Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides.
N-type voltage-gated calcium (Cav2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Cav2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential.



Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Cav2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120.



The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models.



These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3.



This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Aubin
0000-0003-4268-1206
A
Moutal
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Wennan
W
Li
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Yue
Y
Wang
Department of Neurology, The First Hospital of Jilin University, Jilin University, Changchun, Jilin Province, China.|Department of Pharmacology, The First Hospital of Jilin University, Jilin University, Changchun, Jilin Province, China.
Weina
W
Ju
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Shizhen
S
Luo
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Song
S
Cai
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Liberty
L
François-Moutal
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Samantha
0000-0002-9034-3693
S
Perez-Miller
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Jackie
J
Hu
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Erik T
0000-0002-1753-8603
ET
Dustrude
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
Todd W
TW
Vanderah
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, USA.
Vijay
0000-0003-2807-4741
V
Gokhale
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.
May
0000-0002-9989-6374
M
Khanna
Department of Pharmacology, University of Arizona, Tucson, AZ, USA.|Neuroscience Graduate Interdisciplinary Program, College of Medicine, University of Arizona, Tucson, AZ, USA.
Rajesh
0000-0002-9066-2969
R
Khanna
D000700:Analgesics / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D005260:Female; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics*; D008297:Male; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010146:Pain / Q000188:drug therapy* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000700:Analgesics; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C095114:collapsin response mediator protein-2