Figure 4.

D122A Mutation in Ca_V2.2 Prevents the Effect of α₂δ-1 on Ca_V2.2-HA Cell Surface Expression in Hippocampal Neurites and Somata

(A) Representative confocal images showing neurites of hippocampal neurons expressing Ca_V2.2-HA WT together with β1b and mCherry in the presence (top row) or absence (bottom row) of α₂δ-1. Expression of mCherry is shown in red (left). Ca_v2.2-HA (green, center) was stained using rat anti-HA Ab prior to permeabilization, and the rabbit II-III loop Ab (white, right) after permeabilization. DAPI was used to visualize the nucleus (blue). Scale bars, 50 μm.

(B) As for (A) but for hippocampal neurons co-expressing Ca_v2.2-HA D122A with β1b and mCherry in the presence (top row) or absence (bottom row) of α₂δ-1.

(C) Bar chart (mean ± SEM) showing expression of WT and D122A Ca_V2.2-HA, determined by both HA staining prior to permeabilization (green bars) and II-III loop staining after permeabilization (gray bars), together with the expression marker mCherry. Data for 197 (WT + α₂δ-1), 130 (WT − α₂δ-1), 174 (D122A + α₂δ-1), and 211 (D122A − α₂δ-1) neurites from 4 separate transfections in 2 experiments were normalized to the WT Ca_V2.2-HA + α₂δ-1 condition in each experiment. ^∗∗∗∗p < 0.0001 (1-way ANOVA compared with WT Ca_V2.2 + α₂δ-1, with Sidak’s post hoc analysis correcting for multiple comparisons).

(D) Representative confocal images showing hippocampal somata expressing Ca_V2.2-HA WT (top two rows) or Ca_v2.2-HA D122A (bottom two rows) together with β1b and mCherry in the presence (top row) or absence (bottom row, control) of α₂δ-1. Expression of mCherry is shown in red (first panel). Ca_v2.2-HA (green, second panel) was stained using rat anti-HA Ab in non-permeabilized cells, and the rabbit II-III loop Ab (white, third panel) after permeabilization. DAPI was used to visualize the nucleus (blue), and the merged image is shown in the fourth panel. Scale bars, 20 μm.

(E) Bar chart (mean ± SEM) showing expression of WT and D122A Ca_V2.2-HA, determined by HA staining prior to permeabilization (green bars), together with expression marker mCherry (red bars). Data for 32 (WT + α₂δ-1), 26 (WT - α₂δ-1), 37 (D122A + α₂δ-1), and 35 (D122A − α₂δ-1) cell bodies from 4 separate transfections in 2 experiments were normalized to the WT Ca_V2.2-HA + α₂δ-1 condition in each experiment. ^∗∗∗∗p < 0.0001 (1-way ANOVA compared with WT Ca_V2.2 + α₂δ-1, with Sidak’s post hoc analysis correcting for multiple comparisons).