The membrane-associated β2e-subunit of voltage-gated calcium channels translocates to the nucleus and regulates gene expression

Figures

• 

  FIGURE 1. Membrane-targeted Cavβ2e redistributes evenly between the cytosol and nucleus upon PLC activation. (A) Representative confocal fluorescence images of a stable Flp-In™ T-REx™-293 cell line expressing Cavβ2e fused to mRFP and stained with the Nucspot® nuclear marker. Cells were treated with the PLC agonist m-3M3FBS for the indicated time points. The fluorescence signal for Cavβ2e (panels a–e), for the nuclear marker (panels f–j), and merged images (panels k–o) are shown. The scale bar (15 µm) applies to all images. (B) Representative confocal fluorescence images of cells as in A but treated with DMSO as negative control. The fluorescence signal for Cavβ2e (panels a–e), for the nuclear marker (panels f–j), and merged images (panels k–o) are shown. The scale bar (15 µm) applies to all images (C) Plot of the colocalization analysis from (A, B), between Cavβ2e and the Nucspot® nuclear marker based on the Mander´s overlap coefficient (MOC). Values are presented as mean ± S.E. **p < 0.01 vs t = 0 min (two-tailed Student’s t-test).

• 

  FIGURE 2. Ca2+ mediates PLC-induced cellular redistribution of membrane-targeted Cavβ2e. (A) Confocal fluorescence images of tsA201 cells expressing Cavβ2e fused to YFP and untreated or treated with either ionomycin or DMSO (negative control) for 60 min. The fluorescence signal for Cavβ2e (panel a, d, g and j), DRAQ5™ nuclear marker (panel b, e, h and k), and merged images (panels c, f, i and l) are shown. The scale bar (15 µm) is applicable to all images. (B) Plot illustrating the colocalization analysis from panel A between Cavβ2e and the DRAQ5™ marker based on the Mander´s overlap coefficient (MOC). All values are presented as mean ± S.E. **p < 0.01 vs untreated wild-type Cavβ2e (two-tailed Student’s t-test).

• 

  FIGURE 3. Cavβ2e contains a NLS within the NTv domain. (A) Schematic representation of the domain structure of Cavβ2e. NT (N-terminus), SH3 (Src-Homology 3 domain), GK (Guanylate-kinase-like domain), and CT (C-terminus) are indicated. The 23-amino-acid-long sequence of the NTv domain of Cavβ2e that distinguishes Cavβ2e from all other Cavβ2 splice variants is shown. The residues Lys2 and Arg16, which, when mutated to alanine, mimic the effect of PLC activation, are highlighted in bold red. Positively charged amino acids identified as part of the nuclear localization signal are marked in bold blue. Alanine mutations performed to generate the constructs Cavβ2e-PLCmimic, Cavβ2e-ΔNLS, and Cavβ2e-PLCmimic-ΔNLS are also indicated. (B) Representative confocal fluorescence images of tsA201 cells expressing the indicated Cavβ2e mutants fused to YFP and stained with the DRAQ5™ nuclear marker. The fluorescence signal for Cavβ2e (panels a–c), the nuclear marker (panels d–f), and merged images (panels g–i) are shown. The scale bar (15 µm) applies to all images. (C) Plot of the colocalization analysis from panel B, showing the overlap between the Cavβ2e mutants and the DRAQ™ nuclear marker based on…

• 

  FIGURE 4. A NES at Cavβ2e-NTv domain is necessary to export the protein from the nucleus. (A) The 23-amino-acid-long sequence of the NTv region Cavβ2e, corresponding to the variable NTv domain that distinguishes Cavβ2e from all other Cavβ2 splice variants, is shown. The dileucine motif, identified as a nuclear export signal, is highlighted in bold orange. The residues Lys2 and Arg16, which, when mutated to alanine, mimic the effect of PLC activation, are highlighted in bold red. Positively charged amino acids identified as part of the nuclear localization signal are marked in bold blue. Alanine mutations in the constructs Cavβ2e-PLCmimic-L8A, Cavβ2e-PLCmimic-L9A, Cavβ2e-ΔNLS-L8A, and Cavβ2e-ΔNLS-L9A are indicated. (B) Representative confocal fluorescence images of tsA201 cells expressing the indicated Cavβ2e mutants fused to YFP and stained with the DRAQ5™ nuclear marker. The fluorescence images for Cavβ2e mutant proteins (panels a–d), the DRAQ5™ nuclear marker (panels e–h), and merged images (panels i–l) are shown. The scale bar (15 µm) applies to all images. (C) Plot of the colocalization analysis from panel B between the mutants Cavβ2e-PLCmimic-L8A, Cavβ2e-PLCmimic-L9A, and the DRAQ5™…

