Physiological functions of the regulatory domains of the cardiac Na(+)/Ca(2+) exchanger NCX1

Physiologicalfunctions of the intracellular regulatory domains of theNa⁺/Ca²⁺ exchanger NCX1 were studied byexamining Ca²⁺ handling in CCL39 cells expressing alow-affinity Ca²⁺ regulatory site mutant (D447V/D498I), anexchanger inhibitory peptide (XIP) region mutant displaying noNa⁺ inactivation (XIP-4YW), or a mutant lacking most of thecentral cytoplasmic loop (246-672). We found that D447V/D498Iwas unable to efficiently extrude Ca²⁺ from the cytoplasm,particularly during a small rise in intracellular Ca²⁺concentration induced by the physiological agonist -thrombin orthapsigargin. The same mutant took up Ca²⁺ much lessefficiently than the wild-type NCX1 in Na⁺-free medium whentransfectants were not loaded with Na⁺, although itappeared to take up Ca²⁺ normally in transfectantspreloaded with Na⁺. XIP-4YW and, to a lesser extent,246-672, but not NCX1 and D447V/D498I, markedly accelerated theloss of viability of Na⁺-loaded transfectants. Furthermore,XIP-4YW was not activated by phorbol ester, whereas XIP-4YW andD447V/D498I were resistant to inhibition by ATP depletion. The resultssuggest that these regulatory domains play important roles in thephysiological and pathological Ca²⁺ handling by NCX1, aswell as in the regulation of NCX1 by protein kinase C or ATP depletion.

     

Differential inhibition of Na+/Ca2+ exchanger isoforms by divalent cations and isothiourea derivative

We compared the properties of three mammalianNa⁺/Ca²⁺exchanger isoforms, NCX1, NCX2, and NCX3, by analyzing the effects of Ni²⁺ and other cations as well asthe recently identified inhibitor isothiourea derivatives onintracellular Na⁺-dependent⁴⁵Ca²⁺uptake into CCL-39 (Dede) fibroblasts stably expressingeach isoform. All these NCX isoforms had similar affinities for the extracellular transport substratesCa²⁺ andNa⁺.Ni²⁺ inhibited⁴⁵Ca²⁺uptake by competing with Ca²⁺ forthe external transport site, with 10-fold less affinity in NCX3 than inNCX1 or NCX2. Ni²⁺ andCo²⁺ were most efficient in suchdiscrimination of NCX isoforms, although their inhibitory potencieswere less than those of La³⁺ andCd²⁺. The monovalent cationLi⁺ stimulated⁴⁵Ca²⁺uptake rate by all NCX isoforms similarly with low affinity, althoughthe extent of stimulation was somewhat smaller in NCX1. On the otherhand, the isothiourea derivative KB-R7943 was threefold more inhibitoryto NCX3 than to NCX1 or NCX2. Thus distinct differences in the kineticand pharmacological properties were detected between NCX3 and the othertwo isoforms.

     

Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells

High concentrations of cytosolic Na⁺ ions induce the time-dependent formation of an inactive state of the Na⁺/Ca²⁺ exchanger (NCX), a process known as Na⁺-dependent inactivation. NCX activity was measured as Ca²⁺ uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na⁺-dependent inactivation. As expected, 1) Na⁺-dependent inactivation was promoted by high cytosolic Na⁺ concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na⁺-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na⁺-dependent inactivation in the WT exchanger, 3) Na⁺-dependent inactivation did not increase the half-maximal cytosolic Ca²⁺ concentration for allosteric Ca²⁺ activation, 4) Na⁺-dependent inactivation was not reversed by high cytosolic Ca²⁺ concentrations, and 5) Na⁺-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca²⁺ concentration. Thus Na⁺-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na⁺-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca²⁺ influx during ischemia. ischemia; cytosolic calcium concentration; cytosolic sodium concentration; cellular phosphatidylinositol-4,5-bisphosphate     

The topology of the C-terminal sections of the NCX1 Na+/Ca2+ exchanger and the NCKX2 Na+/Ca2+-K+ exchanger

Mammalian Na⁺/Ca²⁺ (NCX) and Na⁺/Ca²⁺-K⁺ exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na⁺/Ca²⁺ exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.     

