Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3)

Three distinctmammalianNa⁺/Ca²⁺exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken adetailed functional comparison of these three exchangers. Eachexchanger was stably expressed at high levels in the plasma membranesof BHK cells. Na⁺/Ca²⁺exchange activity was assessed using three different complementary techniques: Na⁺ gradient-dependent⁴⁵Ca²⁺uptake into intact cells, Na⁺gradient-dependent⁴⁵Ca²⁺uptake into membrane vesicles isolated from the transfected cells, andexchange currents measured using giant patches of excised cellmembrane. Apparent affinities for the transported ionsNa⁺ andCa²⁺ were markedly similar for thethree exchangers at both membrane surfaces. Likewise, generally similarresponses to changes in pH, chymotrypsin treatment, and application ofvarious inhibitors were obtained. Depletion of cellular ATP inhibitedNCX1 and NCX2 but did not affect the activity of NCX3. Exchangeactivities of NCX1 and NCX3 were modestly increased by agents thatactivate protein kinases A and C. All exchangers were regulated byintracellular Ca²⁺. NCX1-inducedexchange currents were especially large in excised patches and, likethe native myocardial exchanger, were stimulated by ATP. Results may beinfluenced by our choice of expression system and specific splicevariants, but, overall, the three exchangers appear to have verysimilar properties.

     

Cellular and subcellular localization of Na+-Ca2+ exchanger protein isoforms, NCX1, NCX2, and NCX3 in cerebral cortex and hippocampus of adult rat

Na(+)-Ca(2+) exchanger (NCX) controls cytosolic Ca(2+) and Na(+) concentrations ([Ca(2+)](i) and [Na(+)](i)) in eukaryotic cells. Here we investigated by immunocytochemistry the cellular and subcellular localization of the three known NCX isoforms, NCX1, NCX2 and NCX3, in adult rat neocortex and hippocampus. NCX1-3 were widely expressed in both brain areas: NCX1 immunoreactivity (ir) was exclusively associated to neuropilar puncta, while NCX2-3 were also detected in neuronal somata and dendrites. NCX1-3 ir was often identified around blood vessels. In both neocortex and hippocampus, all NCX isoforms were prominently expressed in dendrites and dendritic spines contacted by asymmetric axon terminals, whereas they were poorly expressed in presynaptic boutons. In addition, NCX1-3 ir was detected in astrocytes, notably in distal processes ensheathing excitatory synapses. All NCXs were expressed in perivascular astrocytic endfeet and endothelial cells. The robust expression of NCX1-3 in heterogeneous cell types in the brain in situ emphasizes their role in handling Ca(2+) and Na(+) in both excitable and non-excitable cells. Perisynaptic localization of NCX1-3 in dendrites and spines indicates that all isoforms are favourably located for buffering [Ca(2+)](i) in excitatory postsynaptic sites. NCX1-3 expressed in perisynaptic glial processes may participate in shaping astrocytic [Ca(2+)](i) transients evoked by ongoing synaptic activity.     

High levels of synaptosomal Na(+)-Ca(2+) exchangers (NCX1, NCX2, NCX3) co-localized with amyloid-beta in human cerebral cortex affected by Alzheimer's disease

Synaptosomal expression of NCX1, NCX2, and NCX3, the three variants of the Na(+)-Ca(2+) exchanger (NCX), was investigated in Alzheimer's disease parietal cortex. Flow cytometry and immunoblotting techniques were used to analyze synaptosomes prepared from cryopreserved brain of cognitively normal aged controls and late stage Alzheimer's disease patients. Major findings that emerged from this study are: (1) NCX1 was the most abundant NCX isoform in nerve terminals of cognitively normal patients; (2) NCX2 and NCX3 protein levels were modulated in parietal cortex of late stage Alzheimer's disease: NCX2 positive terminals were increased in the Alzheimer's disease cohort while counts of NCX3 positive terminals were reduced; (3) NCX1, NCX2 and NCX3 isoforms co-localized with amyloid-beta in synaptic terminals and all three variants are up-regulated in nerve terminals containing amyloid-beta. Taken together, these data indicate that NCX isoforms are selectively regulated in pathological terminals, suggesting different roles of each NCX isoform in Alzheimer's disease terminals.     

