Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Inhibition of Cav2.1 Ca2+ currents by the R1279X EA2-truncated form of Cav2.1 protein. A, Schematic cartoon representing the main Cav2.1 constructs used in this study. B, The Ca2+ current density in HEK293 cells expressing the Cav2.1 channel protein. The cells were cotransfected with plasmids encoding the human Cav2.1 subunit, the β1b subunit, and the α2/δ1 subunit. Mean ± SEM values of the current density for cells expressing the Cav2.1 subunit alone (white bar; n = 18) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22) are presented (***p < 0.001; Student's t test). The inset shows representative current traces at TP −40 and 0 mV [holding potential (HP), −80 mV]. C, Same experiments as in B performed in the neuroblastoma cell line NG108-15. The HP was −50 mV (***p < 0.001; Student's t test). Cav2.1 subunit alone (white bar; n = 15) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22). D, Measurements of native T-type and HVA Ca2+ current densities in R1279X-transfected NG108-15 cells. The T-current density (left) was measured in proliferative NG108-15 cells (Prolif.; 2–3 d after transfection; n = 9) and in differentiated NG108-15 cells (Diff.; 6 d after transfection; 3–4 d after switching to differentiation medium; n = 11) using HP −100 mV and TP −30 mV. The HVA current density, mainly corresponding to L- and N-type channel activities, was measured using HP −50 mV and TP 0 mV (right).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Inhibition, Construct, Expressing, Mutagenesis, Transfection

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Effect of R1279X mutant on surface expression of Cav2.1 channel. A, HEK293 cells were cotransfected with the HA-tagged wild-type Cav2.1 subunit alone (with free GFP) or together with the R1279X mutant (fused to GFP). Left column, GFP fluorescence. Middle column, Staining of cells with monoclonal rat anti-HA antibody (primary antibody) and Alexa594 (secondary antibody). NP, Nonpermeabilized; P, permeabilized. Images were acquired using a Leica SP2 confocal microscope, with a 63× oil immersion objective. B, Luminometric ELISA assays to quantify surface expression of the HA-tagged Cav2.1 in the presence of the R1279X mutant and auxiliary subunits (**p < 0.01, ***p < 0.001; Student's t test). C, Western blots performed on HEK293 cells expressing Cav2.1-HA alone (−) or in the presence of R1279X (+) without (left) or with (right) auxiliary subunits. β1b-HA was used in these experiments (right).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Mutagenesis, Expressing, Fluorescence, Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Channel misfolding and instability induced by the R1279X mutant. A, B, Pulse-chase experiments were performed on HEK293 cells 48 h after transfection (see Materials and Methods). Cells were labeled with 35S-methionine-cysteine for 20 min. The chase was performed for 0, 1, 2, or 4 h as indicated. After lysis, the Cav2.1 subunit was immunoprecipitated with an anti-HA antibody. The arrow indicates the band corresponding to the EA2 mutant that coimmunoprecipitates with the wild-type Cav2.1 subunit. In B (left), the β1b is detected (arrow). C, Quantification of three independent experiments using GE Healthcare software. D, Pulse-chase analyses performed on HEK293 cells transfected with CD4-AAXX alone or in the presence of R1279X. Quantification was performed as described above. E, Immunoprecipitations performed on cells coexpressing various subunit arrangements (as indicated) in the absence or presence of an untagged R1279X mutant (right) using standard SDS-PAGE (6%). Western blots (WBs) were performed with the indicated antibodies. F, Immunoprecipitations were performed between β1b-HA and GFP-R1279X as described in E. MW, Molecular weight.