New insights into the molecular and cellular workings of the cardiac Na+/Ca2+ exchanger

The cardiac Na⁺/Ca²⁺ exchanger (NCX1) is almost certainly the major Ca²⁺ extrusion mechanism in cardiac myocytes, although the driving force for Ca²⁺ extrusion is quite small. To explain multiple recent results, it is useful to think of the exchanger as a slow Ca²⁺ buffer that can reverse its function multiple times during the excitation-contraction cycle (ECC). An article by the group of John Reeves brings new insights to this function by analyzing the role of regulatory domains of NCX1 that mediate its activation by a rise of cytoplasmic Ca²⁺. It was demonstrated that the gating reactions are operative just in the physiological range of Ca²⁺ changes, a few fold above resting Ca²⁺ level, and that they prevent the exchanger from damping out the influence of mechanisms that transiently increase Ca²⁺ levels. Furthermore, exchangers with deleted regulatory domains are shown to reduce resting Ca²⁺ to lower levels than achieved by wild-type exchangers. A study by the group of Kenneth Philipson demonstrated that the NCX1 regulatory domain can bind and respond to Ca²⁺ changes on the time scale of the ECC in rat myocytes. At the same time, studies of transgenic mice and NCX1 knockout mice generated by the Philipson group revealed that large changes of NCX1 activity have rather modest effects on ECC. Simple simulations predict these results very well: murine cardiac ECC is very sensitive to small changes of the Na⁺ gradient, very sensitive to changes of the sarcoplasmic reticulum Ca²⁺ pump activity, and very insensitive to changes of NCX1 activity. It is speculated that the NCX1 gating reactions not only regulate coupled 3Na⁺:1Ca²⁺ exchange but also control the exchanger’s Na⁺ leak function that generates background Na⁺ influx and depolarizing current in cardiac myocytes. excitation-contraction cycle     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.     

NCX activity generates spontaneous Ca2+ oscillations in the astrocytic leaflet microdomain

The synergy between synaptic Glu release and astrocytic Glu-Na⁺ symport is essential to the signalling function of the tripartite synapse. Here we used kinetic data of astrocytic Glu transporters (EAAT) and the Na⁺/Ca²⁺ exchanger (NCX) to simulate Glu release, Glu uptake and subsequent Na⁺ and Ca²⁺ dynamics in the astrocytic leaflet microdomain following single release event. Model simulations show that Glu-Na⁺ symport differently affect intracellular [Na⁺] in synapses with different extent of astrocytic coverage. Surprisingly, NCX activity alone has been shown to generate markedly stable, spontaneous Ca²⁺ oscillation in the astrocytic leaflet. These on-going oscillations appear when NCX operates either in the forward or reverse direction. We conjecture that intrinsic NCX activity may play a prominent role in the generation of astrocytic Ca²⁺ oscillations.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

Analysis of the Na+/Ca2+ Exchanger Gene Family within the Phylum Nematoda

Na⁺/Ca²⁺ exchangers are low affinity, high capacity transporters that rapidly transport calcium at the plasma membrane, mitochondrion, endoplasmic (and sarcoplasmic) reticulum, and the nucleus. Na⁺/Ca²⁺ exchangers are widely expressed in diverse cell types where they contribute homeostatic balance to calcium levels. In animals, Na⁺/Ca²⁺ exchangers are divided into three groups based upon stoichiometry: Na⁺/Ca²⁺ exchangers (NCX), Na⁺/Ca²⁺/K⁺ exchangers (NCKX), and Ca²⁺/Cation exchangers (CCX). In mammals there are three NCX genes, five NCKX genes and one CCX (NCLX) gene. The genome of the nematode Caenorhabditis elegans contains ten Na⁺/Ca²⁺ exchanger genes: three NCX; five CCX; and two NCKX genes. Here we set out to characterize structural and taxonomic specializations within the family of Na⁺/Ca²⁺ exchangers across the phylum Nematoda. In this analysis we identify Na⁺/Ca²⁺ exchanger genes from twelve species of nematodes and reconstruct their phylogenetic and evolutionary relationships. The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes. Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes. Within the Caenorhabditis and Pristionchus lineages we identify between three and five CCX representatives, whereas in other Clade V and also Clade IV nematode taxa we only observed a single CCX gene in each species, and in the Clade III nematode taxa that we sampled we identify NCX and NCKX encoding genes but no evidence of CCX representatives using our mining approach. We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing. Together, these findings reveal a complex picture of Na⁺/Ca²⁺ transporters in nematodes that suggest an incongruent evolutionary history of proteins that provide central control of calcium dynamics.     

