Characterization and purification of a Na+/Ca2+ exchanger from an archaebacterium

The superfamily of cation/Ca(2+) exchangers includes both Na(+)/Ca(2+) exchangers (NCXs) and Na(+)/Ca(2+),K(+) exchangers (NCKX) as the families characterized in most detail. These Ca(2+) transporters have prominent physiological roles. For example, NCX and NCKX are important in regulation of cardiac contractility and visual processes, respectively. The superfamily also has a large number of members of the YrbG family expressed in prokaryotes. However, no members of this family have been functionally expressed, and their transport properties are unknown. We have expressed, purified, and characterized a member of the YrbG family, MaX1 from Methanosarcina acetivorans. MaX1 catalyzes Ca(2+) uptake into membrane vesicles. The Ca(2+) uptake requires intravesicular Na(+) and is stimulated by an inside positive membrane potential. Despite very limited sequence similarity, MaX1 is a Na(+)/Ca(2+) exchanger with kinetic properties similar to those of NCX. The availability of a prokaryotic Na(+)/Ca(2+) exchanger should facilitate structural and mechanistic investigations.     

The mitochondrial Na(+)/Ca(2+) exchanger

Powered by the steep mitochondrial membrane potential Ca(2+) permeates into the mitochondria via the Ca(2+) uniporter and is then extruded by a mitochondrial Na(+)/Ca(2+) exchanger. This mitochondrial Ca(2+) shuttling regulates the rate of ATP production and participates in cellular Ca(2+) signaling. Despite the fact that the exchanger was functionally identified 40 years ago its molecular identity remained a mystery. Early studies on isolated mitochondria and intact cells characterized the functional properties of a mitochondrial Na(+)/Ca(2+) exchanger, and showed that it possess unique functional fingerprints such as Li(+)/Ca(2+) exchange and that it is displaying selective sensitivity to inhibitors. Purification of mitochondria proteins combined with functional reconstitution led to the isolation of a polypeptide candidate of the exchanger but failed to molecularly identify it. A turning point in the search for the exchanger molecule came with the recent cloning of the last member of the Na(+)/Ca(2+) exchanger superfamily termed NCLX (Na(+)/Ca(2+)/Li(+) exchanger). NCLX is localized in the inner mitochondria membrane and its expression is linked to mitochondria Na(+)/Ca(2+) exchange matching the functional fingerprints of the putative mitochondrial Na(+)/Ca(2+) exchanger. Thus NCLX emerges as the long sought mitochondria Na(+)/Ca(2+) exchanger and provide a critical molecular handle to study mitochondrial Ca(2+) signaling and transport. Here we summarize some of the main topics related to the molecular properties of the Na(+)/Ca(2+) exchanger, beginning with the early days of its functional identification, its kinetic properties and regulation, and culminating in its molecular identification.     

Feedback inhibition of sodium/calcium exchange by mitochondrial calcium accumulation

Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger were subjected to two periods of 5 and 3 min, respectively, during which the extracellular Na(+) concentration ([Na(+)](o)) was reduced to 20 mm; these intervals were separated by a 5-min recovery period at 140 mm Na(+)(o). The cytosolic Ca(2+) concentration ([Ca(2+)](i)) increased during both intervals due to Na(+)-dependent Ca(2+) influx by the exchanger. However, the peak rise in [Ca(2+)](i) during the second interval was only 26% of the first. The reduced rise in [Ca(2+)](i) was due to an inhibition of Na(+)/Ca(2+) exchange activity rather than increased Ca(2+) sequestration since the influx of Ba(2+), which is not sequestered by internal organelles, was also inhibited by a prior interval of Ca(2+) influx. Mitochondria accumulated Ca(2+) during the first interval of reduced [Na(+)](o), as determined by an increase in fluorescence of the Ca(2+)-indicating dye rhod-2, which preferentially labels mitochondria. Agents that blocked mitochondrial Ca(2+) accumulation (uncouplers, nocodazole) eliminated the observed inhibition of exchange activity during the second period of low [Na(+)](o). Conversely, diltiazem, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, increased mitochondrial Ca(2+) accumulation and also increased the inhibition of exchange activity. We conclude that Na(+)/Ca(2+) exchange activity is regulated by a feedback inhibition process linked to mitochondrial Ca(2+) accumulation.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

