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. Author manuscript; available in PMC: 2019 Feb 5.
Published in final edited form as: Pain. 2017 Dec;158(12):2301–2319. doi: 10.1097/j.pain.0000000000001002

Figure 8.

Figure 8.

(S)-LCM normalizes Ca21 and Na1 currents after CRISPR/Cas9 Nf1 editing in rats. (A) Schematic showing how convergent signaling involving CRMP2 and neurofibromin controls channel activity. Our previous work identified a direct binding between CaV2.2 and CRMP2 leading to a CRMP2-mediated increase in Ca2+ current density and increased transmitter release in sensory neurons. In addition, CRMP2 was reported to bind to neurofibromin. Loss of neurofibromin increases CRMP2 phosphorylation, which in turn, can increase its association with CaV2.2 and NaV1.7. The direct association of neurofibromin with non-phosphorylated CRMP2 may protect it from phosphorylation. Here, we propose to control the activity of CaV2.2 via an enantiomer of a clinically available small molecule ((S)-LCM) that interferes with the neurofibromin-CRMP2-channel interactions. (B) Representative Western blot (top) and summary histograms (bottom) of Cdk5-phophorylated (ie, p5222) and total CRMP2 from spinal cords of rats isolated 10 days after intrathecal injection with plasmids harboring control or Nf1 sgRNAs (n = 4 each; *P < 0.05, Student’s t test). (C) Peak CaV2.2 current density, at −10 mV, in dorsal root ganglia (DRG) neurons transfected by Nf1 sgRNA containing plasmid and treated with either 10 µM (S)-LCM or DMSO 0.04%. Line shows peak CaV.2.2 current level in DRG neurons transfected with the empty plasmid. (D) Peak current density for Total, tetrodotoxin-sensitive (TTX-S) or TTX-resistant (TTX-R) VGSC in DRG neurons transfected by Nf1 sgRNA containing plasmid and treated with either 10 µM (S)-LCM or DMSO 0.04%. Line shows peak current level in DRG neurons transfected with the empty plasmid. Representative micrographs (E) of and summary (F) of the relative membrane localization of CaV2.2 and NaV1.7 (compared to cytosol) in DRG neurons transfected by Nf1 sgRNA containing plasmid and treated with either 10 µM (S)-LCM (n = 11) or DMSO 0.04% (n = 12). Asterisks indicate significance compared with control sgRNA transfected cells (*P < 0.05, Student’s t test).