Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides

Abstract

Background and purpose: N-type voltage-gated calcium (Ca_v 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Ca_v 2.2 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 Ca_v 2.2 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 Ca_v 2.2 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.

Experimental approach: 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 Ca_v 2.2 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.

Key results: 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.

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

Linked articles: 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.

Figure 1

Identification of a minimal functional domain within the CBD3 peptide. (A) Predicted α‐helical model of the secondary structure of the parental Ca²⁺ CBD3 peptide. Sequence of the parental and two truncated peptides are shown. (B) Bar graphs show the K⁺‐evoked peak fluorescence response (adjusted for background) of DRG neurons incubated overnight with increasing concentrations (1, 3, 10 or 30 μM) of the indicated peptides or 0.1% DMSO as the vehicle control. Values represent the mean ± SEM, normalized to the DMSO level within each experiment. n = 39 to 58 cells per condition from five independent experiments. *P < 0.05, significantly different from control; one‐way ANOVA with Tukey's post hoc analysis. (C) Bar graph showing normalized CRMP2 binding to the first intracellular loop (Loop 1; left) or the C‐terminus (Ct; right) of Ca_v2.2. The indicated peptides (10 μM) or DMSO (0.1%) was added to CRMP2 prior to their addition on a 96‐well assay plate where Loop 1 or Ct fragments had been immobilized. TAT‐CBD3 and the N‐terminal containing region of CBD3 (i.e. TAT‐CBD3Δ7‐15) inhibited CRMP2 binding to Ca_v2.2 fragments (n = 12). *P < 0.05, significantly different from DMSO; one‐way ANOVA with Tukey's post hoc analysis). (D) Representative pseudocolour inverted micrographs showing PLA signal (fluorescent amplification product; black dots) for CRMP2‐ Ca_v2.2 channel interaction in sensory neurons treated with 0.1% DMSO or 10 μM of TAT‐CBD3Δ7‐15. DAPI (yellow) signal shows the nucleus of the cell. Scale bar is 10 μm. (E) Summary of the number of PLA puncta, normalized to the area of the cell analysed (n = 13–18 cells each from at least five independent experiments). Data shown are means ± SEM. *P < 0.05; significantly different from DMSO;, one‐way ANOVA with Tukey's post hoc analysis.

Figure 2

Homology‐guided design of N‐terminal region of CBD3 peptides. (A) Amino acid alignment of the six N‐terminal residues of the CBD3 peptide across CRMPs 1–4. Asterisks denote amino acids that are conserved/identical between the CRMPs. Residues that differ from the CRMP2 sequence are shown in coloured text. (B) Mutant CBD3 peptides are shown in capped‐stick representation. The coordinates for the six‐amino‐acid fragment were extracted from chain A of human CRMP2 (Protein Data Base ID:2GSE; Stenmark et al., 2007). PyMol (PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC) was used to model each peptide using the mutation wizard and choosing the most common rotamer without clashes with adjacent amino acids. The mutated amino acids are shown in green on the peptide structure. The sequence of each peptide is shown below the model with the coloured residues corresponding to the CRMP isoform that the mutation is originating from (see panel A). (C) Bar graphs showing the melting temperature, measured by DSF, of the indicated peptides. The bars are colour‐coded according to the CRMP protein from which the mutation is derived. No significant difference was observed between any of the peptides (n = 6).

Figure 3

elisa‐based analyses of the ability of CBD3‐derived peptides to inhibit the CRMP2– Ca_v2.2 channel interaction. Bar graph showing normalized CRMP2 binding to Loop 1 (A) or C‐terminus (B) of Ca_v2.2. The indicated peptides (10 μM) or DMSO (0.1%) was added to CRMP2 prior to their addition on a 96‐well assay plate where Loop 1 or Ct fragments had been immobilized. The dotted line shows the level of CRMP2 binding in the presence of 10 μM of TAT‐CBD3. TAT‐CBD3‐S3N, TAT‐CBD3‐S3R, TAT‐CBD3‐R4K, TAT‐CBD3‐L5V and TAT‐CBD3‐L5M inhibited CRMP2 binding to Ca_v2.2 fragments. The bars are colour‐coded according to the CRMP protein from which the mutation is derived. Data shown are means ± SEM (n = 12) *P < 0.05; significantly different from DMSO; Kruskal–Wallis test with pairwise comparison with Dunnett's post hoc analysis).