In dialyzed squid axons oxidative stress inhibits the Na+/Ca2+ exchanger by impairing the Cai2+-regulatory site

The Na(+)/Ca(2+) exchanger, a major mechanism by which cells extrude calcium, is involved in several physiological and physiopathological interactions. In this work we have used the dialyzed squid giant axon to study the effects of two oxidants, SIN-1-buffered peroxynitrite and hydrogen peroxide (H(2)O(2)), on the Na(+)/Ca(2+) exchanger in the absence and presence of MgATP upregulation. The results show that oxidative stress induced by peroxynitrite and hydrogen peroxide inhibits the Na(+)/Ca(2+) exchanger by impairing the intracellular Ca(2+) (Ca(i)(2+))-regulatory sites, leaving unharmed the intracellular Na(+)- and Ca(2+)-transporting sites. This effect is efficiently counteracted by the presence of MgATP and by intracellular alkalinization, conditions that also protect H(i)(+) and (H(i)(+) + Na(i)(+)) inhibition of Ca(i)(2+)-regulatory sites. In addition, 1 mM intracellular EGTA reduces oxidant inhibition. However, once the effects of oxidants are installed they cannot be reversed by either MgATP or EGTA. These results have significant implications regarding the role of the Na(+)/Ca(2+) exchanger in response to pathological conditions leading to tissue ischemia-reperfusion and anoxia/reoxygenation; they concur with a marked reduction in ATP concentration, an increase in oxidant production, and a rise in intracellular Ca(2+) concentration that seems to be the main factor responsible for cell damage.     

Ionic ligand interactions with the intracellular loop of the sodium-calcium exchanger. Modulation by ATP

In the last decade, there has been a large increase in the study of the Na(+)/Ca(2+) exchanger due to its implications in physiological and pathophysiological processes at the cell and organ levels. Key areas of these studies have been molecular biology, regulation and physiology-pathophysiology of the exchanger. There are three main types of regulation that take place at the large intracellular loop of the Na(+)/Ca(2+) exchanger: (i) ionic (sodium inactivation, calcium regulation and proton inhibition), (ii) metabolic (ATP as phosphoryl group donor), and (iii) genetic (alternative splicing). This review analyzes the most recent data on the mutual interactions of regulatory ionic ligands (Ca(2+), Na(+), H(+)) and how they are secondarily modulated by MgATP, emphasizing the importance of the binding of Ca(2+) to its regulatory site as an essential requirement for the exchange function. Intracellular protons and sodium inhibit the Na(+)/Ca(2+) exchanger by reducing the apparent affinity of the Ca(i)-regulatory site for Ca(2+). Although the metabolic pathways are different in the mammalian heart (membrane lipids) and squid nerve cells (soluble cytosolic regulatory protein), the final mechanism for the protective effect of MgATP is the same: a reduction of Na(i)(+)-H(i)(+) binding affinities facilitating the attachment of Ca(2+) to its regulatory site. Kinetic models, which partially analyzed some of these ionic and metabolic interactions, can be integrated into a single scheme where the Ca(i)-regulatory site plays a central role.     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

Calcium transport mechanisms in muskrat and rat hearts

Mammalian hearts experience calcium overload during extreme and prolonged hypoxia and the calcium overload may lead to enzyme activation and cell death. Several calcium transport systems were examined in muskrat hearts and compared to those found in rat hearts to determine if there is a species difference that might be related to the muskrats' superior ability to survive hypoxia. Radiolabeled nitredendipine binding was determined in rat and muskrat hearts to estimate the density of voltage gated calcium channels in surface membranes. There were no species differences. Calcium release channel density in the sarcoplasmic reticulum was estimated by the determination of radiolabeled ryanodine binding in muskrat and rat heart SR membranes. No differences were revealed between species. The SR uptake of calcium was measured in SR membranes from the hearts of the two species. No differences were found in the B(max) values, however, the muskrat SR membranes did have a slightly lower K(m) value. There were large species differences in Na(+)/Ca(2+) exchange in SL membranes with the muskrat heart having approximately 3.5 times the transport capacity of rat SL membranes. During hypoxic conditions in which there is extensive ATP depletion leading to [Na(+)](i) accumulation and discharge of cellular membrane potential, the Na(+)/Ca(2+) exchanger may operate in the reverse mode and import calcium into the cell and accelerate hypoxic damage. Prior to reaching this state a robust Na(+)/Ca(2+) exchange would facilitate the maintenance of normal diastolic calcium levels and calcium cycling. Muskrats hearts are hypoxia tolerant by virtue of their ability to reduce metabolic demand and generate ATP anaerobically thus, maintaining a favorable ATP balance. Therefore, the relative overexpression of Na(+)/Ca(2+) exchangers in muskrat hearts may be beneficial in the preservation of contractile function and calcium homeostasis in this freshwater diving mammal.     

