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Diversity and developmental expression of L-type calcium channel beta2 proteins and their influence on calcium current in murine heart.

ABSTRACT: By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVbeta2 proteins are the major CaVbeta components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVbeta3 proteins are also detectable. At least two CaVbeta2 variants of approximately 68 and approximately 72 kDa are expressed. To identify the underlying CaVbeta2 variants, cDNA libraries were constructed from poly(A)(+) RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVbeta2 cDNA clones coding for four types of CaVbeta2 proteins only differing in their 5' sequences. CaVbeta2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVbeta2 protein pattern in vitro. Significant L-type Ca(2+) currents (I(Ca)) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVbeta2. Current kinetics were determined by the type of CaVbeta2 protein, with the approximately 72-kDa CaVbeta2a-N1 shifting the activation of I(Ca) significantly to depolarizing potentials compared with the other CaVbeta2 variants. Inactivation of I(Ca) was accelerated by CaVbeta2a-N1 and -N4, which also lead to slower activation compared with CaVbeta2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVbeta2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVbeta2a-N1-induced changes of I(Ca) kinetics might be essential in embryonic heart.

SUBMITTER: Link S

PROVIDER: S-EPMC2781568 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Diversity and developmental expression of L-type calcium channel beta2 proteins and their influence on calcium current in murine heart.

Link Sabine S Meissner Marcel M Held Brigitte B Beck Andreas A Weissgerber Petra P Freichel Marc M Flockerzi Veit V

The Journal of biological chemistry 20090901 44

By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVbeta2 proteins are the major CaVbeta components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVbeta3 proteins are also detectable. At least two CaVbeta2 variants of approximately 68 and approximately 72 kDa are expressed. To identify the underlying CaVbeta2 variants, cDNA libraries were constructed from poly(A)(+) RNA isolated from hearts of 7-day-old and ad ...[more]

PMID: 19723630

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Project description:BackgroundCardiomyocyte 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.ResultsCloning 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.ConclusionThese 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.

Project description:Sinoatrial node cardiomyocytes (SANcm) possess automatic, rhythmic electrical activity. SAN rate is influenced by autonomic nervous system input, including sympathetic nerve increases of heart rate (HR) via activation of β-adrenergic receptor signaling cascade (β-AR). L-type calcium channel (LTCC) activity contributes to membrane depolarization and is a central target of β-AR signaling. Recent studies revealed that the small G-protein Rad plays a central role in β-adrenergic receptor directed modulation of LTCC. These studies have identified a conserved mechanism in which β-AR stimulation results in PKA-dependent Rad phosphorylation: depletion of Rad from the LTCC complex, which is proposed to relieve the constitutive inhibition of CaV1.2 imposed by Rad association. Here, using a transgenic mouse model permitting conditional cardiomyocyte selective Rad ablation, we examine the contribution of Rad to the control of SANcm LTCC current (ICa,L) and sinus rhythm. Single cell analysis from a recent published database indicates that Rad is expressed in SANcm, and we show that SANcm ICa,L was significantly increased in dispersed SANcm following Rad silencing compared to those from CTRL hearts. Moreover, cRadKO SANcm ICa,L was not further increased with β-AR agonists. We also evaluated heart rhythm in vivo using radiotelemetered ECG recordings in ambulating mice. In vivo, intrinsic HR is significantly elevated in cRadKO. During the sleep phase cRadKO also show elevated HR, and during the active phase there is no significant difference. Rad-deletion had no significant effect on heart rate variability. These results are consistent with Rad governing LTCC function under relatively low sympathetic drive conditions to contribute to slower HR during the diurnal sleep phase HR. In the absence of Rad, the tonic modulated SANcm ICa,L promotes elevated sinus HR. Future novel therapeutics for bradycardia targeting Rad - LTCC can thus elevate HR while retaining βAR responsiveness.

Dataset Information

Diversity and developmental expression of L-type calcium channel beta2 proteins and their influence on calcium current in murine heart.

Publications

Diversity and developmental expression of L-type calcium channel beta2 proteins and their influence on calcium current in murine heart.

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