Ca2+ signaling in smooth muscle: TRPC6, NCX,and LNats in nanodomains

Following the recent observation of localized cytosolic subplasmalemmal [Na⁺] elevations (LNats) in rat aortic smooth muscle cells, we discuss here the current evidence for the structural and molecular roles of cytosolic nanodomains at close junctions of the plasma membrane (PM) and sarcoplasmic reticulum (SR) in the generation of LNats. These junctions, the loss of which might contribute to vascular aging and disease, provide a platform for ion metabolism signalplexes and the interaction of localized Na⁺ and Ca²⁺ gradients. We moreover suggest the existence in the junctions of a Na⁺ diffusional barrier as a necessary condition for the generation of LNats. LNats are likely a fundamental feature of near membrane ion signaling in many cell types, and their discovery offers new possibilities for elucidating the mechanism, function and pathogenesis of Na⁺ and Ca²⁺ signaling nanodomains.     

Ca2+ signaling in smooth muscle: TRPC6, NCX and LNats in nanodomains

Following the recent observation of localized cytosolic subplasmalemmal [Na+] elevations (LNats) in rat aortic smooth muscle cells, we discuss here the current evidence for the structural and molecular roles of cytosolic nanodomains at close junctions of the plasma membrane (PM) and sarcoplasmic reticulum (SR) in the generation of LNats. These junctions, the loss of which might contribute to vascular aging and disease, provide a platform for ion metabolism signalplexes and the interaction of localized Na+ and Ca2+ gradients. We moreover suggest the existence in the junctions of a Na+ diffusional barrier as a necessary condition for the generation of LNats. LNats are likely a fundamental feature of near membrane ion signaling in many cell types, and their discovery offers new possibilities for elucidating the mechanism, function and pathogenesis of Na+ and Ca2+ signaling nanodomains.     

BHK cells transfected with NCX3 are more resistant to hypoxia followed by reoxygenation than those transfected with NCX1 and NCX2: Possible relationship with mitochondrial membrane potential

The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10 microM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial delta psi collapses and cell death.     

Expression of Na(+)/Ca(2+) exchanger isoforms (NCX1 and NCX3) and plasma membrane Ca(2+) ATPase during osteoblast differentiation

The ability to deliver calcium to the osteoid is critical to osteoblast function as a regulator of bone calcification. There are two known transmembrane proteins capable of translocating calcium out of the osteoblast, the Na(+)/Ca(2+) exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). In this study, we reveal the presence of the NCX3 isoform in primary osteoblasts and examine the expression of NCX1, NCX3, and PMCA1 during osteoblast differentiation. The predominant NCX isoform expressed by osteoblasts is NCX3. NCX1 also is expressed, but at low levels. Both NCX isoforms are expressed at nearly static levels throughout differentiation. In contrast, PMCA expression peaks at 8 days of culture, early in osteoblast differentiation, but declines thereafter. Immunocytochemical co-detection of NCX and PMCA reveal that NCX is positioned along surfaces of the osteoblast adjacent to osteoid, while PMCA is localized to plasma membrane sites distal to the osteoid. The expression pattern and spatial distribution of NCX support a role as a regulator of calcium efflux from osteoblasts required for calcification. The expression pattern and spatial distribution of PMCA makes its role in the mineralization process unlikely and suggests a role in calcium homeostasis following signaling events.     

Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants

The Na(+)-Ca(2+) exchanger (NCX) mediated Ca(2+) fluxes are essential for handling Ca(2+) homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory CBD1 and CBD2 domains, whereas in distinct variants the Ca(2+) binding to Ca3-Ca4 sites of CBD1 results either in sustained activation, inhibition or no effect. CBD2 contains an alternatively spliced segment, which is expressed in a tissue-specific manner although its impact on allosteric regulation remains unclear. Recent studies revealed that the Ca(2+) binding to Ca3-Ca4 sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of "occluded" Ca(2+). Here we investigate the effects of CBD2 variants on Ca(2+) occlusion in the two-domain construct (CBD12). Mutational studies revealed that both sites (Ca3 and Ca4) contribute to Ca(2+) occlusion, whereas after dissociation of the first Ca(2+) ion the second Ca(2+) ion becomes occluded. This mechanism is common for the brain, kidney and cardiac splice variants of CBD12, although the occluded Ca(2+) exhibits 20-50-fold difference in off-rates among the tested variants. Therefore, the spliced exons on CBD2 affect the rate-limiting step of the occluded Ca(2+) dissociation at the primary regulatory sensor to shape dynamic features of allosteric regulation in NCX variants.     

A common Ca2+-driven interdomain module governs eukaryotic NCX regulation

Na(+)/Ca(2+) exchanger (NCX) proteins mediate Ca(2+)-fluxes across the cell membrane to maintain Ca(2+) homeostasis in many cell types. Eukaryotic NCX contains Ca(2+)-binding regulatory domains, CBD1 and CBD2. Ca(2+) binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca(2+). To further elucidate the underlying regulatory mechanisms, we determined the 2.7 ? crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca(2+) binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca(2+)-driven interdomain switch controls slow dissociation of "occluded" Ca(2+) from the primary sensor and thus dictates Ca(2+) sensing dynamics. In the Ca(2+)-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca(2+)-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.     

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.     

Chimeric analysis of Na(+)/Ca(2+) exchangers NCX1 and NCX3 reveals structural domains important for differential sensitivity to external Ni(2+) or Li(+).

Externally applied Ni(2+), which apparently competes with Ca(2+) in all three isoforms of Na(+)/Ca(2+) exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li(+) is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423-C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni(2+) or Li(+) by measuring intracellular Na(+)-dependent (45)Ca(2+) uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal alpha-1 and alpha-2 repeats are individually responsible for the alteration of Ni(2+) sensitivity, both together accounting for approximately 80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the alpha-2 repeat fully accounts for the differential Li(+) sensitivity between the isoforms. The Ni(2+) sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the alpha-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the alpha-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li(+) sensitivity was mimicked by double substitution mutation in the alpha-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn(125) and Val(820) of NCX1 caused significant alterations in the interactions of the exchanger with Ca(2+) and Ni(2+), and Ni(2+) and Li(+), respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the alpha repeats, these regions might form an ion interaction domain in the exchanger.     

Regulation of NCX1 by palmitoylation

Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle.     

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.     

Characterization of zebrafish (Danio rerio) NCX4: a novel NCX with distinct electrophysiological properties

Members of the Na+/Ca2+ exchanger (NCX) family are important regulators of cytosolic Ca2+ in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of Na+-dependent inactivation and requires much higher levels of regulatory Ca2+ to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from Na+-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the Na+-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 microM exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously.     

Positive Feedback Mechanisms among Local Ca Releases,NCX, and ICaL Ignite Pacemaker Action Potentials

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (I_CaL). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to I_CaL activation threshold. During the ignition phase, I_CaL-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and I_CaL, as diastolic LCRs begin to occur below and continue within the voltage range of I_CaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (～0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.     

Model for the allosteric regulation of the Na+/Ca2+ exchanger NCX

The Na⁺/Ca²⁺ exchanger provides a major Ca²⁺ extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca²⁺ concentrations. In Canis familiaris, Na⁺/Ca²⁺ exchanger (NCX) activity is regulated by the binding of Ca²⁺ to two cytosolic Ca²⁺‐binding domains, CBD1 and CBD2, such that Ca²⁺‐binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca²⁺‐regulation remains unclear. It was found previously that for NCX in the absence of Ca²⁺ the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca²⁺‐binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca²⁺‐binding to CBD1 inhibits Ca²⁺ exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca²⁺ modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca²⁺ regulation of the Na⁺/Ca²⁺ exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca²⁺‐binding to CBD1 controlling ion transport across the plasma membrane. Proteins 2016; 84:580–590. © 2016 Wiley Periodicals, Inc.