Intermolecular cross-linking of Na+-Ca2+ exchanger proteins: evidence for dimer formation

The cardiac Na (+)-Ca (2+) exchanger (NCX1) is modeled to contain nine transmembrane segments (TMS) with a pair of oppositely oriented, conserved sequences called the alpha-repeats that are important in ion transport. Residue 122 in the alpha-1 repeat is in proximity to residue 768 in TMS 6, and the two residues can be cross-linked . During studies on the substrate specificity of this intramolecular cross-link, we found evidence that NCX1 can form dimers. At 37 degrees C in the absence of extracellular Na (+), copper phenanthroline catalyzes disulfide bond formation between cysteines at position 122 in adjacent NCX1 proteins. Dimerization was confirmed by histidine tag pull-down experiments that demonstrate the association of untagged NCX1 with histidine-tagged NCX1. Dimerization occurs along a face of the protein that includes parts of the alpha-1 and alpha-2 repeats as well as parts of TMS 1 and TMS 2. We do not see cross-linking between residues in TMS 5, TMS 6, or TMS 7. These data provide the first evidence for dimer formation by the Na (+)-Ca (2+) exchanger.     

Helix packing of functionally important regions of the cardiac Na(+)-Ca(2+) exchanger

In a revised topological model of the cardiac Na(+)-Ca(2+) exchanger, there are nine transmembrane segments (TMSs) and two possible re-entrant loops (Nicoll, D. A., Ottolia, M., Lu, Y., Lu, L., and Philipson, K. D. (1999) J. Biol. Chem. 274, 910-917; Iwamoto, T., Nakamura, T. Y., Pan, Y., Uehara, A., Imanaga, I., and Shigekawa, M. (1999) FEBS Lett. 446, 264-268). The TMSs form two clusters separated by a large intracellular loop between TMS5 and TMS6. We have combined cysteine mutagenesis and oxidative cross-linking to study proximity relationships of TMSs in the exchanger. Pairs of cysteines were reintroduced into a cysteine-less exchanger, one in a TMS in the NH(2)-terminal cluster (TMSs 1-5) and the other in a TMS in the COOH-terminal cluster (TMSs 6-9). The mutant exchanger proteins were expressed in HEK293 cells, and disulfide bond formation between introduced cysteines was analyzed by gel mobility shifts. Western blots showed that S117C/V804C, A122C/Y892C, A151C/T815C, and A151C/A821C mutant proteins migrated at 120 kDa under reducing conditions and displayed a partial mobility shift to 160 kDa under nonreducing conditions. This shift indicates the formation of a disulfide bond between these paired cysteine residues. Copper phenanthroline and the cross-linker N', N'-o-phenylenedimaleimide enhanced the mobility shift to 160 kDa. Our data suggest that TMS7 is close to TMS3 near the intracellular side of the membrane and is in the vicinity of TMS2 near the extracellular surface. Also, TMS2 must adjoin TMS8. This initial packing model of the exchanger brings two functionally important domains in the exchanger, the alpha 1 and alpha 2 repeats, close to each other.     

Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.     

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.     

Na+ entry via TRPC6 causes Ca2+ entry via NCX reversal in ATP stimulated smooth muscle cells

Reversal of the Na+/Ca2+ -exchanger (NCX) has been shown to mediate Ca2+ influx during activation of G-protein linked receptors. Functional coupling between the reverse-mode NCX and the canonical transient receptor potential channels (TRPCs) has been proposed to mediate Ca2+ influx in HEK-293 cells overexpressing TRPC3. In this communication we present evidence for similar functional coupling of NCX to endogenously expressed TRPC6 in rat aorta smooth muscle cells. Selective inhibition of reverse-mode NCX with KB-R7943 and of non-selective cation-channels with SKF-96365 abolished Ca2+ influx in response to agonist stimulation (ATP). Expression of a dominant negative TRPC6 mutant also reduced the Ca2+ influx in proportion to its transfection efficiency. Calyculin A, which is known to disrupt the junctions of the plasma membrane and sarco/endoplasmic reticulum, increased global Na+ elevations and reduced stimulated Ca2+ influx. Together our data provide evidence that localized Na+ elevations are generated by TRPC6 and drive reversal of NCX to mediate Ca2+ influx.     