Upregulation of Na+/Ca2+ exchanger contributes to the enhanced Ca2+ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca²⁺]_cyt in human PASMC. The resting [Ca²⁺]_cyt and Ca²⁺ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na⁺/Ca²⁺ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na⁺/Ca²⁺ exchanger operates in a forward (Ca²⁺ exit) and reverse (Ca²⁺ entry) mode. By activating the reverse mode of Na⁺/Ca²⁺ exchange, removal of extracellular Na⁺ caused a rapid increase in [Ca²⁺]_cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca²⁺ stores using cyclopiazonic acid (10 µM) not only caused a rise in [Ca²⁺]_cyt due to Ca²⁺ influx through store-operated Ca²⁺ channels but also mediated a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange, but did not accelerate Ca²⁺ extrusion via the forward mode of Na⁺/Ca²⁺ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange are an additional mechanism responsible for the elevated [Ca²⁺]_cyt in PASMC from IPAH patients. transient receptor potential channel; reverse and forward mode; proliferation     

Function and Regulation of the Na+-Ca2+ Exchanger NCX3 Splice Variants in Brain and Skeletal Muscle

Isoform 3 of the Na⁺-Ca²⁺ exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca²⁺]_i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca²⁺ extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca²⁺ imaging, we measured the capacity and regulation of the two variants during Ca²⁺ extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na⁺]_i), NCX3-AC has a higher [Na⁺]_i sensitivity, as Ca²⁺ influx is observed in the presence of extracellular Na⁺. This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca²⁺ extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca²⁺. Strikingly, substitution of Glu⁵⁸⁰ in NCX3-B with its NCX3-AC equivalent Lys⁵⁸⁰ recapitulated the functional properties of NCX3-AC regarding Ca²⁺ sensitivity, Lys⁵⁸⁰ presumably acting through a structure stabilization of the Ca²⁺ binding site. The higher Ca²⁺ uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca²⁺ levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca²⁺ handling beyond that of extruding Ca²⁺.     

Cloning, expression, and characterization of the trout cardiac Na+/Ca2+ exchanger

Isoform 1 of the cardiacNa⁺/Ca²⁺exchanger (NCX1) is an important regulator of cytosolicCa²⁺ concentration in contractionand relaxation. Studies with trout heart sarcolemmal vesicles haveshown NCX to have a high level of activity at 7°C, and this uniqueproperty is likely due to differences in protein structure. In thisstudy, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAPII cDNA library constructed from rainbow trout(Oncorhynchus mykiss) heart RNA. TheNCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plotindicates the protein contains 12 hydrophobic segments (of which thefirst is predicted to be a cleaved leader peptide) and a largecytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have ninetransmembrane segments. The sequences demonstrated to be the exchangerinhibitory peptide site and the regulatoryCa²⁺ binding site in thecytoplasmic loop of mammalian NCX1 are almost completely conserved inNCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4days currents were measured by the giant excised patch technique.NCX-TR1 currents measured at ~23°C demonstratedNa⁺-dependent inactivation andCa²⁺-dependent activation in amanner qualitatively similar to that for NCX1 currents.

