Figure 3.

α₂δ-1^G209D does not enhance Ca_V2.2 expression at the cell surface in tsA-201 cells and hippocampal neurons. (A) Representative confocal images of tsA-201 cells transfected with GFP_Ca_V2.2-HA with either α₂δ-1 wild-type (WT) (without tag; top row), empty vector (no α₂δ, middle row) or α₂δ-1^G209D (without tag; bottom row). HA staining was performed in non-permeabilized conditions (white, left), GFP signal shows total Ca_V2.2 (middle). The right panels represent the merged images (HA is shown in red). Scale bar = 20 μm. (B) Bar charts (mean ± SEM with individual data-points representing the mean of at least 50 cells from 12 coverslips) show Ca_V2.2 expressed at the cell surface (HA signal, grey bars) or total Ca_V2.2 (GFP, green bars) in the presence of α₂δ-1 WT, empty vector (no α₂δ-1) or α₂δ-1^G209D. Data obtained from four independent experiments; ns, P > 0.05; ***P = 0.0007, ****P < 0.0001, one-way ANOVA and Bonferroni post hoc test. (C) Representative confocal images of hippocampal somata imaged at ×63 objective and transfected with GFP_Ca_V2.2-HA and either α₂δ-1 WT (top row), empty vector (no α₂δ, middle row) or α₂δ-1^G209D (bottom row) together with β1b and mCherry. HA staining was performed in non-permeabilized conditions (grey, left), GFP signal (middle) and mCherry (transfection marker, right). Scale bar = 10 μm. (D) Bar charts (mean ± SEM with individual data-points) of HA (grey bars) and GFP (green bars) for α₂δ-1 WT (n = 102), no α₂δ (n = 63) and α₂δ-1^G209D (n = 82). Data obtained from three independent experiments; the values are normalized to α₂δ-1 WT condition in each experiment. ns, P > 0.05; ****P < 0.0001, one-way ANOVA and Bonferroni post hoc test. (E) Representative confocal images of hippocampal neurons imaged at ×20 objective and transfected with GFP_Ca_V2.2-HA and either α₂δ-1 WT (top row), empty vector (no α₂δ, middle row) or α₂δ-1^G209D (bottom row) together with β1b and mCherry. HA staining was performed in non-permeabilized conditions (grey, left panels), GFP signal (middle) and mCherry (transfection marker, right). Scale bar = 50 μm. (F) Bar charts (mean ± SEM with individual data-points) of HA (grey bars) and GFP (green bars) for α₂δ-1 WT (n = 114), no α₂δ (n = 50) and α₂δ-1^G209D (n = 117). Data obtained from three independent experiments; the values are normalized to α₂δ-1 WT condition in each experiment. ns, P > 0.05; ****P < 0.0001, one-way ANOVA and Bonferroni post hoc test. Note that for B, D and F, total Ca_V2.2 (GFP) was reduced in both the α₂δ-1^G209D and no α₂δ conditions compared to α₂δ-1 WT condition probably because of its increased degradation when Ca_V2.2 is poorly trafficked out of the endoplasmic reticulum.