The molecular determinants of ionic regulatory differences between brain and kidney Na+/Ca2+ exchanger (NCX1) isoforms

The Na(+)/Ca(2+) exchanger gene NCX1 undergoes alternative splicing leading to several isoforms that differ in a small portion of the large cytoplasmic loop. This loop is involved in many regulatory processes of NCX1, including ionic regulation by the transported substrates Na(+) and Ca(2+). High intracellular Ca(2+) can alleviate intracellular Na(+)-dependent inactivation in exon A (NCX1.4)-containing isoforms but not in those containing the mutually exclusive exon B (NCX1.3). Giant excised patches from Xenopus oocytes expressing various NCX1 constructs were used to examine the specific amino acids responsible for these observed regulatory differences. Using a chimeric approach, the region responsible was narrowed down to the small central part of exon A (IDDEEYEKNKTF). Replacing the second aspartic acid of this sequence with arginine (the corresponding amino acid in exon B) in an exon A background completely prevented the effect of Ca(2+) on intracellular Na(+)-dependent inactivation. Mutating the second lysine to cysteine (exon B) had a similar, but only partial, effect. The converse double mutant, but neither single mutation alone, introduced into an exon B background (arginine to aspartic acid and cysteine to lysine) was able to restore the NCX1.4 regulatory phenotype. These data demonstrate that aspartic acid 610 and lysine 617 (using the rat NCX1.4 numbering scheme) are critical molecular determinants of the unique Ca(2+) regulatory properties of NCX1.4.     

Stoichiometry of the Cardiac Na+/Ca2+ exchanger NCX1.1 measured in transfected HEK cells

The stoichiometry with which the Na+/Ca2+ exchanger, NCX1, binds and transports Na+ and Ca2+ has dramatic consequences for ionic homeostasis and cellular function of heart mycocytes and brain neurons, where the exchanger is highly expressed. Previous studies have examined this question using native NCX1 in its endogenous environment. We describe here whole-cell voltage clamp studies using recombinant rat heart NCX1.1 expressed heterologously in HEK-293 cells. This system provides the advantages of a high level of NCX1 protein expression, very low background ion transport levels, and excellent control over clamped voltage and ionic composition. Using ionic conditions that allowed bi-directional currents, voltage ramps were employed to determine the reversal potential for NCX1.1-mediated currents. Analysis of the relation between reversal potential and external [Na+] or [Ca2+], under a variety of intracellular conditions, yielded coupling ratios for Na+ of 1.9-2.3 ions per net charge and for Ca2+ of 0.45 +/- 0.03 ions per net charge. These data are consistent with a stoichiometry for the NCX1.1 protein of 4 Na+ to 1 Ca2+ to 2 charges moved per transport cycle.     

Anomalous regulation of the Drosophila Na(+)-Ca2+ exchanger by Ca2+

The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1- 10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.     

Fluorescent Na+-Ca+ exchangers: electrophysiological and optical characterization

The cardiac sarcolemmal Na+-Ca2+ exchanger (NCX1) influences cardiac contractility by extruding Ca2+ from myocytes. As a Ca2+ efflux mechanism, the exchanger plays a prominent role in Ca2+ homeostasis. To track NCX1 and study changes in conformation, NCX1 was tagged with derivatives of green fluorescent protein. Cyan (CFP) and yellow (YFP) fluorescent proteins were used for both visualization of the protein in HEK cells and fluorescent resonance energy transfer (FRET). CFP or YFP was inserted at position 266, 371, 467, or 548 of the large intracellular loop of NCX1 located between transmembrane segments 5 and 6. These constructs were tested for functional activity and visualized for cell surface expression. All constructs were targeted to the plasma membrane. Transport properties were assessed by both 45Ca2+ uptake and electrophysiological measurements. The fluorescent-tagged exchangers had similar biophysical properties to the wild type NCX1. Unexpectedly, all constructs retain their sensitivity to regulation by cytoplasmic Na+ and Ca2+ ions. FRET analysis indicates the proximity of NCX1 to plasma membrane phosphatidylinositol 4,5-bisphosphate. These results indicate that insertion of CFP or YFP into the large intracellular loop of NCX1 protein does not impair exchanger properties. These constructs will be useful to further characterize the biological properties of the exchanger in intact cells.     

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.     

