Figure 4. CBD3063 suppresses
Ca v2.2 –CRMP2 interaction and surface
trafficking of the channel. Representative immunoblots (A) and summary
(B) of CRMP2 immunoprecipitation (IP) to detect Cav2.2
from CAD cells treated overnight with CBD3063 (20 µM) (n=3). pvalue as indicated; Unpaired t-test. Representative micrographs of DRG
cells immunolabeled with Cav2.2 (C) and summary (D) of
Cav2.2 membrane/cytosol ratio in DRG neurons treated
overnight with 20 µM of CBD3063 (n=25-38). Error bars show mean ± SEM;p values as indicated; Mann-Whitney test.
Because the cellular functions of CRMP2 are mediated by its
phosphorylation, we next determined whether CBD3063 could alter the
phosphorylation states of CRMP2. Immunoblot analyses of lysates prepared
from CAD cells exposed to overnight incubation of CBD3063 (20 µM) showed
no differences in the expression of phosphorylated CRMP2 at sites 522
(by cyclin dependent kinase 5 (Cdk5); Figure S4A, B ), 514 (by
glycogen synthase kinase-3 beta (GSK-3β); Figure S4C, D ), or
555 (RhoK; Figure S4E, F ). These results rule out a potential
side effect of CBD3063 on the phosphorylation status CRMP2.