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Mutagenesis, Pulse Chase, Transfection, Labeling, Lysis, Immunoprecipitation, Software, SDS Page, Western Blot, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: EA2 missense mutants act in a dominant-negative manner by misfolding and instability induction. A, Histograms of the mean Ca2+ current density (± SEM) obtained in a representative batch of NG108-15 cells expressing wild-type subunit alone (Cav2.1-HA, n = 18); the EA2 mutants alone (Cav2.1-HA-G293R, n = 33, 5 with detectable current; Cav2.1-HA-AY1593/94D, n = 6); and the wild-type and EA2 mutants together (Cav2.1-HA+Cav2.1-HA-G293R, n = 23, 3 with detectable current; Cav2.1-HA+Cav2.1-HA-AY1593/94D, n = 10). These experiments were conducted in the presence of the auxiliary β1b and α2/δ1 subunits. B, Normalized native T-type Ca2+ current densities (mean ± SEM) in the NG108-15 cells analyzed in A. The differences are not statistically significant between the various conditions tested (Student's t test). C, Western blots with the indicated antibodies (WB) were performed on HEK293 cells coexpressing Cav2.1-HA alone or in the presence of G293R and AY1593/94D mutants together with auxiliary subunits. The bottom panel shows the Western blot from cells expressing missense mutants alone. D, Pulse-chase analyses were performed on HEK293 cells transfected with wild-type Cav2.1 alone or in the presence of G293R. Quantification of three independents experiment was performed as described above. MW, Molecular weight.

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Dominant Negative Mutation, Expressing, Western Blot, Pulse Chase, Transfection, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Endoplasmic reticulum retention and proteasomal degradation of the dominant-negative Cav mutants. A, Confocal images of nonpermeabilized NG108-15 cells expressing EA2 mutants and truncated Cav3.2 subunits. Alexa 594-coupled CT was used as plasma membrane marker (0.5 μg/ml). B, Confocal images of immunofluorescence staining performed on permeabilized NG108-15 cells expressing indicated EA2 mutants and truncated Cav3.2. Polyclonal anti-protein disulfide isomerase (Assay Designs, Ann Arbor, MI) was used as ER marker. C, Pulse-chase experiments were performed as in Figure 3 on cells transfected with EA2 mutants (R1279X and G293R) and Cav3.2 truncated forms. Chase was done for the indicated time (hours) with or without MG-132 proteasome inhibitor (50 μm). After lysis, the truncated Cav channels were immunoprecipitated with anti-GFP antibody. D, Representative pulse chase performed on HEK293 cells transfected with Cav2.1 and the R1279X mutant and the corresponding quantification (n = 3). During the chase, cells were treated with MG-132 (50 μm), leupeptin (20 μm), and NH4Cl (10 mm).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Dominant Negative Mutation, Expressing, Marker, Immunofluorescence, Staining, Pulse Chase, Transfection, Lysis, Immunoprecipitation, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Stroke-Like Episodes and Cerebellar Syndrome in Phosphomannomutase Deficiency (PMM2-CDG): Evidence for Hypoglycosylation-Driven Channelopathy