• 

  FIGURE 5. The dileucine motif at the NTv domain of Cavβ2e is involved in the nuclear export and the plasma membrane anchoring of the protein. (A) Representative confocal fluorescence images of tsA201 cells expressing the indicated Cavβ2e mutants fused to YFP and stained with the DRAQ5™ nuclear marker. The fluorescence images for Cavβ2e mutant proteins (panels a, d and g), DRAQ5™ nuclear marker (panels b, e, and h), and merged images (panels c, f, and i) are shown. The scale bar (15 µm) applies to all images. (B) Plot displaying the colocalization analysis results from panel A between the Cavβ2e mutants and the DRAQ5™ nuclear marker, based on Mander’s overlap coefficient (MOC). The green dashed line represents the average value obtained for the untreated wild-type Cavβ2e (WT), as shown in Figure 2B. (C) Representative SDS-PAGE gels from protein-liposome cosedimentation assays using wild-type and mutants Cavβ2e-NTv fused to GST (GST-Cavβ2e-NTv). Supernatant (S) and pellet (P) fractions after centrifugation of the corresponding GST-Cavβ2e-NTv protein in assay buffer only (Ø) or preincubated with either neutral (PC) or negatively charged (PS/PC) liposomes. Controls assays using the GST moiety…

• 

  FIGURE 6. Cavβ2e N-terminus suffices to control the plasma membrane-nuclear shuttling of the protein. (A) Representative confocal fluorescence images of tsA201 cells expressing either wild-type or the indicated mutant Cavβ2e-NTv domain fused to a three-tandem YFP molecules (YFP3). Cells expressing YFP3 alone were used as controls. The fluorescence signal for YFP3-fused proteins (panels a–d), nucleus (panels e–h), and merged images (panels i–l) are shown. The scale bar (15 µm) applies to all images. (B) Plot displaying the colocalization analysis from panel B between YFP3-fused proteins and the DRAQ5™ nuclear marker based on Mander’s overlap coefficient (MOC). Values are presented as mean ± S.E. **p < 0.01 vs. YFP3 (two-tailed Student’s t-test).

• 

  FIGURE 7. Quantitative MS-analysis. Volcano plot representing the results of a two-tailed Student’s t-test comparing 2,567 protein LFQ intensities between cells expressing Cavβ2e L9A (B2eL9A) and wild-type Cavβ2e (B2eWT). Among these, 459 proteins were regulated with p < 0.01 (shown in blue), and 8 proteins were significantly regulated (FDR-corrected p < 0.01, fold change (FC) > 2) (shown in red).

• 

  FIGURE 8. Schematic representation illustrating how PLC signaling potentially exerts a dual physiological effect via Cavβ2e. PLC activation (red arrows) leads to the hydrolysis of PIP2 molecules at the plasma membrane, generating IP3, which triggers Ca2+ release from the endoplasmic reticulum through IP3 receptors. This Ca2+ release diminishes the electrostatic interactions between the Cavβ2e membrane targeting domain at the NTv region (depicted as red “+++”) and lipids, resulting in two distinct outcomes. First, Cavβ2e molecules that are membrane-associated but not interacting with VGCC are released from the plasma membrane. They then translocate to the nucleus via an NLS at the NTv domain of the protein, and participate in the regulation of gene expression. Subsequently, they can exit the nucleus through an NES, also located within the NTv domain. Second, Cavβ2e molecules that interact with both the plasma membrane and VGCCs contribute to a slower inactivation rate of these channels. In this context, PLC-mediated release of the Cavβ2e-NTv domain from the plasma membrane leads to a faster inactivation rate of the VGCCs as reported by Kim et al. (2015b).