     

Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca²⁺ stores opens store-operated Ca²⁺ channels (SOC) and causes Ca²⁺ entry. Transient receptor potential (TRP) cation channels that are permeable to Na⁺ and Ca²⁺ are believed to form functional SOC. Because sarcolemmal Na⁺/Ca²⁺ exchanger has also been implicated in regulating [Ca²⁺]_cyt, this study was designed to test the hypothesis that the Na⁺/Ca²⁺ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca²⁺]_cyt by contributing to store depletion-mediated Ca²⁺ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na⁺, which switches the Na⁺/Ca²⁺ exchanger from the forward (Ca²⁺ exit) to reverse (Ca²⁺ entry) mode, significantly increased [Ca²⁺]_cyt, whereas inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 (10 µM) markedly attenuated the increase in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Store depletion also induced a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Removal of extracellular Na⁺ or inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 attenuated the store depletion-mediated Ca²⁺ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange contributes to store depletion-mediated increase in [Ca²⁺]_cyt, and that blockade of the Na⁺/Ca²⁺ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension. sodium-calcium exchange; calcium homeostasis; vascular smooth muscle     

Calmodulin Interacts with the Sodium/Calcium Exchanger NCX1 to Regulate Activity

Changes in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) are an important signal for various physiological activities. The Na⁺/Ca²⁺ exchangers (NCX) at the plasma membrane transport Ca²⁺ into or out of the cell according to the electrochemical gradients of Na⁺ and Ca²⁺ to modulate [Ca²⁺]_i homeostasis. Calmodulin (CaM) senses [Ca²⁺]_i changes and relays Ca²⁺ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca²⁺ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM₁₂₃₄ (a Ca²⁺-binding deficient mutant) in HEK293T cells and measured the increase in [Ca²⁺]_i contributed by the influx of Ca²⁺ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM₁₂₃₄. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3^rd or 4^th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2^nd and 3^rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca²⁺]_i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.     

Lanthanum is transported by the sodium/calcium exchanger and regulates its activity

La³⁺ uptake was measured in fura 2-loaded Chinese hamster ovary cells expressing the bovine cardiac Na⁺/Ca²⁺ exchanger (NCX1.1). La³⁺ was taken up by the cells after an initial lag phase of 50-60 s and achieved a steady state within 5-6 min. Neonatal cardiac myocytes accumulated La³⁺ in a similar manner. La³⁺ uptake was due to the activity of the exchanger, because no uptake was seen in nontransfected cells or in transfected cells that had been treated with gramicidin to remove cytosolic Na⁺. The low rate of La³⁺ uptake during the lag period resulted from insufficient cytosolic Ca²⁺ to activate the exchanger at its regulatory sites, as shown by the following observations. La³⁺ uptake occurred without a lag period in cells expressing a mutant of NCX1.1 that does not exhibit regulatory activation by cytosolic Ca²⁺. The rate of La³⁺ uptake by wild-type cells was increased, and the lag phase was reduced or eliminated, when the cytosolic Ca²⁺ concentration was increased before initiating La³⁺ uptake. La³⁺ could substitute for Ca²⁺ at very low concentrations to activate exchange activity. Thus preloading cells expressing NCX1.1 with a small quantity of La³⁺ increased the rate of exchange-mediated Ca²⁺ influx by 20-fold; in contrast, cytosolic La³⁺ partially inhibited Ca²⁺ uptake by the regulation-deficient mutant. With an estimated K_D of 30 pM for the binding of La³⁺ to fura 2, we conclude that cytosolic La³⁺ activates exchange activity at picomolar concentrations. We speculatively suggest that endogenous trace metals might activate exchange activity under physiological conditions. fura 2; NCX1.1; myocyte     

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation

NGF induces neuronal differentiation by modulating [Ca²⁺]_i. However, the role of the three isoforms of the main Ca²⁺-extruding system, the Na⁺/Ca²⁺ exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca²⁺]_i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca²⁺ content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na⁺ currents and 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na⁺]_i significantly increased in cells overexpressing NCX1.4 as well as ER Ca²⁺ content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca²⁺]_i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca²⁺ content and PI3K signaling.     

Determinants of cardiac Na(+)/Ca(2+) exchanger temperature dependence: NH(2)-terminal transmembrane segments

The cardiacNa⁺/Ca²⁺ exchanger (NCX) in troutexhibits profoundly lower temperature sensitivity in comparison to themammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout andcanine NCX cDNA and expressed in Xenopus oocytes.NCX-mediated currents were measured at 7, 14, and 30°C using thegiant excised-patch technique. By using this approach, the differentialtemperature dependence of NCX was found to reside within theNH₂-terminal region of the molecule. Specifically, we foundthat ~75% of the Na⁺/Ca²⁺ exchangedifferential energy of activation is attributable to sequencedifferences in the region that include the first four transmembranesegments, and the remainder is attributable to transmembrane segmentfive and the exchanger inhibitory peptide site.

