Phylogeny of Na+/Ca2+ exchanger (NCX) genes from genomic data identifies new gene duplications and a new family member in fish species

Christian R. Marshall1,2,3, Joanne A. Fox4, Stefanie L. Butland4, B. F. Francis Ouellette4, Fiona S. L. Brinkman1 and Glen F. Tibbits1,2,3

1 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby
2 Cardiovascular Sciences, British Columbia Research Institute for Children’s and Women’s Health, Vancouver
3 Cardiac Membrane Research Laboratory, Simon Fraser University, Burnaby
4 University of British Columbia, Bioinformatics Centre, University of British Columbia, Vancouver, British Columbia, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The Na+/Ca2+ exchanger (NCX) is a member of the cation/Ca2+ antiporter (CaCA) family and plays a key role in maintaining cellular Ca2+ homeostasis in a variety of cell types. NCX is present in a diverse group of organisms and exhibits high overall identity across species. To date, three separate genes, i.e., NCX1, NCX2, and NCX3, have been identified in mammals. However, phylogenetic analysis of the exchanger has been hindered by the lack of nonmammalian NCX sequences. In this study, we expand and diversify the list of NCX sequences by identifying NCX homologs from whole-genome sequences accessible through the Ensembl Genome Browser. We identified and annotated 13 new NCX sequences, including 4 from zebrafish, 4 from Japanese pufferfish, 2 from chicken, and 1 each from honeybee, mosquito, and chimpanzee. Examination of NCX gene structure, together with construction of phylogenetic trees, provided novel insights into the molecular evolution of NCX and allowed us to more accurately annotate NCX gene names. For the first time, we report the existence of NCX2 and NCX3 in organisms other than mammals, yielding the hypothesis that two serial NCX gene duplications occurred around the time vertebrates and invertebrates diverged. In addition, we have found a putative new NCX protein, named NCX4, that is related to NCX1 but has been observed only in fish species genomes. These findings present a stronger foundation for our understanding of the molecular evolution of the NCX gene family and provide a framework for further NCX phylogenetic and molecular studies.

sodium/calcium exchanger; membrane transporter; phylogeny; molecular evolution


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
TRANSPORT ACROSS BIOLOGICAL membranes is fundamental to cellular processes, and thus it is not surprising that ~400 families of transport proteins have evolved to manage this diverse and complex task (10, 57). Ca2+ is a ubiquitous and highly versatile intracellular messenger used in practically all cell types spanning plant (64), animal (5), protist (43), and bacterial (50) kingdoms. Cytosolic Ca2+ concentrations are tightly regulated and differentially interpreted to allow modulation of a variety of cellular functions (38). This Ca2+ concentration plasticity is absolutely required for fine tuning of intracellular signaling, and hence Ca2+ is in constant flux across the membrane. Nature has evolved multiple systems to regulate intracellular Ca2+, including Na+/Ca2+ exchange. The Na+/Ca2+ exchanger (NCX) is a polytopic membrane transporter that catalyzes the countertransport of three Na+ for one Ca2+ (7, 53). Forming an essential part of the Ca2+ efflux system, the NCX competes with other Ca2+ transport systems to restore resting cytosolic levels, thereby maintaining Ca2+ homeostasis. The molecular cloning of the mammalian NCX greatly accelerated our understanding of Ca2+ exchange (47). This provided the impetus for the biochemical and functional characterization of the NCX at the molecular level and further led to the discovery and cloning of NCX homologs in various species (22, 60, 65). The current era of informatics is marked by the availability of an increasing number of genome sequences from prokaryotes and from metazoan organisms (63). Mining of genomic databases affords tremendous potential in discovering new NCX homologs, which in turn would offer insights into NCX phylogeny and the evolution of the protein from a molecular and genetic perspective (4).

NCX is a member of the cation/Ca2+ antiporter (CaCA) family, which corresponds to family 2.A.19 of the Transport Classification Database (TCDB) (see http://tcdb.ucsd.edu/index.php) (10). Recently, Cai and Lytton (11) performed an extensive sequence comparison and phylogenetic analysis of 147 sequences in the CaCA family. They defined the NCX group as one of five major subgroups of the CaCA family, and furthermore, found the group to be almost exclusively composed of sequences with animal origins (11). The NCX genes are classified as members of the CaCA family based on two defining characteristics: conserved {alpha}-repeats in the transmembrane segments (TMS) (60), and hydrophobicity plots that predict ~10 TMS (53). The presence of intramolecular homology and relative conservation of TMS throughout the CaCA family suggests its members arose from an ancient intragenic gene duplication event, in which a primordial gene encoding a protein with 5–6 TMS duplicated internally to give one protein with twice the TMS (57). To date, no primordial "half" exchanger has been found, and it appears that, at least for NCX, both the NH2- and COOH-terminal hydrophobic domains are needed for proper membrane trafficking and function (51).