Increased expression of the Na(+)/Ca(2+) exchanger in the rat heart after immobilization stress is not induced by cortisol.

Calcium homeostasis is crucial for the proper function of cardiac cells. Since the Na(+)/Ca(2+) exchanger is an important modulator of calcium homeostasis especially in the heart, the objective of this study was to investigate the effect of immobilization stress on the high capacity Na(+)/Ca(2+) exchanger in rat heart ventricles and atria. Repeated immobilization stress increased both the mRNA and the protein level and the activity of the Na(+)/Ca(2+) exchanger in the left, but not the right ventricle of rat heart. Since corticosterone is rapidly increased during the stress stimulus, it might be assumed that mRNA of the Na(+)/Ca(2+) exchanger is increased through a glucocorticoid responsive element. However, we have found that cortisol did not change the Na(+)/Ca(2+) exchanger at the mRNA or protein levels. These results clearly show that this effect of stress is not mediated via cortisol.     

Electrogenic Na(+)/Ca(2+) exchange. A novel amplification step in squid olfactory transduction

Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca(2+) concentrations ([Ca(2+)](i)). To directly asses the effects of increasing [Ca(2+)](i) in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca(2+) from internal stores. We observed an inward current response to caffeine. Monovalent cation replacement of Na(+) from the external bath solution completely and selectively inhibited the caffeine-induced response, and ruled out the possibility of a Ca(2+)-dependent nonselective cation current. The strict dependence on internal Ca(2+) and external Na(+) indicated that the inward current was due to an electrogenic Na(+)/Ca(2+) exchanger. Block of the caffeine-induced current by an inhibitor of Na(+)/Ca(2+) exchange (50-100 microM 2',4'-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na(+)/Ca(2+) exchange contributed to odor responses by applying the aquatic odor l-glutamate in the presence and absence of 2', 4'-dichlorobenzamil. We found that electrogenic Na(+)/Ca(2+) exchange was responsible for approximately 26% of the total current associated with glutamate-induced odor responses. Although Na(+)/Ca(2+) exchangers are known to be present in ORNs from numerous species, this is the first work to demonstrate amplifying contributions of the exchanger current to odor transduction.     

Dual effect of Nai+ on Ca2+ influx through the Na+/Ca2+ exchanger in dialyzed squid axons. Experimental data confirming the validity of the squid axon kinetic model

We propose a steady-state kinetic model for the squid Na(+)/Ca(2+) exchanger that differs from other current models of regulation in that it takes into account, within a single kinetic scheme, all ionic [intracellular Ca(2+) (Ca(i)(2+))-intracellular Na(+) (Na(i)(+))-intracellular H(i)(+)] and metabolic (ATP) regulations of the exchanger in which the Ca(i)(2+)-regulatory pathway plays the central role in regulation. Although the integrated ionic-metabolic model predicts all squid steady-state experimental data on exchange regulation, a critical test for the validity of it is the predicted dual effect of Na(i)(+) on steady-state Ca(2+) influx through the exchanger. To test this prediction, an improved technique for the estimation of isotope fluxes in squid axons was developed, which allows sequential measurements of ion influx and effluxes. With this method, we report here two novel observations of the squid axon Na(+)/Ca(2+) exchanger. First, at intracellular pH (7.0) and in the absence of MgATP, Na(i)(+) has a dual effect on Ca(2+) influx: inhibition at low concentrations followed by stimulation at high Na(i)(+) concentrations, reaching levels higher than those seen without Na(i)(+). Second, in the presence of MgATP, the biphasic response to Na(i)(+) disappears and is replaced by a sigmoid activation. Furthermore, the model predicts that Ca(2+) efflux is monotonically inhibited by Na(i)(+), more pronouncedly without than with MgATP. These results are predicted by the proposed kinetic model. Although not fully applicable to all exchangers, this scheme might provide some insights on expected net Ca(2+) movements in other tissues under a variety of intracellular ionic and metabolic conditions.     