Figure 4

CBD3‐derived peptides inhibit depolarization‐evoked Ca²⁺ influx in DRG neurons. Bar graphs showing the K⁺‐evoked peak fluorescence response (adjusted for background) of DRGs incubated overnight with increasing concentrations (1, 3, 10 or 30 μM) of the indicated peptides or DMSO (0.1%) as the vehicle control. The dotted line shows the peak fluorescence response in the presence of 30 μM of TAT‐CBD3 (from Figure 1B). Values represent the average ± SEM, normalized to the DMSO level within each experiment. n = 37 to 68 cells per condition from five independent experiments. *P < 0.05, significantly different from control; one‐way ANOVA with Tukey's post hoc analysis).

Figure 5

CBD3‐derived peptides inhibit Ca²⁺ currents and the CRMP2– Ca_v2.2 channel interaction in sensory neurons. (A) Representative family of current traces are illustrated from sensory neurons treated with DMSO (0.1%; vehicle control) or treated with 10 μM of the indicated peptides. (B) Peak current density, at +10 mV, for the indicated conditions. n = 8–32 cells from five independent experiments. Data shown are means ± SEM. *P < 0.05, significantly different from DMSO; one‐way ANOVA with Tukey's post hoc analysis. (C) Summary of the number of PLA dots, normalized to the area of the cell analysed (n = 11–23 cells each from five independent experiments). The dotted line shows the mean PLA signal in the presence of 10 μM of TAT‐CBD3 or TAT‐CBD3Δ7‐15 (from Figure 1E). Data shown are means ± SEM. *P < 0.05, significantly different from DMSO; one‐way ANOVA with Tukey's post hoc analysis.

Figure 6

TAT‐CBD3‐L5M peptide reduces incision‐ and gp120‐induced nociceptive behaviours. Rats received a plantar incision on the left hind paw. Paw withdrawal latencies (PWLs) were significantly decreased 24 h after incision. (A) TAT‐CBD3‐L5M (20 μg/5 μL) or vehicle (saline) was injected into the i.t. space and PWLs measured. Paw withdrawal latencies were significantly reversed at the indicated times after injection of TAT‐CBD3‐L5M. Data shown are means ± SEM (n = 5–6). *P < 0.05; significantly different from pre‐incision value; two‐way ANOVA with a Student–Neuman–Keuls post hoc test) where time was treated as ‘within subjects’ factor, whereas treatment was treated as ‘between subjects’ factor. Likewise, paw withdrawal thresholds (PWTs) were significantly decreased 24 h after incision. (C) Injection of TAT‐CBD3‐L5M significantly reversed PWTs at the indicated times. Data shown are means ± SEM (n = 5–6). *P < 0.05; significantly different from pre‐incision value; two‐way ANOVA with a Student–Neuman–Keuls post hoc test). AUC, using the trapezoid method, for PWL (B; summary for data shown in A) and PWT (D; summary for data shown in C) are shown. Data shown are means ± SEM. *P < 0.05, significantly different as indicated; one‐way ANOVA with Tukey's post hoc analysis. (E) PWTs were significantly reduced 15 days after three injections of gp120 in the i.t. space. (F) Injection of TAT‐CBD3‐L5M (20 μg in 5 μL) significantly reversed PWTs at the indicated times. Data shown are means ± SEM (n = 5). *P < 0.05; significantly different from pre‐incision value; two‐way ANOVA with a Student–Neuman–Keuls post hoc test). (G) Bar graph showing the AUC, using the trapezoid method for the data shown in (F). *P < 0.05, significantly different from vehicle; one‐way ANOVA with Tukey's post hoc analysis.

Figure 7

Pharmacophore models for the N‐terminal six‐mer peptide of CBD3. Two models, based on the biochemical and functional data on mutant peptides, are illustrated. P17 (blue) is a positively charged site, H15 (green) is a hydrophobic region and A2 (orange) is a hydrogen bond acceptor. The yellow sphere in the right panel represents excluded volume corresponding to alanine at the first position of the six‐mer N‐terminal CBD3 peptide.