The architecture of the NCX protein is shown in Fig. 1. Based on the topological models of Iwamoto et al. (28) and Nicoll et al. (48), the mature NCX is modeled to have nine putative TMS organized in NH2- and COOH-terminal hydrophobic domains of five and four TMS, respectively. A signal sequence of ~30 residues is cleaved during initial processing (15, 25), which makes the mature NCX protein ~900 residues in length, depending upon the isoform. Within the hydrophobic domains are the opposing {alpha}-1 and {alpha}-2 repeats, which are critical for ion translocation (46). The intracellular loop comprises more than half the protein and contains sites important for Ca2+ regulation, Na+-dependent inactivation, and alternative splicing. So far, little information on NCX tertiary structure is available, although an extracellular disulfide bond has been shown to exist between cysteines in the NH2- and COOH-terminal hydrophobic domains (59). In addition, Qui et al. (54) found the helix packing of TMS2, TMS3, TMS7, and TMS8 is such that the two {alpha}-repeats may be adjacent to each other in the tertiary configuration.



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Fig. 1. Topology of the Na+/Ca2+ exchanger. The Na+/Ca2+ exchanger (NCX) is modeled to have 9 putative transmembrane segments (TMS), based on experimental evidence of Nicoll et al. (48) and Iwamoto et al. (28). The TMS are shown as light gray cylinders, with 5 in the NH2-terminal (NH2) domain and 4 in the COOH-terminal domain (COOH). The NCX has an ~32-residue signal peptide (SP) that is cleaved during processing and is indicated by an open cylinder. On opposite sides of the membrane between TMS 2–3 and TMS 7–8 are the respective {alpha}-1 and {alpha}-2 repeats, shown in dark gray. These regions display intramolecular similarity and are important in ion translocation. The NH2- and COOH-terminal TMS clusters are separated by a large hydrophilic intracellular loop (~550 residues) that is important in exchanger regulation. The XIP ("exchanger inhibitory peptide") site is implicated in Na+-dependent inactivation, and Ca2+ binding sites are implicated in Ca2+-dependent activation. In addition, the COOH-terminal end of the hydrophilic loop contains an alternative splice region.

 
NCX is present in most plasma membranes where its relative abundance is correlative with the importance of Na+/Ca2+ exchange in that cell type. Expression of NCX is especially high in heart, brain, and kidney tissue, where NCX clearly plays an important role in Ca2+ homeostasis (31, 55). The NCX family contains three separate gene products exhibiting differential expression, NCX1 (47), NCX2 (36), and NCX3 (49). NCX1 is highly expressed in the heart but is virtually ubiquitous, whereas NCX2 and NCX3 are found exclusively in brain and skeletal muscle (55). The NCX1 gene is organized into 12 exons, although most of the protein is coded by exon 2 (33). In mammalian isoforms, the exon boundaries of all three NCX genes are identical, except that the long coding exon 2 found in NCX1 and NCX3 is split into three exons in NCX2 (18, 33, 35). The protein products of all three NCX genes display an overall identity of ~70%; however, no major functional differences occur between genes (37). It appears that two sequential gene replication events gave rise to the three NCX genes, but the date of these replications is unknown. In addition to having three separate gene products, tissue-specific expression of the NCX is further diversified by alternative splicing. Alternative splice variants of NCX arise from a region in the intracellular loop where six small exons, designated A-F, are encoded. Exons A and B are mutually exclusive and are used in combination with cassette exons C through F to express splice variants in a tissue-specific manner (32). The functional significance of NCX alternative splicing remains unclear and requires further investigation.

The early evolutionary emergence of NCX predicts that all metazoans should express some exchanger isoform. To date, most cloned NCX genes are from mammalian origins; however, NCX1 orthologs have been cloned and characterized from lower vertebrates such as trout (Oncorhynchus mykiss) (65) and frog (Xenopus laevis) (29) and the invertebrates fruit fly (Drosophila melanogaster) (56, 60), squid (Loligo opalescens) (22), and nematode (Caenorhabditis elegans) (33). Previously, no NCX2 or NCX3 orthologs have been found in nonmammalian species, leading to the hypothesis that nonmammalian exchangers diverged before mammalian exchangers split into NCX1, NCX2, and NCX3 (11). Phylogenetic analysis of the NCX family has been hindered by the lack of nonmammalian NCX sequences available in public databases. The increase in availability of whole genomes from both vertebrates and nonvertebrates provides the opportunity to increase the number of NCX sequences available for phylogenetic analysis.

In this study, we have expanded the phylogenetic examination of NCX by increasing the number and diversity of full-length sequences available for analysis. Combined with NCX sequences currently present in the protein databases, these new NCX sequences were subjected to a comprehensive sequence comparison and phylogenetic analysis. The inclusion of 13 new NCX sequences from 6 different organisms provides novel insights into the molecular evolution and function of NCX.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Sequence data.
A complete list of all the NCX sequences used in this study is shown in Table 1 (see Supplemental Fig. S1 for FASTA amino acid sequences; the Supplemental Material for this article is available at the Physiological Genomics web site).1 All sequences used in this study were obtained from either the National Center for Biotechnology Information (NCBI, Bethesda, MD) nonredundant (nr) protein database or the Ensembl Genome Browser (Wellcome Trust Genome Campus, Hinxton, Cambridge, UK) (6, 26). BLASTP (1) was used to screen the nr protein database at NCBI, using canine NCX1.1 (GenBank GI no. 127793) or trout NCX-TR1.0 (GenBank GI no. 6273849) amino acid sequences as queries. Default parameters for BLASTP were used, except the descriptions option was set to 1,000 to increase the number of reported hits and to ensure all NCX sequences were found. We ensured that other members of the CaCA family (e.g., NCKX) were identified in our search, providing further confidence that no NCX sequences were missed. The frog NCX1 full-length gene was constructed by splicing together the NH2- and COOH-terminal portions of the exchanger, GenBank GI nos. 1019101 and 109099, respectively.


