CBD3063 disrupts Cav2.2–CRMP2 binding and impairs membrane trafficking of Cav2.2 channels without effecting CRMP2 phosphorylation
We previously reported that tat-CBD3 (Brittain et al., 2011b) and myr-tat-CBD3 (Francois-Moutal et al., 2015) peptides inhibit the Cav2.2–CRMP2 interaction and reduced surface expression of Cav2.2. Therefore, we next asked if CBD3 peptidomimetic CBD3063 could interfere with Cav2.2–CRMP2 binding and affect the membrane localization of Cav2.2 channels. For this, catecholamine A differentiated (CAD) cells – a mouse neuronal cell line expressing both CRMP2 and Cav2.2 – were incubated overnight with 0.1% DMSO or 20 µM CBD3063. Immunoprecipitation assays revealed an ~35% reduction in the level of Cav2.2 protein immunoprecipitated by CRMP2 in cells treated with CBD3063 versus control (Figure 4A, B ; DMSO: 1.00 ± 0.05; CBD3063: 0.64 ± 0.02). These results provide evidence that CBD3063 inhibits the association between Cav2.2 and CRMP2.
We reported previously that CRMP2 facilitates the trafficking of Cav2.2 to the plasma membrane (Brittain, Piekarz, Wang, Kondo, Cummins & Khanna, 2009; Chi et al., 2009) and that cell-penetrant CBD3 peptides (Brittain et al., 2011b; Francois-Moutal et al., 2015; Xie et al., 2016) decrease surface trafficking of Cav2.2. Having established that CBD3063 can inhibit Cav2.2–CRMP2 interaction in vitro , we next asked if CBD3063 alters the subcellular localization of Cav2.2 channels. To evaluate this, DRG neurons were incubated with vehicle (0.1% DMSO) or CBD3063 (20 µM) overnight and subjected to immunofluorescent microscopy to assess the membrane expression of these channels. In control DRGs treated with vehicle, the fluorescent signal for Cav2.2 presents as an annulus at the plasma membrane (Figure 4C ). As illustrated in Figure 4D , Cav2.2 expression in the membrane was significantly decreased when cells were incubated with CBD3063 (DMSO: 3.59 ± 0.42; CBD3063: 1.64 ± 0.10). These data suggests that by uncoupling Cav2.2–CRMP2 interaction, CBD3063 reduces surface trafficking of Cav2.2 channels to the plasma membrane of sensory neurons.