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BMC Molecular Biology

, 9:38

First Online: 17 April 2008Received: 24 December 2007Accepted: 17 April 2008

Abstract

BackgroundCardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β 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 β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts.

ResultsCloning of two zebrafish β2 subunit genes β2.1 and β2.2 indicated they are membrane-associated guanylate kinase MAGUK-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the -core- of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 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 β2 subunit gene loci. Comparative analysis of the teleost and human β2 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 β2 transcript variants is detected in the embryonic heart compared to the adult.

ConclusionThese studies refine our understanding of β2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of β2 subunit diversity in the embryonic heart.

AbbreviationsReverse-transcriptase polymerase chain reaction RT-PCRmembrane-associated guanylate-kinase protein MAGUK, voltage-dependent L-type Ca channel V-LTCC, rapid amplification of cDNA ends RACE, basepairs bp, cardiac myosin light chain cmlc, green fluorescent protein GFP, days post-fertilization dpf, hours post-fertilization hpf, maximum parsimony bootstrap proportion MPBP, maximum likelihood quartet puzzling MLQP.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2199-9-38 contains supplementary material, which is available to authorized users.

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Autor: Alicia M Ebert - Catherine A McAnelly - Ashok Srinivasan - Rachel Lockridge Mueller - David B Garrity - Deborah M Garri

Fuente: https://link.springer.com/







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