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Differential neuronal targeting of a new and 2 known calcium channel β4 subunit splice variants correlates with their regulation of gene expression

ABSTRACT: The β subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits, and thus contribute to neuronal excitability, neurotransmitter release and calcium-induced gene regulation. In addition certain β subunits are targeted into the nucleus, where they directly interact with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β4 subunit lacking the variable N-terminus (β4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGC) and modulates P/Q-type calcium currents in tsA cells and CaV2.1 surface expression in neurons. Compared to the other two known full-length β4 variants (β4a, β4b) β4e is most abundantly expressed in the distal axon, but lacks nuclear targeting properties. To examine the importance of nuclear targeting of β4 subunits for transcriptional regulation, we performed whole genome expression profiling of CGCs from lethargic mice individually reconstituted with β4a, β4b, and β4e. Notably, the number of genes regulated by each β4 splice variant correlated with the rank order of their nuclear targeting properties (β4b> β4a> β4e). Together these findings support isoform-specific functions of β4 splice variant in neurons, with β4b playing a dual role in channel modulation and gene regulation, while the newly detected β4e variant serves exclusively in calcium channel-dependent functions. We used microarrays to identify gene expression changes caused by β4 splice variants (β4a, β4b and β4e) of the voltage gated calcium channel in cultured cerebellar granule cells of lethargic mice

ORGANISM(S): Mus musculus

PROVIDER: GSE50822 | GEO | 2013/12/31

SECONDARY ACCESSION(S): PRJNA219091

REPOSITORIES: GEO

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Project description:The M-NM-2 subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 M-NM-11 subunits, and thus contribute to neuronal excitability, neurotransmitter release and calcium-induced gene regulation. In addition certain M-NM-2 subunits are targeted into the nucleus, where they directly interact with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of M-NM-2 isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual M-NM-2 variants in specific neuronal functions. In the present study, an alternatively spliced M-NM-24 subunit lacking the variable N-terminus (M-NM-24e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGC) and modulates P/Q-type calcium currents in tsA cells and CaV2.1 surface expression in neurons. Compared to the other two known full-length M-NM-24 variants (M-NM-24a, M-NM-24b) M-NM-24e is most abundantly expressed in the distal axon, but lacks nuclear targeting properties. To examine the importance of nuclear targeting of M-NM-24 subunits for transcriptional regulation, we performed whole genome expression profiling of CGCs from lethargic mice individually reconstituted with M-NM-24a, M-NM-24b, and M-NM-24e. Notably, the number of genes regulated by each M-NM-24 splice variant correlated with the rank order of their nuclear targeting properties (M-NM-24b> M-NM-24a> M-NM-24e). Together these findings support isoform-specific functions of M-NM-24 splice variant in neurons, with M-NM-24b playing a dual role in channel modulation and gene regulation, while the newly detected M-NM-24e variant serves exclusively in calcium channel-dependent functions. We used microarrays to identify gene expression changes caused by M-NM-24 splice variants (M-NM-24a, M-NM-24b and M-NM-24e) of the voltage gated calcium channel in cultured cerebellar granule cells of lethargic mice Cultured cerebellar granule cells from lethargic (129/SvJ background) mice reconstituted with the M-NM-24 splice variants (M-NM-24a, M-NM-24b and M-NM-24e) were compared to eGFP transfected controls

Project description:Voltage gated calcium channels play a central role in regulating the electrical and biochemical properties of neurons and muscle cells. Cells coordinate the expression of voltage gated calcium channels with the expression of other proteins that regulate membrane potential and calcium homeostasis. We report that the C-terminus of CaV 1.2, an L-type calcium channel (LTC) contains a C-terminal fragment that translocates to the nucleus and regulates transcription. This calcium channel associated transcription factor (CCAT) associates with transcriptional co-regulators such as p54nrb/NonO and binds to endogenous promoters. CCAT regulates the expression of gap junctions, sodium calcium exchangers, NMDA receptors, potassium channels and other proteins that regulate neuronal signaling. Electrical activity and developmental processes regulate the nuclear localization of CCAT, suggesting that the CCAT integrates information about the number of LTCs with information about the developmental history and electrical activity of a cell. These findings provide the first evidence that voltage gated calcium channels can directly activate transcription and suggest a novel mechanism linking voltage gated channels to the function and differentiation of excitable cells. Experiment Overall Design: The goal of these experiments was to identify the genes that are regulated by CCAT, a novel transcription factor derived from the C-terminus of CaV1.2. Neuro2A neuroblastoma cells were transfected with the last 503 AA of CaV1.2 which is full length CCAT (CCAT FL) or with the last 280 AA of CaV1.2, a form of CCAT that lacks the transcriptional activation domain (CCAT DTA). The mRNA from either CCAT FL or CCAT DTA expressing cells was hybridized to Agilent mouse genome microarrays along with mRNA from untransfected neuro2A cells (CCAT FL or DTA A series). Subsequent investigation revealed that transfection with the PA1 plasmid by itself increases the expression of some genes. To control for this effect we compared Neuro2A cells transfected with full length CCAT (in PA1) with Neuro2A cells transfectected with PA1 alone (CCAT FL B series). The microarray data was analyzed with the Rossetta Luminator gene expression data analysis system.

Project description:Expression data from the hippocampus of epileptic mice carrying two different pathogenic de novo mutations in the hyperpolarization-activated, cyclic nucleotide-gated, non-selective cation channel gene Hcn1 (p.G391D in human corresponding to G380D in mouse; p.M153I in human corresponding to M142I in mice) in comparison to wildtype controls. De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo mutations in the HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Animals replicating the p.G391D variant had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from patients with pathogenic HCN1 sequence variations, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an effect to further impair inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the need to tailor effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool towards reaching this objective. This dataset contains a comparison of the gene expression profile of the hippocampus of epileptic mice carrying either the pathogenic G380D (GD) variant (Hcn1GD/+ mice), or the M142I (MI) variant (Hcn1MI/+ mice) in Hcn1 in comparison to wildtype (WT) controls (Hcn1+/+ mice).

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Differential neuronal targeting of a new and 2 known calcium channel β4 subunit splice variants correlates with their regulation of gene expression

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