coupling in neonatal cardiomyocytes. The expression of voltage gated sodium channels was determined using Western blot analysis at different developmental time points. In this study, we investigate the regulation of neuronal (Nav1.1, Nav1.3, Nav1.6), skeletal ((Nav1.4) and the cardiac Nav1.5 isoforms and their respective intermolecular interactions with NCX in developing hearts. Immunoblot analysis of heart samples isolated from rabbits at 3, 10, 20 and 56 days after birth revealed a robust expression of skeletal muscle (Nav1.4) in the neonates and decreases significantly in 56 day old rabbit. The neuronal isotypes Nav 1.1 and Nav1.3 were found to have low levels of expression through development. Cardiac isoform (Nav 1.5) expression was similar to Nav 1.4 in the neonatal heart homogenates but the protein levels decreased in the 56 day heart homogenate. In isolated cardiomyocytes, skeletal isoform protein expression was significantly more prominent in neonates (3 days) compared to the adult (56 day). Our preliminary results suggest that in the neonate heart Nav1.4 may dictate the role of NCX in regulating Ca2+ influx during contraction.

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP−/− Mice

BackgroundThe role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.Principal FindingsWe used intact olfactory epithelium obtained from WT and OMP−/− mice to monitor the Ca2+ dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca2+ channels, or Ca2+ stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca2+-homeostasis in these neurons by influencing the activity of the plasma membrane Na+/Ca2+-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca2+ elevation by stimulating the reverse mode of NCX in both WT and OMP−/− mice. The resulting Ca2+ responses indicate that OMP facilitates NCX activity and allows rapid Ca2+ extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).ConclusionsSince CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions.

Neuronal injury in NCX3 knockout mice following permanent focal cerebral ischemia and in NCX3 knockout cortical neuronal cultures following oxygen-glucose deprivation and glutamate exposure

In this study we subjected NCX3 knockout and wildtype mice to permanent focal cerebral ischemia by intraluminal middle cerebral artery occlusion. Analysis of brain sections by 2,3,5-Triphenyl-2H-tetrazolium chloride staining, 12 hours after middle cerebral artery occlusion revealed no difference in infarct volume between NCX3 knockout and wildtype mice. In addition, we evaluated the effect of NCX3 protein knockdown on neuronal viability in primary cortical neuronal cultures following in vitro ischemia (oxygen-glucose deprivation) and L-glutamate (glutamate) exposure. In vitro experiments revealed that neuronal viability was higher in NCX3 knockout neuronal cultures than in the wildtype cultures following ischemic and glutamate insults. The reduced sensitivity of neurons from NCX3 knockout mice to in vitro ischemia and excitotoxicity indicates that NCX3 calcium entry mode activity contributes to calcium overload and neuronal death. However our in vivo finding of a lack of differential sensitivity on infarct volume in NCX3 knockout and wildtype mice suggests that any benefit of reduced NCX3 activity is overridden following permanent focal cerebral ischemia. Taken together, these findings suggest that reduced NXC3 activity limits calcium neurotoxicity during severe transient, but not during severe sustained ischemic insults. These results have important implications for the development of the NCX3 protein as a therapeutic target to reduce ischemic brain injury.