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.