Project description:To find Cacnb3-dependent transcriptomic changes after eATP stimulation, we performed RNA-sequening of WT and Cacnb3-KO BMCs in steady-state and after eATP stimulation.
Project description: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
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:Fungal infections, especially candidiasis and aspergillosis, claim an unacceptably high fatality rate. The energy ATP that is necessary for fungal cell growth and function is synthesized mainly through oxidative phosphorylation, with the key enzyme being F1Fo-ATP synthase. But it remains unknown how this enzyme affects fungal pathogenicity. Here, we show that F1Fo-ATP synthase δ subunit deletion abrogates lethal Candida albicans infection without affecting intracellular ATP concentrations or growth. Mechanistically, δ subunit deletion reduces Pfk1 activity by interrupting Pfk1 phosphorylation to trigger its conformation shifts, decreases downstream FBP level, blocks Ras1-dependent and -independent cAMP-PKA pathways, and curtails virulence factors. Based on these findings, we engineer a small molecule compound targeting δ subunit that effectively protects mice from succumbing to invasive candidiasis. In summary, our findings reveal that F1Fo-ATP synthase δ subunit determines lethal infection from pathogenic fungi and represents a potential therapeutic target.
Project description:L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Cav1 pore-forming subunit and the accessory subunits Cav, Cav2 and Cav. Cav is a cytosolic soluble protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failure, depends on the activation of calcium-dependent pro-hypertrophic signaling cascades; however, the role of LTCCs in this pathology remains controversial. Here, by using shRNA-mediated Cav silencing, we demonstrate that Cav2 downregulation enhances 1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy in an LTCC-independent manner. We report that a pool of Cav2 is targeted to the nucleus in cardiomyocytes and that the expression of this nuclear fraction decreases during in vitro and in vivo induction of cardiac hypertrophy. Moreover, the overexpression of nucleus-targeted Cav2 in cardiomyocytes inhibits in vitro-induced hypertrophy. Quantitative proteomic analyses showed that Cav2 knockdown leads to changes in the expression of diverse myocyte proteins, including reduction of calpastatin, an endogenous inhibitor of the calcium-dependent protease calpain. Accordingly, Cav2-deficient cardiomyocytes had a two-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cav2-deficient cells abolished the enhanced 1-adrenergic receptor agonist-induced hypertrophy observed in these cells. Our findings indicate that in cardiomyocytes, a nuclear pool of Cav2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cav2 during cardiac hypertrophy promotes the activation of calpain-dependent hypertrophic pathways.
Project description:We performed RNA-Seq analysis of neoatal rat ventricular cardiomyocytes (NRVCs) and human pluripotent stem cells derived cardiomyocytes (hPSC-CMs) which were treated with Nimodipine (NM) to investigate the moleclular mechanism of inhibiting L-type calcium channel (LTCC) to promote cardiomyocyte proliferation.
Project description:Pheochromocytoma (PCC) and abdominal paraganglioma (aPGL) (together abbreviated PPGL) frequently present with an underlying genetic event in a PPGL driver gene, and additional susceptibility genes are anticipated. Here we re-analysed whole-exome sequencing data for PCC patients, and identified two patients with very rare missense variants in the calcium voltage-gated channel subunit 1H gene (CACNA1H). CACNA1H variants were also found in the clinical setting in PCC patients using targeted sequencing and analysis of The Cancer Genome Atlas database. In total, CACNA1H variants were found in 7 PCC cases. Four of these were constitutional and two are known to have functional consequences on hormone production and gene expression in primary aldosteronism and aldosterone-producing adrenocortical adenoma. In general, PPGL exhibited reduced CACNA1H mRNA expression as compared to normal adrenal. Immunohistochemistry showed strong CACNA1H (CaV3.2) staining in adrenal medulla while PPGL typically had weak staining or were negative. Furthermore, reduced CACNA1H gene expression was especially pronounced in PCC as compared to aPGL, in cases with normal norepinephrine levels and in PPGL with Cluster 2 kinase signalling phenotype. Moreover, TCGA data revealed a correlation between CACNA1H methylation density and gene expression. Expression of rCacna1h in PC12 cells induced differential protein expression profiles determined by mass spectrometry as well as a shift in the membrane potential where maximum calcium currents were observed as determined by electrophysiology. The findings add a possible novel genetic event to the growing palette of PPGL susceptibility genes and establish a potential link between the aetiology of adrenomedullary and adrenocortical tumor development.
Project description:The Moonwalker (Mwk) mouse is a model of dominantly inherited cerebellar ataxia caused by a gain-of-function mutation in the transient receptor potential (TRP) channel TRPC3. We report impairments in dendritic growth and synapse formation early on during Purkinje cell development in the Mwk cerebellum that are accompanied by alterations in calcium signaling.
Project description:Crohn’s disease is a chronic, debilitating inflammatory bowel disease. Here we report a critical role for phospholipase C-β3 (PLC-β3) in intestinal homeostasis. In PLC-β3- deficient mice, exposure to oral dextran sodium sulfate induced lethality and severe inflammation in the small intestine. The lethality was due to PLC-β3 deficiency in multiple non-hematopoietic cell types. PLC-β3 deficiency resulted in reduced Wnt/β-catenin signaling, which is essential for homeostasis and regeneration of the intestinal epithelium. PLC-β3 regulated the Wnt/β-catenin pathway in small intestinal epithelial cells (IECs) at transcriptional, epigenetic, and potentially protein-protein interaction levels. PLC-β3- deficient IECs were unable to respond to stimulation by R-spondin 1, an enhancer of Wnt/β-catenin signaling. Reduced expression of PLC-β3 and its signature genes was found in biopsies of ileal Crohn’s disease patients. PLC-β regulation of Wnt signaling was evolutionally conserved in Drosophila. Our data indicate that reduction of PLC-β3- mediated Wnt/β-catenin signaling contributes to the pathogenesis of ileal Crohn’s disease.