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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 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.
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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.

Mentions: Alternative splicing also occurs internally for one zebrafish β2 subunit gene. β2.1 encodes three alternatively spliced HOOK domain exons (exons 7, 8 and 9; Figs. 1 and 4). The high conservation of β2.1 exons 8 and 9 with mammalian counterparts (Fig. 2A, lines 15 and 16), and exon 8 with several other teleost genes (Fig. 4), suggests that these internal sequences could have functional relevance. The β2.1 exon 7, which appears to be unique to zebrafish, includes a premature in-frame stop codon expected to truncate the protein in the HOOK domain (Fig. 2A, line 14). In β2.1 transcripts that contain both exon 8 and 9, the reading frame is altered such that a premature in-frame stop codon truncates the protein in exon 10.


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

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

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.
Mentions: Alternative splicing also occurs internally for one zebrafish β2 subunit gene. β2.1 encodes three alternatively spliced HOOK domain exons (exons 7, 8 and 9; Figs. 1 and 4). The high conservation of β2.1 exons 8 and 9 with mammalian counterparts (Fig. 2A, lines 15 and 16), and exon 8 with several other teleost genes (Fig. 4), suggests that these internal sequences could have functional relevance. The β2.1 exon 7, which appears to be unique to zebrafish, includes a premature in-frame stop codon expected to truncate the protein in the HOOK domain (Fig. 2A, line 14). In β2.1 transcripts that contain both exon 8 and 9, the reading frame is altered such that a premature in-frame stop codon truncates the protein in exon 10.

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