Role of cysteine residues in the NCKX2 Na+/Ca(2+)-K+ Exchanger: generation of a functional cysteine-free exchanger

Cysteine residues play an important role in many proteins, either in enzymatic activity or by mediating inter- or intramolecular interactions. The Na(+)/Ca(2+)-K(+) exchanger plays a critical role in Ca(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by extruding Ca(2+) that enters rod and cone cells via the cGMP-gated channels. NCKX1 and NCKX2 contain five highly conserved cysteine residues. The objectives of this study were threefold: (1) to examine the importance of cysteine residues in NCKX2 protein function; (2) to examine their role in the interaction between NCKX2 and the CNGA subunit of the cGMP-gated channel; and (3) to generate a functional cysteine-free NCKX2 protein. The latter will facilitate structural studies taking advantage of the unique chemistry of the thiol group following insertion of cysteine residues at specific positions in the cysteine-free background. We generated a cysteine-free NCKX2 mutant protein that showed normal protein synthesis and processing and approximately 50% wild-type cation transport function. Cysteine residues were also not critical for the formation of NCKX2 homo-oligmers or NCKX2 hetero-oligomers with the CNGA subunit of the cGMP-gated channel. Our results appear to rule out a critical importance of an intramolecular disulfide linkage in NCKX2 protein synthesis and folding as had been reported before.     

Mutational analysis of the alpha-1 repeat of the cardiac Na(+)-Ca2+ exchanger

The Na(+)-Ca2+ exchanger contains internal regions of sequence homology known as the alpha repeats. The first region (alpha-1 repeat) includes parts of transmembrane segments (TMSs) 2 and 3 and a linker modeled to be a reentrant loop. To determine the involvement of the reentrant loop and TMS 3 portions of the alpha-1 repeat in exchanger function, we generated a series of mutants and examined ion binding and transport and regulatory properties. Mutations in the reentrant loop did not substantially modify transport properties of the exchanger though the Hill coefficient for Na+ and the rate of Na(+)-dependent inactivation were decreased. Mutations in TMS 3 had more striking effects on exchanger activity. Of mutations at 10 positions, 3 behaved like the wild-type exchanger (V137C, A141C, M144C). Mutants at two other positions expressed no activity (Ser139) or very low activity (Gly138). Six different mutations were made at position 143; only N143D was active, and it displayed wild-type characteristics. The highly specific requirement for an asparagine or aspartate residue at this position may indicate a key role for Asn143 in the transport mechanism. Mutations at residues Ala140 and Ile147 decreased affinity for intracellular Na+, whereas mutations at Phe145 increased Na+ affinity. The cooperativity of Na+ binding was also altered. In no case was Ca2+ affinity changed. TMS 3 may form part of a site that binds Na+ but not Ca2+. We conclude that TMS 3 is involved in Na+ binding and transport, but previously proposed roles for the reentrant loop need to be reevaluated.     

Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)-K(+)-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.     

Substitution of a single residue, Asp575, renders the NCKX2 K+-dependent Na+/Ca2+ exchanger independent of K+

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na(+) gradient and the outward K(+) gradient to drive Ca(2+) extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K(+) dependence of NCKX-mediated Ca(2+) transport. Charge-removing replacement of Asp(575) by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K(+), whereas the charge-conservative substitution of Asp(575) to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp(575) is conserved in the NCKX1-5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K(+)-independent Na(+)/Ca(2+) exchanger.     

Functional differences of Na+/Ca2+ exchanger expression in Ca2+ transport system of smooth muscle of guinea pig stomach

The plasma membrane ATP-dependent Ca2+ pump and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion in smooth muscle. However, little is known regarding distribution and function of the NCX in guinea pig gastric smooth muscle. The expression pattern and distribution of NCX isoforms suggest a role as a regulator of Ca2+ transport in cells. Na+ pump inhibition and the consequent to removal of K+ caused gradual contraction in fundus. In contrast, the response was significantly less in antrum. Western blotting analysis revealed that NCX1 and NCX2 are the predominant NCX isoforms expressed in stomach, the former was expressed strongly in antrum, whereas the latter displayed greater expression in fundus. Isolated plasma membrane fractions derived from gastric fundus smooth muscle were also investigated to clarify the relationship between NCX protein expression and function. Na+-dependent Ca2+ uptake increased directly with Ca2+ concentration. Ca2+ uptake in Na+-loaded vesicles was markedly elevated in comparison with K+-loaded vesicles. Additionally, Ca2+ uptake by the Na+- or K+-loaded vesicles was substantially higher in the presence of A23187 than in its absence. The result can be explained based on the assumption that Na+ gradients facilitate downhill movement of Ca2+. Na+-dependent Ca2+ uptake was abolished by the monovalent cationic ionophore, monensin. NaCl enhanced Ca2+ efflux from vesicles, and this efflux was significantly inhibited by gramicidin. Results documented evidence that NCX2 isoform functionally contributes to Ca2+ extrusion and maintenance of contraction-relaxation cycle in gastric fundus smooth muscle.     

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.     

