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.