title: A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors.
abstract: Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2-CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3-the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, USA Department of Pharmacology, Norman Bethune College of Medicine, Changchun, Jilin Province, China, Neuroscience Graduate Interdisciplinary Program, College of Medicine, University of Arizona, Tucson, AZ, USA.
authors.forename: Liberty
authors.initials: L
authors.lastname: François-Moutal
authors.forename: Yue
authors.initials: Y
authors.lastname: Wang
authors.forename: Aubin
authors.initials: A
authors.lastname: Moutal
authors.forename: Karissa E
authors.initials: KE
authors.lastname: Cottier
authors.forename: Ohannes K
authors.initials: OK
authors.lastname: Melemedjian
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Yang
authors.forename: Yuying
authors.initials: Y
authors.lastname: Wang
authors.forename: Weina
authors.initials: W
authors.lastname: Ju
authors.forename: Tally M
authors.initials: TM
authors.lastname: Largent-Milnes
authors.forename: May
authors.initials: M
authors.lastname: Khanna
authors.forename: Todd W
authors.initials: TW
authors.lastname: Vanderah
authors.forename: Rajesh
authors.initials: R
authors.lastname: Khanna
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D052158:Aptamers, Peptide / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism; D007249:Inflammation / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D010149:Postoperative Pain / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D052158:Aptamers, Peptide; C503279:Cacna1b protein, rat; D020864:Calcium Channels, N-Type; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; C095114:collapsin response mediator protein-2