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Table 1. Organism name, common name, gene name, and GI numbers of NCX sequences used for analyses in this study

 
The Ensembl Genome Browser was used to search for new NCX sequences within six complete genomes (five nonmammalian) including zebrafish (Danio rerio) (version 23.3c.1), Japanese pufferfish (Fugu rubripes) (version 23.2c.1), honeybee (Apis mellifera) (version 1 pre), chimpanzee (Pan troglodytes) (version 23.1.1), chicken (Gallus gallus) (version 23.1a.1), and mosquito (Anopheles gambiae) (version 23.2b.1). The genomes at Ensembl are from sequencing projects from various institutions. Zebrafish sequence data were produced by the Zebrafish Genome Sequencing Group at the Sanger Institute (Hinxton) and can be obtained from the Ensembl web site (http://www.ensembl.org/Danio_rerio/exportview). Fugu Ensembl is a joint project between the IMCB (Singapore) and the EMBL-EBI to produce and maintain an automatic annotation of the Fugu Genome, using data sequenced by the Fugu Consortium. The honeybee genome sequence was determined by whole-genome shotgun at the Human Genome Sequencing Center at Baylor College of Medicine (Houston, TX). The chimpanzee genome was sequenced by the Chimpanzee Sequencing Consortium headed by the Genome Sequencing Center at Washington University (St. Louis, MO) and the Broad Institute at MIT (Boston, MA). The chicken genome sequence was determined by the Genome Sequencing Center at Washington University. Finally, assembly of the mosquito genome was prepared by The International Anopheles Genome project.

TBLASTN was used to search the above genomes with varying NCX protein sequences as queries, depending on the genome being searched. All vertebrate genomes were searched with mammalian NCX1–NCX3, whereas the fruit fly CALX (GenBank GI no. 2266953) was used to TBLASTN the insect genomes. Because of the high degree of sequence similarity among all NCX isoforms, all NCX queries produced similar TBLASTN results. Segments of genomic DNA that encompassed both the NH2 and COOH terminus were obtained (~10–100 kb), and coding exons were manually identified using a combination of the original TBLASTN results, the Ensembl Contig view, and previously cloned NCX sequences. Full-length proposed NCX protein and cDNA sequences were then constructed by splicing together the coding exons, and then these sequences were added to our list of NCX sequences identified from the NCBI nr database.

Sequence alignments.
All acquired NCX sequences were initially aligned using the default parameters of ClustalX (version 1.83) (62). The resulting alignment was then imported into GeneDoc (version 2.6.002) (44) for manual examination and editing. Partial, duplicated, and alternatively spliced isoforms were discarded to ensure that only one NCX gene of each type from each species was used and, further, that this gene was a full-length and nonredundant sequence. The variable alternative splice site region of NCX (illustrated schematically in Fig. 1) was not included in subsequent phylogenetic analyses, because of the high potential for homoplasy in that region.

Phylogenetic analysis.
Phylogenetic analyses were performed using ClustalX (primarily for initial tree constructions and manipulations) and the PHYLIP package for verification of the initially derived trees (version 3.6b; Joe Felsenstein, Department of Genome Sciences, University of Washington, Seattle, WA) (17). Included in the alignments were two out-group sequences, AtMHX from mouse-ear cress (Arabidopsis thaliana) (GenBank GI no. 6492237) and an Na+/Ca2+ exchanger from the bacterium Pirellula sp. (GenBank GI no. 32475149). These sequences appear at the base of the NCX subgroup in the unrooted tree of the recent phylogenetic analysis of the CaCA family by Cai and Lytton (11).

Neighbor-joining (NJ) trees (58) were generated using ClustalX, followed by tree evaluation with bootstrap resampling (1,000 times). Further phylogenetic analysis (including confirmation of the ClustalX-based NJ tree topologies) was performed using the PHYLIP package, where trees were also constructed using maximum parsimony (MP) and maximum likelihood (ML) methods. Bootstrapping was performed with 100 replicates in such cases. The program TreeView (version 1.6.6) (52) was used to examine and display all trees.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
NCX is characterized by two transmembrane spanning domains separated by a large intracellular hydrophilic loop (53). Signifying its importance in regulating intracellular Ca2+ concentrations, NCX is present and highly conserved across phyla within the animal kingdom. Studies of the molecular phylogeny of NCX have used limited sequences or have been part of larger studies on the CaCA family in general. In 1996, Kraev et al. (33) found an NCX homolog in C. elegans and compared its genomic structure to human NCX1, and a year later, Schwarz and Benzer (60) first noted the intramolecular homology of NCX in their multiple alignment of 20 exchangers related to fruit fly CALX. Philipson and Nicoll (53) performed BLAST searches of the GenBank protein database and designated the NCX family as one of four subfamilies of the exchanger superfamily. However, this analysis contained only 8 NCX sequences and 29 in total. This study was expanded in the first comprehensive phylogenetic analysis of the CaCA family, which was recently undertaken by Cai and Lytton (11). They categorized 147 members of the CaCA family into five subfamilies, with the NCX subfamily containing 22 proteins. Of these NCX proteins, only seven were nonmammalian, including two from nonmammalian vertebrates. Cloning and functional characterization of the NCX has always had a strong mammalian bias despite the protein’s early emergence in evolution. From our initial searches, ~75% of all publicly available NCX protein sequences were from mammalian origins. The dearth of sequence data from nonmammalian species has greatly impaired evolutionary analyses of the NCX family. This study builds on previous research through inclusion of additional NCX sequences derived from sequenced genomes and represents the first phylogenetic analysis specifically focused on NCX.

