(a) Images showing cell-surface CaV2.2-BBS (upper row, white), and total CaV2.2 (II-III loop Ab, lower row, red), for CaV2.2-BBS/β1b in N2A cells, with empty vector (panel 1) or α2δ-3-HA (panel 2). Scale bar 5 µm. (b) Quantification (box and whisker plots) of effect α2δ-3 on cell-surface CaV2.2-BBS following expression of CaV2.2-BBS/β1b with empty vector (open bar, n = 188) or WT α2δ-3 (red bar, n = 188). Statistical difference determined by Student’s t test, **p=0.0028. (c) Alignment of α2δ-3 sequence around the predicted cleavage site with α2δ-1, showing weak homology. Underlined sequence (MTAKAQ) mutated to V6 or HRV-3C cleavage motif. (d) α2δ-3-HA (lanes 1, 2) and α2(3C)δ-3-HA (lanes 3, 4) expressed in tsA-201 cells, with either inactive (C147V, lanes 1, 3) or WT 3C-protease (WT, lanes 2, 4), cell-surface biotinylated and deglycosylated. Full WB and corresponding WCL in Figure 4—figure supplement 3 (e) Quantification of cell-surface expression of WT α2δ-3-HA (red speckled bar) and α2(3C)δ-3-HA (blue speckled bar), with 3C-protease, normalized relative to inactive 3C-protease (C147V) for n = 3 experiments. Data are mean (± SEM) and individual data points: p=0.4721 for WT α2δ-3-HA and p=0.9513 for α2(3C)δ-3 (1 sample t-test compared to respective control). (f) Example traces (−30 to +5 mV steps) for CaV2.2/β1b-GFP with no α2δ (black traces), WT α2δ-3 (red traces) or α2(3C)δ-3 (blue traces). Gmax: 0.24 ± 0.03, 1.46 ± 0.22 and 0.21 ± 0.03 nS/pF respectively. V50,act: 0.91 ± 1.015, 1.01 ± 0.85 and 4.03 ± 1.04 mV, respectively. (g) Example traces (−30 to +10 mV steps) for CaV2.2/β1b-GFP/α2(3C)δ-3 and no protease (black traces), 3C-protease (blue traces) or inactive 3C-protease (C147V) (cyan traces). For (f) and (g), charge carrier 1 mM Ba2+, scale bars refer to all traces. (h) Mean (± SEM) IV curves for CaV2.2/β1b-GFP/α2(3C)δ-3 without protease (black open circles, n = 28), with 3C-protease (blue squares, n = 29) or inactive 3C-(C147V)-protease (cyan triangles, n = 24). Gmax: 0.28 ± 0.05, 0.70 ± 0.11 and 0.30 ± 0.08 nS/pF, respectively. Gmax for 3C-protease condition larger than other two conditions (Kruskal-Wallis test with Dunn’s post-hoc test, p<0.05). V50, act: 4.0 ± 0.7, 0.3 ± 0.7 and 1.5 ± 0.6 mV, respectively.