Figure 4. CBD3063 suppresses Ca v2.2CRMP2 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.