NCX is present in a diverse group of organisms.
All NCX sequences identified and used in this study are listed in Table 1 (see Supplemental Fig. S1 for FASTA amino acid sequences). A BLASTP search of the NCBI nr database produced almost identical results when dog NCX1.1 or trout NCX-TR1.0 was used as a query. Eighty NCX amino acid sequences were initially obtained from the search and pared down to 25 full-length nonredundant NCX sequences. An additional 13 previously unidentified NCX sequences were obtained from analysis of 6 genome sequences available through the Ensembl Genome Browser. These included four NCX sequences from zebrafish, four from Japanese pufferfish, two from chicken, and one each from honeybee, mosquito, and chimpanzee. For some of the above species, additional NCX genes were found on other chromosomes but were incomplete or lacked sufficient identity to be included. In total, 38 full-length nonredundant NCX sequences from 23 species were used in our analyses, including 16 mammalian NCX sequences and 22 nonmammalian sequences. Of the 22 nonmammalian NCX sequences, 8 were from invertebrates and 14 were from lower vertebrates. These NCX proteins range in size from 861 to 973 amino acids, because of gene isoform and splice variant differences.

It should be noted that not all of the NCX sequences obtained from the Ensembl Genome Browser were annotated correctly, illustrating the difficulties currently faced in whole-genome annotation (9, 12, 27) and gene prediction (20, 39). Genes at Ensembl are computationally annotated based on gene prediction and comparison to protein, cDNA, and expressed sequence tag (EST) databases. These methods result in some annotation errors that can be resolved by manual re-annotation. In general, NCX coding exons displaying high identity across many species were annotated correctly, whereas erroneous annotation of intron-exon boundaries tended to occur in less conserved regions or in the alternative splice region in which individual coding exons are small. In most cases the start codon and portions of the signal peptide were also missing, since this region is poorly conserved among NCX isoforms. The NCX sequences were manually re-annotated using a combination of strategies, including TBLASTN searches and manual translation of coding areas to find intron/exon boundaries. Because of incomplete genome sequencing, some final NCX sequences contained gaps (e.g., zebrafish NCX2 is only 747 amino acids). The addition of 13 (12 nonmammalian) previously unidentified NCX sequences is crucial to expanding the phylogenetic analyses of the exchanger.

NCX identity in the ion translocation and regulatory regions is high.
The conservation of the NCX throughout evolution is evidenced by the overall high identity of the full sequence alignment (see Supplemental Fig. S2 for full multiple sequence alignment). The relatively high identity in the TMS of NCX is a feature consistent throughout the CaCA family, in which the intracellular loop is poorly conserved between independent clades (11). The data are consistent with the findings that only the NH2- and COOH-terminal hydrophobic domains are required for ion transport (42, 51) and further supports the idea that the hydrophilic loop has evolved to gain specific regulatory properties.

The {alpha}-1 and {alpha}-2 repeats, together with the immediate surrounding TMS, are central to ion translocation function of the NCX. An alignment of these regions using a representative list of 26 NCX sequences is shown in Fig. 2. An immediate and striking observation is the high identity across all NCX species and isoforms (35/55 identical residues for {alpha}-1 region and 32/53 identical residues for {alpha}-2 region), particularity for those residues modeled to be within the membrane. Previous studies have shown that residues in these regions are extremely sensitive to mutation, especially the acidic (Glu and Asp), polar (Thr, Ser, Asn), and flexible (Gly, Pro, Ala) groups of amino acids (13, 45, 53).



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Fig. 2. Multiple sequence alignments of the {alpha}-1 (A) and {alpha}-2 (B) internal repeats and surrounding TMS. This region is important for ion translocation. The predicted secondary structure profile as described in Fig. 1 is shown above the alignment, with the {alpha}-repeats shown in dark gray cylinders. The mammalian NCX sequences in these regions display 100% identity and are therefore represented by human isoforms of NCX1, NCX2, and NCX3. GI numbers for all NCX sequences are shown in Table 1. NCX sequences are grouped based on NCX isoform, with the residues flanking the alignment numbered from the start codon. The asterisk in the zebrafish NCX2 sequence signifies a gap in sequence due to incomplete sequencing of the zebrafish genome.