钠钙交换在心脏发育早期自主膜电活动发生中的作用

钠钙交换是小鼠心脏发育中最早有功能性表达的通道基因。它的功能主要是通过泵出1个钙,泵入3个钠位置细胞内的钙稳态,此外可能参与兴奋收缩偶联。但是,至今钠钙交换在心脏发育过程中的功能性表达及其在细胞早期兴奋形成中的作用还不是很清楚。采用胚胎干细胞分化的心肌细胞为研究对象,发现在发育极早期,电压钳制在35mV的条件下,10mmol／L咖啡因诱导的内向电流的80％能被灌流液中Na^＋被等浓度的Li^＋取代（n=8）。此为钠钙交换电流。所有钳制的细胞单细胞RT-PCR都检测到了NCX1亚型的mRNA表达。进一步研究了钠钙交换的功能,发现等浓度Li^＋取代灌流液中Na^＋及应用高浓度Ni^2＋阻断了膜电位震荡及与震荡相间的动作电位（早期膜兴奋形式）。因此认为钠钙交换（NCX1亚型）在心脏发育极早期的心肌细胞中已有大量功能性表达,它对于早期自主性兴奋活动的发生起着关键性的作用。     

The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity

We examined the membrane topology and functional importance of residues in regions of the Na(+)/Ca(2+) exchanger NCX1 encompassing the conserved internal alpha repeats by substituted cysteine scanning analysis and kinetic analysis of site-directed mutants. The results suggest that both the alpha-1 repeat and a region encompassing the alpha-2 repeat and its immediately C-terminal segment contain reentrant loop domains, each oriented in an opposite direction with respect to the membrane. We found that single or multiple mutations of six residues including Asn-125 and conserved aspartates Asp-130, Asp-825, and Asp-829 in the alpha repeat reentrant domains reduce the apparent affinity of the exchanger for extracellular Ca(2+) by up to 6-fold. In contrast, the triple cysteine mutation D130C/D825C/D829C did not influence the current-voltage (I-V) relationship of the exchange current. Cysteine accessibility scanning with different thiol modifiers suggested that N125C, D130C, and D825C may be located in a restricted aqueous space in the membrane accessible only to ions when examined with external probes, although N125C and D825C were previously shown to be internally accessible during exchange reaction. The results suggest that these reentrant domains in the alpha repeats may participate in the formation of the ion transport pathway in the exchanger with some of the aspartates possibly lining it or located close to it.     

Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport

Mammalian Na+/Ca2+ exchangers are members of three branches of a much larger family of transport proteins [the CaCA (Ca2+/cation antiporter) superfamily] whose main role is to provide control of Ca2+ flux across the plasma membranes or intracellular compartments. Since cytosolic levels of Ca2+ are much lower than those found extracellularly or in sequestered stores, the major function of Na+/Ca2+ exchangers is to extrude Ca2+ from the cytoplasm. The exchangers are, however, fully reversible and thus, under special conditions of subcellular localization and compartmentalized ion gradients, Na+/Ca2+ exchangers may allow Ca2+ entry and may play more specialized roles in Ca2+ movement between compartments. The NCX (Na+/Ca2+ exchanger) [SLC (solute carrier) 8] branch of Na+/Ca2+ exchangers comprises three members: NCX1 has been most extensively studied, and is broadly expressed with particular abundance in heart, brain and kidney, NCX2 is expressed in brain, and NCX3 is expressed in brain and skeletal muscle. The NCX proteins subserve a variety of roles, depending upon the site of expression. These include cardiac excitation-contraction coupling, neuronal signalling and Ca2+ reabsorption in the kidney. The NCKX (Na2+/Ca2+-K+ exchanger) (SLC24) branch of Na+/Ca2+ exchangers transport K+ and Ca2+ in exchange for Na+, and comprises five members: NCKX1 is expressed in retinal rod photoreceptors, NCKX2 is expressed in cone photoreceptors and in neurons throughout the brain, NCKX3 and NCKX4 are abundant in brain, but have a broader tissue distribution, and NCKX5 is expressed in skin, retinal epithelium and brain. The NCKX proteins probably play a particularly prominent role in regulating Ca2+ flux in environments which experience wide and frequent fluctuations in Na+ concentration. Until recently, the range of functions that NCKX proteins play was generally underappreciated. This situation is now changing rapidly as evidence emerges for roles including photoreceptor adaptation, synaptic plasticity and skin pigmentation. The CCX (Ca2+/cation exchanger) branch has only one mammalian member, NCKX6 or NCLX (Na+/Ca2+-Li+ exchanger), whose physiological function remains unclear, despite a broad pattern of expression.