Figure 9. Expression of RAP alone enhances Ca_V2.2 current density and Ca_V2.2 cell surface expression.

(a) Mean (±SEM) IV curves for I_Ba recorded from tsA-201 cells transfected with Ca_V2.2/β1b/α₂δ-1 (normalised to mean controls within each experiment), for control cells (open circles, n = 23) and cells transfected additionally with RAP (red circles, n = 25), from 2 independent experiments. The charge carrier is 1 mM Ba²⁺. (b) Comparison of normalised peak I_Ba density at +10 mV (mean ± SEM with individual data points) for control cells (open bar) and RAP-transfected cells (red bar). One data point was removed as an outlier (4.1-fold greater than the mean, P < 0.01 Grubb’s test). Statistical difference, Student’s t test: *P = 0.0193. (c) tsA-201 cells transfected with BBS-Ca_V2.2/β1b/HA-α₂δ-1 and cell-surface labelled with α-bungarotoxin AF-488. Top panel: control, transfected with empty vector; middle and bottom panels: co-transfected with 0.75 and 1.5 μg RAP, respectively. Scale bar 10 μm. (d) Bar chart (mean ± SEM) quantifying effect of the two RAP concentrations on cell surface BBS-Ca_V2.2 signal (blue bar, 0.75 μg RAP, n = 246; red bar, 1.5 μg RAP, n = 285), normalised to control BBS-Ca_V2.2 cell surface expression (open bar, n = 176), from three independent transfections, including that shown in (c). ****P < 0.0001, 1-way ANOVA and Bonferroni’s post-hoc test. (e) Example western blot from tsA-201 cells transfected with HA-α₂δ-1, without RAP (lane 1) or with RAP (0.5, 0.75 and 1.5 μg, lanes 2–4), showing WCL (left panel) and corresponding cell surface biotinylation (right panel) for HA-α₂δ-1. Ca_v2.2 and β1b subunits were co-transfected for consistency with the experiments shown in parts (a–d) of this figure, but were not visualised by antibody staining. (f) Bar chart (mean ± SEM) and individual data points, for 0.75 μg (blue bar, ○, n = 6) and 1.5 μg RAP (red bar, ∆, n = 3)), showing effect of RAP on α₂δ-1 cell surface expression determined by biotinylation from experiments including that in (e) normalised to control (open bar). *P = 0.0313, Wilcoxon’s matched pairs signed rank test. Combining all data, for both RAP concentrations, P < 0.0039 compared to control. Full blots for all figure parts are shown in Supplementary information.