Genomic organization, expression, and phylogenetic analysis of Ca2+ channel beta4 genes in 13 vertebrate species.

The Ca(2+) channel beta-subunits, encoded by CACNB genes 1-4, are membrane-associated guanylate kinase (MAGUK) proteins. As auxiliary subunits of voltage-gated Ca(2+) channels, the beta-subunits facilitate membrane trafficking of the pore-forming alpha1 subunits and regulate voltage-dependent channel gating. In this report, we investigate whether two zebrafish beta4 genes, beta4.1 and beta4.2, have diverged in structure and function over time. Comparative expression analyses indicated that beta4.1 and beta4.2 were expressed in separable domains within the developing brain and other tissues. Alternative splicing in both genes was subject to differential temporal and spatial regulation, with some organs expressing different subsets of beta4.1 and beta4.2 transcript variants. We used several genomic tools to identify and compare predicted cDNAs for eight teleost and five tetrapod beta4 genes. Teleost species had either one or two beta4 paralogs, whereas each tetrapod species contained only one. Teleost beta4.1 and beta4.2 genes had regions of sequence divergence, but compared with tetrapod beta4s, they exhibited similar exon/intron structure, strong conservation of residues involved in alpha1 subunit binding, and similar 5' alternative splicing. Phylogenetic results are consistent with the duplicate teleost beta4 genes resulting from the teleost whole genome duplication. Following duplication, the beta4.1 genes have evolved faster than beta4.2 genes. We identified disproportionately large second and third introns in several beta4 genes, which we propose may provide regulatory elements contributing to their differential tissue expression. In sum, both mRNA expression data and phylogenetic analysis support the evolutionary divergence of beta4.1 and beta4.2 subunit function.

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

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.