 
There are several conserved acidic residues in the {alpha}-repeats and surrounding TMS. Glu148 in TMS2 and Asp849 in TMS7 in human NCX1.1 are completely conserved throughout the CaCA family (11) and are absolutely required for NCX ion translocation function (45). TMS2 has an additional conserved glutamate Glu155, which would place the charged residue on the same side of the helix as Glu148. Mutational studies have shown that mutation of Glu155 reduces NCX activity by 50% (45). TMS3 lacks the presence of charged amino acids, but the proximal end of the re-entrant loop between TMS7 and TMS8 has two conserved aspartate residues: Asp860 and Asp864. Asp860 is very sensitive to mutation, whereas Asp864 is not (53). These conserved acidic residues throughout the region are likely involved in neutralizing the positive charges of Na+ and Ca2+, thereby allowing transport of the cations through the membrane (11).

In addition to negatively charged residues, the {alpha}-repeats and surrounding TMS have a number of conserved polar (Ser, Thr, Asn) amino acids (Fig. 2). In TMS2, TMS3, and TMS7, the polar residues are spaced at regular intervals of 3–4 residues, making these helices amphipathic. The conservation of polar residues in this region is consistent with expected functional modifications based on previous mutational analyses (45). The hydrophilic faces of the amphipathic TMS2, TMS3, and TMS7 are believed to form a portion of the ion translocation pathway (54) and may help coordinate cation binding or provide the hydrophilic environment necessary for ion translocation (11).

The {alpha}-repeat regions are also rich in conserved glycine and proline residues, even within the regions modeled to be TMS helices. The {alpha}-1 region has three proline and two glycine residues that are conserved across all species and isoforms. These residues do not appear to be absolutely required for ion translocation, but not all have been tested (Fig. 2). In contrast, the {alpha}-2 region has one conserved proline and five conserved glycine residues, with mutation of the glycines at positions 844, 881, and 883 modifying transport properties. The latter two glycine residues flank a G(I/L)G sequence that is similar to the GYG motif in the P-loop of K+ channels (14) and the GIG motif in the pore region of the sarcoplasmic Ca2+ release channels (RyR) (3, 19). However, other than being a tight turn within the re-entrant loop, this motif in NCX has not been shown to have any functional significance. The presence of proline and glycine residues in the middle of TMS2 and TMS7 may provide the helix flexibility needed for the conformational changes that occur upon ion binding and translocation (11).

In addition to being substrates, Na+ and Ca2+ also have a role in the regulation of the NCX in the forms of Na+-dependent inactivation and Ca2+-dependent activation, respectively. The regions responsible for this regulation are located in the large intracellular loop, and an alignment of these regions is shown in Fig. 3. Na+-dependent inactivation causes inactivation of NCX current to a steady-state level (23) and is attributed, at least in part, to the XIP ("exchanger inhibitory peptide") site. Immediately apparent in Fig. 3A is the lack of sequence similarity of the nematode XIP sites compared with all other species, despite high identity in the TMS5. It is not known whether nematode NCX isoforms display Na+-dependent inactivation, but based on lack of sequence similarity in the XIP site, it is unlikely. Even among the rest of the species, sequence identity is low except for the start of the XIP site beginning with the consensus RRLL(X)nG. Na+-dependent inactivation has been demonstrated in different NCX isoforms (37) and in NCX from different species including squid (22), fruit fly (24), and trout (16). Consistent with low XIP identity across all species, mutations in this region do not affect ion translocation of the NCX but rather have modulating effects on inactivation (21, 40). The relatively low evolutionary conservation in the XIP site may be due to indirect binding of Na+ to the XIP site.



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Fig. 3. Multiple sequence alignment of regulatory regions from selected NCX sequences: alignments of the TMS5 and XIP site (A) and Ca2+-regulatory region (B). The XIP site has been implicated in Na+-dependent inactivation, and the Ca2+-binding sites have been implicated in Ca2+-dependent activation. The predicted secondary structure profile as described in Fig. 1 is shown above the alignment. The mammalian NCX sequences in these regions display nearly 100% identity and are therefore represented by human isoforms of NCX1, NCX2, and NCX3. GI numbers for all NCX sequences are shown in Table 1. NCX sequences are grouped based on NCX isoform, with the residues flanking the alignment numbered from the start codon. The asterisk in the zebrafish NCX2 sequence signifies a gap in sequence due to incomplete sequencing of the zebrafish genome.

 
Conversely, the Ca2+-dependent activation does require the binding of Ca2+, and this is reflected in two highly conserved binding sites located in the intracellular loop (Fig. 3B). Regulatory Ca2+ is not translocated but is required for exchange activity, binding with high affinity at two aspartate- and glutamate-rich sites in the regulatory loop (34). The zebrafish NCX2 sequence shown in Fig. 3B is missing the first binding site due to incomplete genomic sequencing, and the tilapia NCX1.1 has a frame shift in this region due to sequencing error (unpublished observations, C. R. Marshall). Mutations in this region (especially the first trio of aspartates) do not affect ion translocation but do affect Ca2+ binding and regulation properties (41). Interestingly, regulatory Ca2+ binding in the fruit fly CALX decreases exchanger activity, which is opposite to the observed effect in other NCX isoforms. It is not known whether this is the case in the related exchangers from the honeybee and mosquito, but it must be due to an allosteric effect in the protein since the Ca2+ binding sites themselves have high identity.

