The calcium channel beta2 (CACNB2) subunit repertoire in teleosts

Abstract

Background: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts.

Results: Cloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.

Conclusion: These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

Figure 1

Structure of zebrafish β2.1 and β2.2 MAGUK genes. Exons are shown as black boxes; introns are not drawn to scale. Alternative splicing is indicated in the N-terminus and HOOK domain-encoding regions. * indicates the site of a premature stop codon in β2.1 transcripts that include exon 7. Labeled arrows indicate the locations and names of primers used in RACE and RT-PCR. Human exon structure was adapted from [57]. See Additional File 2A for primer sequences.

Figure 2

Comparative gene structure in teleost β2 subunits. A) The exons identified for each of four teleost β2 genes are listed vertically with indication of their size in bp. Exons on each horizontal line of the table are homologous in sequence (see later figures for alignments). Exons are color coded to indicate what portion of the 5-domain MAGUK protein (diagrammed in B) they comprise. ^ATG, indicates the presence of an initiation codon in the exon. *, indicates the exon contains a premature in-frame termination codon. C) Percent amino acid identity among vertebrate β2 subunits within the SH3 through GK sequences. D) The transcript variant numbers assigned to the alternatively spliced transcripts are listed on the left; transcript variant composition is depicted by exon numbers.

Figure 3

Sequences and alignments of alternatively spliced 5' regions. (A -E) Alignments show the entire N-terminal portion of the protein (sequences prior to the SH3 domain) for vertebrate β2 sequences culled from public databases. An overall percent amino acid identity, calculated for the various pairwise combinations, is listed in parentheses for each peptide alignment. Where appropriate, homology to previously described human transcript variants is indicated [57]. E) In humans, exon 1A or exon 1B is spliced to exon 2A. No exon 1A or1B-like exons could be identified in the current databases for teleosts, but an exon homologous to human 2A is present in several species. DR, Danio rerio; FR, Fugu rubripes; GA, Gasterosteus aculeatus (three-spined stickleback); GG, Gallus gallus; HS, Homo sapiens; MM, Mus musculus; OL, Oryzias latipes (Medaka killifish); OM, Onchorhynchus mykiss (trout); RN, Rattus norvegicus, TN, Tetraodon nigroviridis; XT, Xenopus tropicalis. In this and other alignments, conceptual translations were used if protein accession numbers were not available. * denotes a single genomic contig which contains the predicted exons shown. 6 denotes an exon border, numbered with reference to (A-D) zebrafish or (E) pufferfish exons. ##, indicates the location of two conserved cysteine residues which are palmitoylated in human β2 proteins.

Figure 4

Sequences and alignments of alternatively spliced exons contributing to the HOOK domain. Four distinct exons occur in zebrafish β2 transcript variants which differentially join to exon 6 to encode the HOOK domain (see Fig. 1C). (A-C) Three of these exons are alternatively spliced in β2.1, whereas the sequence in (D) was the sole sequence found in all β2.2 transcripts. Species names are abbreviated as in Figure 3.

Figure 5

Large intron sizes in β2 genomic loci. Boxes denote the location of exons that encode the 5' region of β2 subunit genes. The intervening regions denote the relative sizes of introns, labeled in bp. The scale is expanded for the teleost gene diagrams relative to human. The various N-terminal (grey) exons splice to the solid black exon, which begins with amino acids GSAD...The following genomic contigs were used for the analysis: Human β2 (NT_008705.15), Danio β2.2 (NC_007113), Danio β2.1 (NC_007133), Tetraodon β2 (CAAE01015017.1), Fugu β2 (CAAB01000004.1), Gasterosteus β2 (AANH01005391.1, using the EST DW608729, which resembles zebrafish β2.2).

Figure 6

Phylogeny of β2 subunit genes. Phylogenetic tree showing the relationships among β2 subunit core domain (SH3 – GK) sequences. Numbers above the nodes indicate maximum likelihood quartet puzzling support values; numbers below the nodes are maximum parsimony bootstrap proportions. "---" indicates a node that was unresolved in the maximum parsimony analysis. The long branch associated with zebrafish β2.2 reflects an elevated rate of amino acid substitution throughout the core domain, particularly at the 5' end. See Methods for accession numbers of sequences used.

Figure 7

Expression of β2 subunit transcript variants in the embryo and adult. RT-PCR analysis using transcript variant-specific primers (located in the 5' exons 1 or 2 and exon 10) was performed on RNA samples from A) whole embryos at various developmental stages, B) cardiac tissue dissected from cmlc2:GFP embryos or from adult fish, and C) adult organs and tissues. Expression of a housekeeping gene, EF1α, was used as a control for RNA integrity. In B, 72 hpf or adult RNA reactions were run on single gels, subsequently subdivided to multiple panels for clarity in presentation. Transcript variant numbers are listed to the right of panels; refer to Fig. 1D.