N-type Ca(2+) channels (CaV2.2) are a nidus for neurotransmitter release and nociceptive transmission. However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects resulting from inhibition of the physiological functions of CaV2.2 within the CNS. We identified an anti-nociceptive peptide (Brittain, J. M., Duarte, D. B., Wilson, S. M., Zhu, W., Ballard, C., Johnson, P. L., Liu, N., Xiong, W., Ripsch, M. S., Wang, Y., Fehrenbacher, J. C., Fitz, S. D., Khanna, M., Park, C. K., Schmutzler, B. S., Cheon, B. M., Due, M. R., Brustovetsky, T., Ashpole, N. M., Hudmon, A., Meroueh, S. O., Hingtgen, C. M., Brustovetsky, N., Ji, R. R., Hurley, J. H., Jin, X., Shekhar, A., Xu, X. M., Oxford, G. S., Vasko, M. R., White, F. A., and Khanna, R. (2011) Suppression of inflammatory and neuropathic pain by uncoupling CRMP2 from the presynaptic Ca(2+) channel complex. Nat. Med. 17, 822-829) derived from the axonal collapsin response mediator protein 2 (CRMP2), a protein known to bind and enhance CaV2.2 activity. Using a peptide tiling array, we identified novel peptides within the first intracellular loop (CaV2.2(388-402), "L1") and the distal C terminus (CaV1.2(2014-2028) "Ct-dis") that bound CRMP2. Microscale thermophoresis demonstrated micromolar and nanomolar binding affinities between recombinant CRMP2 and synthetic L1 and Ct-dis peptides, respectively. Co-immunoprecipitation experiments showed that CRMP2 association with CaV2.2 was inhibited by L1 and Ct-dis peptides. L1 and Ct-dis, rendered cell-penetrant by fusion with the protein transduction domain of the human immunodeficiency virus TAT protein, were tested in in vitro and in vivo experiments. Depolarization-induced calcium influx in dorsal root ganglion (DRG) neurons was inhibited by both peptides. Ct-dis, but not L1, peptide inhibited depolarization-stimulated release of the neuropeptide transmitter calcitonin gene-related peptide in mouse DRG neurons. Similar results were obtained in DRGs from mice with a heterozygous mutation of Nf1 linked to neurofibromatosis type 1. Ct-dis peptide, administered intraperitoneally, exhibited antinociception in a zalcitabine (2'-3'-dideoxycytidine) model of AIDS therapy-induced and tibial nerve injury-related peripheral neuropathy. This study suggests that CaV peptides, by perturbing interactions with the neuromodulator CRMP2, contribute to suppression of neuronal hypersensitivity and nociception.