In overall sequence identity, there is a core of consensus residues that are needed for full NCX function and regulation. Surprisingly, not all residues that are conserved across all species are essential to NCX function; however, any residue that is known to be very sensitive to mutation is conserved across all species. NCX sequence similarity follows that of the CaCA family in general, with high relative sequence identity in the TMS and lower sequence identity in the cytoplasmic regulatory loop (11). This in turn is consistent with the expected sequence-function relationship: the TMS of NCX are absolutely required for ion translocation and have therefore remained relatively intact throughout evolution, whereas regions in the hydrophilic loop have evolved separately among NCX isoforms to confer species specific regulatory properties.

NCX gene families have similar genomic structure and intron-exon boundaries.
In searching for new NCX sequences in sequenced genomes, we found multiple NCX isoforms in three of the six organisms: chicken, zebrafish, and Japanese pufferfish. The genomic organization of the coding exons for the 13 NCX sequences found at Ensembl is shown in Fig. 4 and can be a useful tool in examining NCX evolution. Human NCX1, NCX2, and NCX3 genes have been previously mapped to chromosomes 2p22.1 (33), 19q13.2 (30), and 14q24.2 (18), respectively. We found the three human NCX genes at Ensembl and used them as reference sequences in our analysis (Fig. 4). A couple of features regarding the human NCX genes are notable. First, the NCX sequences contain only the coding exons and do not contain portions of 5'- and 3'-untranslated regions. Previously, it has been shown that the start codon of NCX1 and NCX3 is in exon 2, a large 1.8-kb exon that extends to the alternative splice site (18, 33). In Fig. 4, the untranslated exon 1 is not shown, and exon numbering begins with the first coding exon. Human NCX2 displays the same exon boundaries as NCX1 and NCX3, except the long initial coding exon is split into three exons. Second, the coding regions of human NCX1 and NCX3 genes have been reported to stretch over ~200 kb (33) and ~126 kb (18) of genomic DNA, respectively. According to the human NCX genes at Ensembl, these distances are ~316 kb for NCX1 and ~126 kb for NCX3. The reason for this length discrepancy in the NCX1 gene is unknown, but is solely attributable to differences in intron length and is not due to miscalculation of the gene boundaries. There are a couple of possible reasons for this discrepancy. First, it is possible that there are errors in the assembly of the human genome sequence at Ensembl; however, examination of the human genomic sequence encompassing the NCX1 gene is of high quality, and it is unlikely that errors in genome assembly exist. Supporting the genomic assembly of human NCX1 at Ensembl is the fact that the closely related chimpanzee genome yields an NCX1 gene that spans a distance similar to that of the human NCX1 gene. Second, Kraev et al. (33) did not sequence the introns completely, and therefore it is possible that their length was underestimated.



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Fig. 4. Gene structure of NCX sequences derived from whole genomes. The gene structures of the 13 new NCX sequences and representative human NCX1, NCX2, and NCX3 were constructed from intron-exon data at Ensembl. The genes are grouped based on NCX isoform, with the invertebrate NCX isoforms being unclassified. Exons are denoted by black boxes, beginning with the start codon and terminating with the stop codon. Exons only represent the coding sequence of the NCX gene and are numbered accordingly from the start codon. Previously, it has been shown that exon 1 of the NCX genes is not translated, and therefore we have not included it in this schematic. The start codon is in exon 2, which is designated exon 1, here. Introns are indicated by lines connecting the exons. The exon and intron widths are drawn approximately to scale based on their nucleotide size over 80 kb of genomic DNA. Genes that extend beyond 80 kb have the longest intron given simply by their length so as to fit the scale bar. Total genomic distance each gene stretches is given to the right of the last exon in kilobases. Exons belonging to the alternative splice region are underlined and lettered accordingly. It should be noted that not all of the alternative splice exons predicted to exist in these species were found. The mutually exclusive exons A and B are both shown in the schematic in cases where they could both be found in the genomic data. Because of sequencing errors in the chimpanzee genome, the first exon of NCX1 was split into three exons, each separated by a single nucleotide. Since the actual chimpanzee NCX1 gene would not contain these insertions, we have numbered the exons the same as human NCX1. Exon 6 in both the chicken NCX1 and the zebrafish NCX2 contain gaps and are indicated by open boxes. Exons 2 and 3 from zebrafish NCX2 were not present, due to incomplete sequencing of the zebrafish genome, and are indicated by open boxes and a question mark.

 
In addition to having a different initial coding exon structure, the human NCX2 gene is very compact, stretching over only ~36 kb of genomic DNA. In general, the NCX isoforms from other species followed these trends, with NCX2 having more compact genomic DNA compared with NCX1 and NCX3. NCX2 also has a greater number of exons before the alternative splice region, a trend which is also common in the invertebrates’ NCX. In contrast, NCX1 and NCX3 typically have a single large initial coding exon separated from the alternative splice site by a very long intron. This suggests that the NCX1 and NCX3 genes of vertebrates have undergone some form of intron loss, effectively fusing together all their coding exons up to the alternative splice site. Based on similarities in exon structures and genomic organization, NCX1 and NCX3 appear to be more closely related to each other than either gene is to NCX2.