Discovery of a novel potent Na+/Ca2+ exchanger inhibitor: design, synthesis and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives

Design, synthesis and structure-activity relationships for 3,4-dihydro-2(1H)-quinazolinone derivatives with the inhibitory activities of the Na(+)/Ca(2+) exchanger are discussed. These studies based on lead compound 1a lead to the discovery of a structurally novel and highly potent inhibitor against the Na(+)/Ca(2+) exchanger 4f (SM-15811), which directly inhibited the Na(+)-dependent Ca(2+) influx via the Na(+)/Ca(2+) exchanger in cardiomyocytes with a high potency.     

Intracellular and plasma membrane-initiated pathways involved in the [Ca2+]i elevations induced by iodothyronines (T3 and T2) in pituitary GH3 cells

The role of 3,5,3'-triiodo-l-thyronine (T3) and its metabolite 3,5-diiodo-l-thyronine (T2) in modulating the intracellular Ca(2+) concentration ([Ca(2+)](i)) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH(3) cells in the absence or presence of extracellular Ca(2+). When applied in Ca(2+)-free solution, T2 and T3 increased [Ca(2+)](i), in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by N(G)-nitro-l-arginine methyl ester and l-n(5)-(1-iminoethyl)ornithine hydrochloride significantly reduced the [Ca(2+)](i) increase induced by T2 and T3. However, while depletion of inositol trisphosphate-dependent Ca(2+) stores did not interfere with the T2- and T3-induced [Ca(2+)](i) increases, the inhibition of phosphatidylinositol 3-kinase by LY-294002 and the dominant negative form of Akt mutated at the ATP binding site prevented these effects. Furthermore, the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prevented the increases in both [Ca(2+)](i) and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca(2+)](i) increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na(+)/Ca(2+) exchanger by CGP37157 significantly reduced the [Ca(2+)](i) increases induced by T2 and T3. In the presence of extracellular calcium (1.2 mM), under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, T2 and T3 increased both [Ca(2+)](i) and intracellular Na(+) concentration; nimodipine reduced the [Ca(2+)](i) increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na(+)/H(+) pump by 5-(N-ethyl-N-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na(+)/Ca(2+) exchanger, PKC, and phosphatidylinositol 3-kinase, respectively) failed to modify the T2- and T3-induced effects. Collectively, the present results suggest that T2 and T3 exert short-term nongenomic effects on intracellular calcium and NO by modulating plasma membrane and mitochondrial pathways that differ between these iodothyronines.     

Protein kinase C alpha enhances sodium-calcium exchange during store-operated calcium entry in mouse platelets

A rise in intracellular calcium concentration ([Ca(2+)](i)) is necessary for platelet activation. A major component of the [Ca(2+)](i) elevation occurs through store-operated Ca(2+) entry (SOCE). The aim of this study was to understand the contribution of the classical PKC isoform, PKCα to platelet SOCE, using platelets from PKCα-deficient mice. SOCE was reduced by approximately 50% in PKCα(-/-) platelets, or following treatment with bisindolylmaleimide I, a PKC inhibitor. However, TG-induced Mn(2+) entry was unaffected, which suggests that divalent cation entry through store-operated channels is not directly regulated. Blocking the autocrine action of secreted ADP or 5-HT on its receptors did not reproduce the effect of PKCα deficiency. In contrast, SN-6, a Na(+)/Ca(2+) exchanger inhibitor, did reduce SOCE to the same extent as loss of PKCα, as did replacing extracellular Na(+) with NMDG(+). These treatments had no further effect in PKCα(-/-) platelets. These data suggest that PKCα enhances the extent of SOCE in mouse platelets by regulating Ca(2+) entry through the Na(+)/Ca(2+) exchanger.     

