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Activity and calcium regulate nuclear targeting of the calcium channel beta4b subunit in nerve and muscle cells.

ABSTRACT: Auxiliary beta subunits are critical determinants of membrane expression and gating properties of voltage-gated calcium channels. Mutations in the beta(4) subunit gene cause ataxia and epilepsy. However, the specific function of beta(4) in neurons and its causal relation to neurological diseases are unknown. Here we report the localization of the beta(4) subunit in the nuclei of cerebellar granule and Purkinje cells. beta(4b) was the only beta isoform showing nuclear targeting when expressed in neurons and skeletal myotubes. Its specific nuclear targeting property was mapped to an N-terminal double-arginine motif, which was necessary and sufficient for targeting beta subunits into the nucleus. Spontaneous electrical activity and calcium influx negatively regulated beta(4b) nuclear localization by a CRM-1-dependent nuclear export mechanism. The activity-dependent shuttling of beta(4b) into and out of the nucleus indicates a specific role of this beta subunit in neurons, in communicating the activity of calcium channels to the nucleus.

SUBMITTER: Subramanyam P

PROVIDER: S-EPMC2853709 | biostudies-literature | 2009 Sep-Oct

REPOSITORIES: biostudies-literature

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Activity and calcium regulate nuclear targeting of the calcium channel beta4b subunit in nerve and muscle cells.

Subramanyam Prakash P Obermair Gerald J GJ Baumgartner Sabine S Gebhart Mathias M Striessnig Jörg J Kaufmann Walter A WA Geley Stephan S Flucher Bernhard E BE

Channels (Austin, Tex.) 20090903 5

Auxiliary beta subunits are critical determinants of membrane expression and gating properties of voltage-gated calcium channels. Mutations in the beta(4) subunit gene cause ataxia and epilepsy. However, the specific function of beta(4) in neurons and its causal relation to neurological diseases are unknown. Here we report the localization of the beta(4) subunit in the nuclei of cerebellar granule and Purkinje cells. beta(4b) was the only beta isoform showing nuclear targeting when expressed in ...[more]

PMID: 19755859

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Project description:BACKGROUND:Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K+ channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. METHODS:We transduced C2C12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student's t test or two-way ANOVAs and SAS software as indicated. RESULTS:Expression of human ERG1A in C2C12 myotubes increased basal intracellular calcium concentration 51.7% (p?<?0.0001; n?=?177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p?<?0.08; n?=?24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p?<?0.05; n?=?6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p?<?0.05; n?=?6) in calpastatin which could contribute to enhanced calpain activity. CONCLUSIONS:Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

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.

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Activity and calcium regulate nuclear targeting of the calcium channel beta4b subunit in nerve and muscle cells.

Publications

Activity and calcium regulate nuclear targeting of the calcium channel beta4b subunit in nerve and muscle cells.

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