Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2365960&req=5

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.

Mentions: TBLASTN searches of the GenBank database at NCBI using human β2 subunit sequences suggested that zebrafish encode two β2 homologues [43]. We therefore designed primers to highly conserved sequences within the SH3 and GK protein domains, and performed 5' and 3' RACE-PCR on RNA extracted from embryos aged 1–3 dpf. Using RACE and reverse-transcriptase PCR (RT-PCR), we isolated cDNAs representing two zebrafish β2 genes, termed β2.1 and β2.2 (see Figs. 1, 2 and Additional File 1). The β2.1 gene showed a near perfect match with genomic sequences located within Genbank zebrafish chromosome 22 (NC_007133.1), whereas β2.2 matched genomic sequences found on chromosome 2 (NC_007113.1) [43-45]. Alternative splicing occurred at the N-terminus for both genes and within the HOOK domain for β2.1 (see Figs. 1 and 2D for descriptions of transcript variants). In the initial RACE analysis for β2.1, we recovered four β2.1_tv1 clones, and five β2.1_tv6 clones, suggesting these may be the most abundant transcript variants. One clone each was found for the β2.1_tv2, 3, 4, 5, 7 and 8 transcripts, suggesting they may be less abundant. For β2.2, we recovered two β2.2_tv1 and three β2.2_tv2 5' RACE clones. In additional RT-PCR analysis using primers closely flanking the HOOK domain, we confirmed that β2.1 transcript variants containing more than one exon between exons 6 and 10 (i.e. β2.1_tv3, 4, 7 and 8) could be detected in the RNA samples (see Additional file 2). Conversely, we confirmed that no alternative splicing occurred in the β2.2 HOOK domain (see Additional File 2). Several 3' RACE clones were of a single variant for β2.1 and a single variant for β2.2, suggesting that no alternative splicing occurred in the GK or C-terminal regions of the genes.


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)

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

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

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.
Mentions: TBLASTN searches of the GenBank database at NCBI using human β2 subunit sequences suggested that zebrafish encode two β2 homologues [43]. We therefore designed primers to highly conserved sequences within the SH3 and GK protein domains, and performed 5' and 3' RACE-PCR on RNA extracted from embryos aged 1–3 dpf. Using RACE and reverse-transcriptase PCR (RT-PCR), we isolated cDNAs representing two zebrafish β2 genes, termed β2.1 and β2.2 (see Figs. 1, 2 and Additional File 1). The β2.1 gene showed a near perfect match with genomic sequences located within Genbank zebrafish chromosome 22 (NC_007133.1), whereas β2.2 matched genomic sequences found on chromosome 2 (NC_007113.1) [43-45]. Alternative splicing occurred at the N-terminus for both genes and within the HOOK domain for β2.1 (see Figs. 1 and 2D for descriptions of transcript variants). In the initial RACE analysis for β2.1, we recovered four β2.1_tv1 clones, and five β2.1_tv6 clones, suggesting these may be the most abundant transcript variants. One clone each was found for the β2.1_tv2, 3, 4, 5, 7 and 8 transcripts, suggesting they may be less abundant. For β2.2, we recovered two β2.2_tv1 and three β2.2_tv2 5' RACE clones. In additional RT-PCR analysis using primers closely flanking the HOOK domain, we confirmed that β2.1 transcript variants containing more than one exon between exons 6 and 10 (i.e. β2.1_tv3, 4, 7 and 8) could be detected in the RNA samples (see Additional file 2). Conversely, we confirmed that no alternative splicing occurred in the β2.2 HOOK domain (see Additional File 2). Several 3' RACE clones were of a single variant for β2.1 and a single variant for β2.2, suggesting that no alternative splicing occurred in the GK or C-terminal regions of the genes.

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