Gene structure evolution of the Na+-Ca2+ exchanger (NCX) family

Abstract

Background: The Na+-Ca2+ exchanger (NCX) is an important regulator of cytosolic Ca2+ levels. Many of its structural features are highly conserved across a wide range of species. Invertebrates have a single NCX gene, whereas vertebrate species have multiple NCX genes as a result of at least two duplication events. To examine the molecular evolution of NCX genes and understand the role of duplicated genes in the evolution of the vertebrate NCX gene family, we carried out phylogenetic analyses of NCX genes and compared NCX gene structures from sequenced genomes and individual clones.

Results: A single NCX in invertebrates and the protochordate Ciona, and the presence of at least four NCX genes in the genomes of teleosts, an amphibian, and a reptile suggest that a four member gene family arose in a basal vertebrate. Extensive examination of mammalian and avian genomes and synteny analysis argue that NCX4 may be lost in these lineages. Duplicates for NCX1, NCX2, and NCX4 were found in all sequenced teleost genomes. The presence of seven genes encoding NCX homologs may provide teleosts with the functional specialization analogous to the alternate splicing strategy seen with the three NCX mammalian homologs.

Conclusion: We have demonstrated that NCX4 is present in teleost, amphibian and reptilian species but has been secondarily and independently lost in mammals and birds. Comparative studies on conserved vertebrate homologs have provided a possible evolutionary route taken by gene duplicates subfunctionalization by minimizing homolog number.

Figure 1

Maximum Likelihood NCX Phylogenetic Tree. The ML phylogenetic tree was generated with the PHYML program with 500 replicates using all known and postulated NCX protein sequences. Pirellula bacterial strain (Rhodopirellula baltica) was used to root this tree. The five-point stars () indicate NCX duplication after Ciona intestinalis divergence and the four-point stars () indicate teleost-specific NCX duplication. Branches are colour coded for vertebrate species, in which red designates mammals, purple indicates birds/aves, teal branches are for reptiles, green are amphibians, and blue are teleosts/fish. The dashed branch (---) indicates the division between invertebrate and vertebrate NCX sequences. The asterisk (*) indicates complete transcripts and those without an asterisk are genomic sequences. The letters 'a' and 'b' correspond to the teleost duplicate versions of NCX1, NCX2 and NCX4. Underlined species are new NCX sequences added in comparison to a previous paper [20].

Figure 2

Summaries of Phylogeny Trees. Non-scaled, simplified representations of NCX evolution, rooted with a bacterial sequence is demonstrated with two manners of duplication: A. serial (Neighbour Joining) and B. parallel (Maximum Parsimony) duplication.

Figure 3

Synteny Alignment Diagram at NCX4 Loci. Synteny diagram among zebrafish, fugu, frog, tetraodon and human. NCX4 (a or b) gene is represented as a circle and all other surrounding genes are represented as ovals and orthologs are associated with a line.

Figure 4

Sample Species of NCX Homologs Exon Splicing Pattern. Three sample organisms for mammalian (Homo sapiens), reptilian (Anolis carolinensis), amphibian (Xenopus tropicalis) and teleost (Danio rerio) are shown aligned at exon A (blue)/B (orange). Light blue rectangles belong to NCX N-terminus before the alternative spliced exons and yellow rectangles represent the C-terminus after the alternative spliced region. Cassette exons (C-F) are indicated in light green, purple, red and dark green where it is applicable and respectively. Distances between exons are scaled as indicated on the legend but not the length of the exons.

Figure 5

NCX Gene Homologs and Localization. Teleost, amphibian and aves list of NCX genes and localization with scaled distances in between exons. Open rectangles represent the exons at the N-terminus located before the alternative spliced exons and the dashed rectangles represent the exons after the alternative spliced exons at the C-terminus. Alternative exons A (black), B (light grey) and the rest of the cassette exons (dark grey) are shown where it is applicable. NCX duplicate genes are separated with NCXa and NCXb at the top and bottom of the list, respectively.