Nonmammalian vertebrates have multiple NCX genes.
In addition to insights into the evolution of the NCX gene, similarities in gene structure can be used to annotate new NCX genes. Using the human NCX genes as a reference, the 13 NCX isoforms from Ensembl were annotated based on an examination of the sequence alignment (see Supplemental Fig. S2) and genomic organization of exons (Fig. 4). The chimpanzee NCX1 sequence shares 99% amino acid identity with human NCX1 and has an almost identical exon structure. Frame shifts due to sequencing errors in the chimpanzee genome gave different NCX exon boundaries compared with human NCX in the first exon, but correction would yield identical NCX exons for human and chimpanzee. Both NCX2 and NCX3 are present in the chimpanzee genome, but contained too many gaps to warrant inclusion in our sequence lists. The mosquito and honeybee NCX sequences did not display high amino acid or intron-exon boundary similarity to NCX1, NCX2, or NCX3 and are therefore unclassified. This is consistent with the literature, in which no invertebrate NCX isoform has been classified as one of NCX1, NCX2, or NCX3. The chicken genome had NCX1, NCX2, and NCX3 genes, but as was the case for chimpanzee isoforms, NCX2 was not added to the list because of the presence of partial sequence only. Both zebrafish and Japanese pufferfish genomes contained NCX1, NCX2, and NCX3. For all NCX genes, the Japanese pufferfish isoforms had the shortest introns, consistent with this species having a genome that is ~7.5 times as compact as the human genome (2, 8). The NCX isoforms from chicken, zebrafish, and Japanese pufferfish represent the first NCX2 and NCX3 sequences from nonmammalian species.

To further confirm the annotation of the NCX sequences from Ensembl, the alternative splice site was aligned and examined (Fig. 5). The exons expressed in this region, both in terms of number and sequence, can offer insight into the identity of the NCX gene. In vertebrate NCX genes, exons A and B are mutually exclusive and produce tissue-specific variants when expressed in combination with exons C, D, E, and F. The NCX1 gene has exons A through F, whereas NCX2 has only A and C and NCX3 only A, B, and C (55). The NCX sequences of invertebrates do not have alternative splicing to the same extent as isoforms from vertebrate species, but do have a variant of exon A/B. For all sequences except fugu NCX3, either exon A or B was found and showed significant sequence identity for it to be categorized as NCX1, NCX2, or NCX3 specific. Interestingly, both exons A and B were found for chicken and Japanese pufferfish NCX1, which to our knowledge is the first time both exons have been shown in nonmammalian species. Exon A likely exists in the zebrafish NCX1 gene, but was not found, because of genomic sequence gaps. Some of the smaller cassette exons (C, D, and E) present in NCX1 could not be found in nonmammalian species because they were either too small (5–7 amino acids) or lacked sufficient similarity to be picked up by BLAST analysis. These exons (C, D, E, and F) are predicted to exist in both zebrafish and Japanese pufferfish NCX1 isoforms, since all are present in trout NCX1.0 and tilapia NCX1.1 with fairly high similarity to the mammalian exons. However, it is possible that the cassette exons have been lost in some species or have diverged to a point that they cannot be identified using sequence similarity methods. This may be the case for NCX3, where exon C, which is present in mammalian NCX genes, could not be found in chicken, zebrafish, or Japanese pufferfish. Conversely, exon C in the NCX2 subfamily was found in both zebrafish and Japanese pufferfish and appears to be highly conserved in all species. In general, the alternative splice region within NCX subfamilies displays high similarity in both exon structure and exon sequence. The fact that this region has maintained a high degree of conservation throughout evolution implicates its role in creating functionally different NCX splice variants through the differential expression of exons, a strategy that is employed in species ranging from lower vertebrates to mammals.



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Fig. 5. Multiple sequence alignment of alternative splice region from NCX sequences derived from whole genomes. The alternative splice regions of NCX genes derived in this study were aligned with human NCX splice variants for reference. The sequences are grouped by isoform, with boxes above the alignment denoting mutually exclusive (A or B, gray boxes) and cassette (C, D, E, and F; white boxes) exons. In instances where exons A and B were found in a species’ genome, both are shown. The asterisks denote exons that should be present in the species but were not found, whereas the question mark indicates that it is not known whether homologous cassettes exist in NCX4. The "X" marks in exon B of chicken NCX3 denote amino acids that cannot be identified, due to incomplete genome sequencing.