Ca2+ regulation of ion transport in the na+/ca2+ exchanger

The binding of Ca(2+) to two adjacent Ca(2+)-binding domains, CBD1 and CBD2, regulates ion transport in the Na(+)/Ca(2+) exchanger. As sensors for intracellular Ca(2+), the CBDs form electrostatic switches that induce the conformational changes required to initiate and sustain Na(+)/Ca(2+) exchange. Depending on the presence of a few key residues in the Ca(2+)-binding sites, zero to four Ca(2+) ions can bind with affinities between 0.1 to 20 μm. Importantly, variability in CBD2 as a consequence of alternative splicing modulates not only the number and affinities of the Ca(2+)-binding sites in CBD2 but also the Ca(2+) affinities in CBD1.     

Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange and triggers Ca(i)2+-induced Ca2+ release in rat cerebellar Type-1 astrocytes

We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing Na(+) entry through the electrogenic Na(+)-Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.     

Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes

We have examined the distribution of ryanodine receptors, L-type Ca(2+) channels, calsequestrin, Na(+)/Ca(2+) exchangers, and voltage-gated Na(+) channels in adult rat ventricular myocytes. Enzymatically dissociated cells were fixed and dual-labeled with specific antibodies using standard immunocytochemistry protocols. Images were deconvolved to reverse the optical distortion produced by wide-field microscopes equipped with high numerical aperture objectives. Every image showed a well-ordered array of fluorescent spots, indicating that all of the proteins examined were distributed in discrete clusters throughout the cell. Mathematical analysis of the images revealed that dyads contained only ryanodine receptors, L-type Ca(2+) channels, and calsequestrin, and excluded Na(+)/Ca(2+) exchangers and voltage-gated Na(+) channels. The Na(+)/Ca(2+) exchanger and voltage-gated Na(+) channels were distributed largely within the t-tubules, on both transverse and axial elements, but were not co-localized. The t-tubule can therefore be subdivided into at least three structural domains; one of coupling (dyads), one containing the Na(+)/Ca(2+) exchanger, and one containing voltage-gated Na(+) channels. We conclude that if either the slip mode conductance of the Na(+) channel or the reverse mode of the Na(+)/Ca(2+) exchanger are to contribute to the contractile force, the fuzzy space must extend outside of the dyad.     

Reversed Na+/Ca2+ exchange contributes to Ca2+ influx and respiratory burst in microglia

Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca(2+)) influx. However, the specific Ca(2+) entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca(2+)-permeable ion channels or Na(+)/Ca(2+) exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na(+)/Ca(2+) exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na(+)/Ca(2+) exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na(+)/Ca(2+) exchanger activity in these cells. To investigate conditions under which Na(+)/Ca(2+) exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca(2+) indicator (Fura-2) and a Na(+) indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na(+)/Ca(2+) exchange with candidate Ca(2+)-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na(+)/Ca(2+) exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca(2+) channels or L-type voltage-gated Ca(2+) channels had no effect. These results provide evidence that Na(+)/Ca(2+) exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.     

Sodium accumulation in SERCA knockout-induced heart failure

In cardiomyocytes, a major decrease in the level of sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) can severely impair systolic and diastolic functions. In mice with cardiomyocyte-specific conditional excision of the Serca2 gene (SERCA2 KO), end-stage heart failure developed between four and seven weeks after gene deletion combined with [Na(+)](i) elevation and intracellular acidosis. In this study, to investigate the underpinning changes in Ca(2+) dynamics and metabolic homeostasis, we developed data-driven mathematical models of Ca(2+) dynamics in the ventricular myocytes of the control, four-week, and seven-week SERCA2 knockout (KO) mice. The seven-week KO model showed that elevated [Na(+)](i) was due to increased Na(+) influxes through the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/H(+) exchanger, with the latter exacerbated by intracellular acidosis. Furthermore, NCX upregulation in the seven-week KO model resulted in increased ATP consumption for ion transport. Na(+) accumulation in the SERCA KO due to NCX upregulation and intracellular acidosis potentially play a role in the development of heart failure, by initiating a reinforcing cycle involving: a mismatch between ATP demand and supply; an increasingly compromised metabolism; a decreased pH(i); and, finally, an even greater [Na(+)](i) elevation.