doi: 10.3390/ijms19020619

Figure Lengend Snippet: N283Q glycosylation site mutation reduces Ca 2+ current density through Ca V 2.1 channels heterologously expressed in HEK293 cells, without altering their voltage-dependent activation. ( A ) Current traces elicited by 20 ms depolarizing pulses from −80 mV to the indicated voltages (inset) illustrating the decrease in Ca 2+ current density through Ca V 2.1 channels containing the mutation at the α 1A glycosylation site N283, which exhibit voltage dependence of activation similar to that of WT channels. Dotted lines mark the zero current level. Average Ca 2+ current density-voltage relationships ( B ) and normalized I-V curves ( C ) for WT (open circles, n = 13) and N283Q (filled dark cyan triangles, n = 19) Ca V 2.1 channels. N283Q reduces maximal Ca 2+ current density (obtained by membrane depolarization to +15 mV) from −69.4 ± 12.9 pA/pF ( n = 13) to −19.5 ± 3.2 pA/pF ( n = 19) ( p < 0.001, Mann-Whitney U -test). ( D ) N283Q mutation has no significant effect on the V 1/2 for Ca V 2.1 channel activation (estimated from normalized I-V curves shown in C as indicated in Materials and Methods, p = 0.798, Student’s t -test). ( E ) Current traces elicited by 20 ms depolarizing pulses from −80 mV to the indicated voltages (inset) illustrating the shift of activation to lower depolarization induced by 0.6 μg/mL tunicamycin on N283Q mutant Ca V 2.1 channels containing β 3 and α 2 δ 1 subunits. The zero current level is indicated by dotted lines. Average Ca 2+ current density-voltage relationships ( F ) and normalized I-V curves ( G ) for N283Q mutant Ca V 2.1 channels expressed in DMSO-treated cells (filled cyan triangles, n = 10) and in cells treated with 0.6 μg/mL tunicamycin (filled green circles, n = 8). Peak Ca 2+ current density through N283Q Ca V 2.1 channels after vehicle (DMSO) and tunicamycin treatments were −23.3 ± 5.6 pA/pF ( n = 10) and −11.6 ± 3.6 pA/pF ( n = 8), respectively ( p = 0.06, Student’s t -test). ( H ) Reduction in V 1/2 for activation of Ca V 2.1 channels composed by N283Q mutant α 1A , β 3 and α 2 δ 1 subunits (estimated from normalized I-V curves shown in ( G ) as indicated in Materials and Methods) produced by 0.6 μg/mL tunicamycin ( n = 8). ** p < 0.01 versus the control condition (vehicle, n = 10; Student’s t -test).

Article Snippet: Ca V 2.1 N283Q mutant channel was generated by site-directed mutagenesis of the human α 1A cDNA (GenScript Corporation, Piscatway, NJ, USA).

Techniques: Glycoproteomics, Mutagenesis, Activation Assay, Membrane, MANN-WHITNEY, Produced, Control

Journal: International Journal of Molecular Sciences

Article Title: Stroke-Like Episodes and Cerebellar Syndrome in Phosphomannomutase Deficiency (PMM2-CDG): Evidence for Hypoglycosylation-Driven Channelopathy

doi: 10.3390/ijms19020619

Figure Lengend Snippet: N283Q glycosylation site mutation lessens Ca V 2.1 channel inactivation. Ca 2+ current traces (normalized to the corresponding peak amplitude) illustrating differential inactivation of WT (black traces) and N283Q (cyan traces) Ca V 2.1 channels, in response to a 3 s depolarizing pulse to +20 mV ( A ) or 0 mV ( D ). Dotted lines indicate the zero current level. ( B , E ) Average Ca 2+ current inactivation (in %) at the end of these 3s depolarizing pulses obtained from HEK293 cells expressing either WT or N283Q Ca V 2.1 channels. Data are expressed as the mean ± SEM of the number of experiments shown in brackets (*** p < 0.001 and ** p < 0.01 versus WT, Mann-Whitney U -test). ( C , F ) Average τ inactivation values of Ca 2+ currents through WT (open bars) and N283Q (cyan bars) Ca V 2.1 channels expressed in HEK293 cells, elicited by a 3 s depolarizing pulse to +20 mV or 0 mV, as indicated. Data are expressed as the mean ± SEM of the number of experiments shown in brackets (*** p < 0.001 and ** p < 0.01 versus WT, Mann-Whitney U -test).

Article Snippet: Ca V 2.1 N283Q mutant channel was generated by site-directed mutagenesis of the human α 1A cDNA (GenScript Corporation, Piscatway, NJ, USA).

Techniques: Glycoproteomics, Mutagenesis, Expressing, MANN-WHITNEY

Journal: Cell Reports

Article Title: PRRT2 modulates presynaptic Ca 2+ influx by interacting with P/Q-type channels

doi: 10.1016/j.celrep.2021.109248

Figure Lengend Snippet:

Article Snippet: Anti - Ca v 2.1 alpha-1A Rabbit Polyclonal , Synaptic Systems , Cat. #152203; RRID: AB_2619841.

Techniques: Recombinant, Saline, Protease Inhibitor, BIA-KA, Bradford Assay, Magnetic Beads, Cell Culture, Software