 
NCX gene duplication events occurred before emergence of mammals.
Phylogenetic analysis of all 38 NCX sequences offered further insight into the evolution of NCX (Fig. 6 and Supplemental Fig. S3). Phylogenetic analysis using different tree-building methods have often shown a lack of uniformity in their results (61); therefore, we have used three different methods in the phylogenetic reconstruction of NCX. Figure 6 shows the rooted NJ tree for all 38 NCX sequences, together with the 2 out-group sequences. There is clear delineation between the invertebrate NCX sequences, which further branch into groups containing the nematodes, insects, and squid. The vertebrate NCX sequences are distinctly grouped into clades, including the three familiar NCX1, NCX2, and NCX3 proteins. Within the NCX1, NCX2, and NCX3 groups, mammalian and nonmammalian sequences are separated and grouped in a manner that seems to be consistent with the evolution of the organisms. In their analysis, Cai and Lytton (11) determined that invertebrate and nonmammalian vertebrate exchangers diverged before mammalian exchangers split into NCX1, NCX2, and NCX3. Increasing the number of NCX sequences for phylogenetic analysis has allowed us to fashion a rudimentary account of the molecular evolution of the NCX. The divergence of NCX into three genes occurred after the divergence of invertebrate exchangers but before the divergence of vertebrate exchangers, indicating these duplication events took place at least 450 million years ago. After the separation of the invertebrate NCX clade, the ancestor to the vertebrate NCX gene family underwent two separate gene duplication events. From our analysis it appears the vertebrate NCX2 gene evolved from the first duplication event, whereas the other gene underwent a subsequent duplication giving rise to NCX1 and NCX3. The actual timing of these serial duplications cannot be estimated due to the lack of NCX sequences from organisms spanning the evolutionary gap between invertebrate and lower vertebrate fish species (~100–150 million year period). Sequencing of genomes from species spanning this time frame (e.g., hagfish, lampreys) should help pinpoint the actual timing of NCX gene duplications. This theory of NCX evolution is supported by the MP method in Supplemental Fig. S3A, as it produced virtually the same tree topology as the NJ approach. The ML tree (Supplemental Fig. S3B) is similar to the above trees with a few exceptions that offer an alternative theory to the evolution of NCX. The tree topology shows the split in the invertebrate and vertebrate NCX isoforms and groups the vertebrate isoforms in the same NCX groups as the NJ and MP trees. However, the ML tree shows that the NCX1 group was the first to diverge, and then NCX2 and NCX3 arose from gene duplication. Although this theory is plausible, it is not supported by the similarity of exon structures shared between NCX1 and NCX3, compared with NCX2. Expanding the number and diversity of NCX sequences for phylogenetic analysis should resolve these discrepancies and create a clearer picture of the evolution of NCX.



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Fig. 6. Phylogenetic analysis of the NCX family. The neighbor-joining (NJ) tree was constructed with resampling (bootstrap, 1,000 data sets) using ClustalX. Vertebrate and invertebrate sequences are enclosed with brackets, whereas vertebrate NCX genes are labeled with vertical lines. The tree is rooted with a Na+/Ca2+ exchanger from Pirellula sp., and rooting with AtMHX from A. thaliana did not change the tree topology. NCX4 (bold) refers to our putative NCX group that is unique to the fish species, and asterisks denote NCX isoforms that where derived from whole genomes.

 
Fish species have a fourth NCX gene related to NCX1.
Both zebrafish and Japanese pufferfish have a fourth NCX isoform that has high identity to an exchanger from the green spotted pufferfish (GenBank GI no. 47219419). Collectively, these putative genes are distinct from NCX1–3, and therefore we have tentatively named them NCX4. So far, NCX4 is present in only in fish genomes, as orthologs could not be found in any nonfish species. Based on sequence similarity, fish NCX4 is most closely related to NCX1; however, it is not known whether this gene is expressed in fish or functions as an exchanger. In terms of exon structure, the fugu NCX4 is more similar to NCX2, whereas the zebrafish NCX4 has an exon structure more similar to both NCX1 and NCX3 (Fig. 4). At the NCX4 alternative splice site, only exon A/B was found and it is not known whether any of the cassette exons exist. This new NCX isoform appears to be a result of a separate gene duplication of NCX. NJ and MP trees both suggest that it evolved from a gene duplication of the ancestor of NCX1 and NCX4 and that the duplication may have occurred after the divergence of fish from other vertebrates (Fig. 6 and Supplemental Fig. S3A). The date of this duplication is unknown but appears to have occurred early in the evolution of NCX, because of the degree of divergence between NCX4 and NCX1 and the fact that NCX4 is not present in mammalian or avian species. The ML tree is not consistent, suggesting instead that NCX4 evolved from an older gene duplication that may be ancestral to the divergence of NCX1, NCX2, and NCX3 (Supplemental Fig. S3A). However, it should be noted that bootstrap values for this component of the tree are not very reliable. In addition, the fish NCX4 shares its highest identity with NCX1, meaning these proteins would have diverged at a much slower rate than NCX2 and NCX3 if the ML tree was a representation of true NCX evolution.

In summary, this study has built on previous work to present the first comprehensive sequence and phylogenetic analysis of the NCX family, which now includes 13 new NCX sequences derived from whole-genome sequencing projects. Integration of sequence alignment, gene structure, and phylogenetic data has generated a solid framework for future analyses and has provided novel insights into the molecular evolution of the NCX. For the first time, NCX2 and NCX3 have been shown to exist in nonmammalian species, and we propose that an NCX4 gene exists in fish species.


    ACKNOWLEDGMENTS
 
We thank Dr. Deb Nicoll of UCLA for helpful comments on the manuscript.

This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada to G. F. Tibbits. C. R. Marshall was supported by PhD research trainee scholarship from the Michael Smith Foundation for Health Research (MSFHR). F. S. L. Brinkman is an MSFHR Scholar, and G. F. Tibbits is the recipient of Tier I Canada Research Chair.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: G. F. Tibbits, Simon Fraser Univ., 8888 Univ. Drive, Burnaby, BC, Canada V5A 1S6 (E-mail: tibbits{at}sfu.ca).

10.1152/physiolgenomics.00286.2004.

1 The Supplemental Material (Supplemental Figs. S1–S3) for this article is available online at http://physiolgenomics.physiology.org/cgi/content/full/00286.2004/DC1. Back


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