     

Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA

Isoform 3 of the Na⁺-Ca²⁺ exchanger (NCX3) participates in the Ca²⁺ fluxes across the plasma membrane. Among the NCX family, NCX3 carries out a peculiar role due to its specific functions in skeletal muscle and the immune system and to its neuroprotective effect under stress exposure. In this context, proper understanding of the regulation of NCX3 is primordial to consider its potential use as a drug target. In this study, we demonstrated the regulation of NCX3 by protein kinase A (PKA) and C (PKC). Disparity in regulation has been previously reported among the splice variants of NCX3 therefore the activity of Ca²⁺ uptake and extrusion of the two murine variants was measured using fura-2-based Ca²⁺ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca²⁺ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca²⁺]_i or [Na⁺]_i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca²⁺ uptake after a rise in [Ca²⁺]_i. The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca²⁺ extrusion capacity remained unaffected under activation of PKC. Next, using site-directed mutagenesis, the sensitivity of NCX3 to PKC was abolished by singly mutating its predicted phosphorylation sites T529 or S695. The sensitivity to PKC might be due to the influence of T529 phosphorylation on the Ca²⁺-binding domain 1. Additionally, we showed that stimulation of NCX3 by PKA occurred through residue S524. This effect may well participate in the fight-or-flight response in skeletal muscle and the long-term potentiation in hippocampus.     

Nuclear localization of NCX: Role in Ca2+ handling and pathophysiological implications

Numerous lines of evidence indicate that nuclear calcium concentration ([Ca²⁺]_n) may be controlled independently from cytosolic events by a local machinery. In particular, the perinuclear space between the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) of the nuclear envelope (NE) likely serves as an intracellular store for Ca²⁺ ions. Since ONM is contiguous with the endoplasmic reticulum (ER), the perinuclear space is adjacent to the lumen of ER thus allowing a direct exchange of ions and factors between the two organelles. Moreover, INM and ONM are fused at the nuclear pore complex (NPC), which provides the only direct passageway between the nucleoplasm and cytoplasm. However, due to the presence of ion channels, exchangers and transporters, it has been generally accepted that nuclear ion fluxes may occur across ONM and INM. Within the INM, the Na⁺/Ca²⁺ exchanger (NCX) isoform 1 seems to play an important role in handling Ca²⁺ through the different nuclear compartments. Particularly, nuclear NCX preferentially allows local Ca²⁺ flowing from nucleoplasm into NE lumen thanks to the Na⁺ gradient created by the juxtaposed Na⁺/K⁺-ATPase. Such transfer reduces abnormal elevation of [Ca²⁺]_n within the nucleoplasm thus modulating specific transductional pathways and providing a protective mechanism against cell death. Despite very few studies on this issue, here we discuss those making major contribution to the field, also addressing the pathophysiological implication of nuclear NCX malfunction.     

Anoctamin-6 Controls Bone Mineralization by Activating the Calcium Transporter NCX1

Anoctamin-6 (Ano6, TMEM16F) belongs to a family of putative Ca²⁺-activated Cl⁻ channels and operates as membrane phospholipid scramblase. Deletion of Ano6 leads to reduced skeleton size, skeletal deformities, and mineralization defects in mice. However, it remains entirely unclear how a lack of Ano6 leads to a delay in bone mineralization by osteoblasts. The Na⁺/Ca²⁺ exchanger NCX1 was found to interact with Ano6 in a two-hybrid split-ubiquitin screen. Using human osteoblasts and osteoblasts from Ano6^−/− and WT mice, we demonstrate that NCX1 requires Ano6 to efficiently translocate Ca²⁺ out of osteoblasts into the calcifying bone matrix. Ca²⁺-activated anion currents are missing in primary osteoblasts isolated from Ano6 null mice. Our findings demonstrate the importance of NCX1 for bone mineralization and explain why deletion of an ion channel leads to the observed mineralization defect: Ano6 Cl⁻ currents are probably required to operate as a Cl⁻ bypass channel, thereby compensating net Na⁺ charge movement by NCX1.