Inhibition of transmitter release and attenuation of anti-retroviral-associated and tibial nerve injury-related painful peripheral neuropathy by novel synthetic Ca2+ channel peptides.
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.
Department of Program in Medical Neurosciences, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Sarah M
SM
Wilson
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Brian S
BS
Schmutzler
Department of Program in Medical Neurosciences, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Joel M
JM
Brittain
Department of Program in Medical Neurosciences, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Erik T
ET
Dustrude
Department of Anesthesia, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Matthew S
MS
Ripsch
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Jessica J
JJ
Pellman
Department of Anesthesia, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Tae-Sung
TS
Yeum
Department of Program in Medical Neurosciences, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Joyce H
JH
Hurley
Department of Program in Medical Neurosciences, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Neurology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Cynthia M
CM
Hingtgen
Department of Program in Medical Neurosciences, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Anesthesia, Indiana University School of Medicine, Indianapolis, Indiana 46202.
Fletcher A
FA
White
Department of Program in Medical Neurosciences, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202; Sophia Therapeutics LLC, Indianapolis, Indiana 46202. Electronic address: khanna5@iu.edu.
Rajesh
R
Khanna
D016263:AIDS-Associated Nephropathy / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D005727:Ganglia, Spinal / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D009456:Neurofibromatosis 1 / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D025542:Neurofibromin 1 / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D018377:Neurotransmitter Agents / Q000235:genetics / Q000378:metabolism*; D059225:Nociception / Q000187:drug effects; D010455:Peptides / Q000235:genetics / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D020429:Tibial Neuropathy / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000494:pharmacology
C500545:CACNA1B protein, human; C500963:Cacna1b protein, mouse; D020864:Calcium Channels, N-Type; D025542:Neurofibromin 1; D018377:Neurotransmitter Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D015740:Calcitonin Gene-Related Peptide; D002118:Calcium