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The calcium channel beta2 (CACNB2) subunit repertoire in teleosts.

Ebert AM, McAnelly CA, Srinivasan A, Mueller RL, Garrity DB, Garrity DM - BMC Mol. Biol. (2008)

Bottom Line: Moreover, phenotypes may be obscured by secondary effects of hypoxia.Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. amebert@lamar.colostate.edu

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.

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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.
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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.

Mentions: In contrast, the more divergent zebrafish β2.2 core region shares only ~62% amino acid identity (see Additional File 4 for alignment of core regions). The most extreme sequence divergence in the zebrafish β2.2 gene occurs at the 5' end of the gene, both 5' of and within the SH3 domain, although high levels of divergence exist throughout the entire protein. We identified a single EST, termed DW608729, from three-spined stickleback as a possible ortholog to the zebrafish β2.2 gene. Within the core domain, DW608729 is 74% identical to zebrafish β2.2 (EU301442) but only 52% identical to zebrafish β2.1 (EU301434). In addition, DW608729 maps to a genomic contig, AANH01005391.1, which contains sequences homologous to zebrafish β2.2 exon 1 (encoding MFCCGLGHWRREQSTY) and β2.2 exon 2 (encoding MPPKKK)(Fig. 3). In the β2.2 gene(s), regions of high divergence encompass sequences both within and outside of inferred secondary structures [32]. Nevertheless, 19 of 21 β subunit residues noted for interaction with the α subunit AID are conserved in zebrafish β2.2 (see Additional File 4) [48-50]. High sequence divergence in β2.2 relative to other vertebrate β genes is reflected in the branch lengths on the phylogeny (Fig. 6). This pattern contrasts with that seen in the β4 group, where the two zebrafish paralogs have experienced similar rates of amino acid substitution.


The calcium channel beta2 (CACNB2) subunit repertoire in teleosts.

Ebert AM, McAnelly CA, Srinivasan A, Mueller RL, Garrity DB, Garrity DM - BMC Mol. Biol. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2365960&req=5

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.
Mentions: In contrast, the more divergent zebrafish β2.2 core region shares only ~62% amino acid identity (see Additional File 4 for alignment of core regions). The most extreme sequence divergence in the zebrafish β2.2 gene occurs at the 5' end of the gene, both 5' of and within the SH3 domain, although high levels of divergence exist throughout the entire protein. We identified a single EST, termed DW608729, from three-spined stickleback as a possible ortholog to the zebrafish β2.2 gene. Within the core domain, DW608729 is 74% identical to zebrafish β2.2 (EU301442) but only 52% identical to zebrafish β2.1 (EU301434). In addition, DW608729 maps to a genomic contig, AANH01005391.1, which contains sequences homologous to zebrafish β2.2 exon 1 (encoding MFCCGLGHWRREQSTY) and β2.2 exon 2 (encoding MPPKKK)(Fig. 3). In the β2.2 gene(s), regions of high divergence encompass sequences both within and outside of inferred secondary structures [32]. Nevertheless, 19 of 21 β subunit residues noted for interaction with the α subunit AID are conserved in zebrafish β2.2 (see Additional File 4) [48-50]. High sequence divergence in β2.2 relative to other vertebrate β genes is reflected in the branch lengths on the phylogeny (Fig. 6). This pattern contrasts with that seen in the β4 group, where the two zebrafish paralogs have experienced similar rates of amino acid substitution.

Bottom Line: Moreover, phenotypes may be obscured by secondary effects of hypoxia.Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. amebert@lamar.colostate.edu

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.

Show MeSH
Related in: MedlinePlus