The peptide Phα1β, from spider venom, acts as a TRPA1 channel antagonist with antinociceptive effects in mice

Abstract

Background and purpose: Peptides from venomous animals have long been important for understanding pain mechanisms and for the discovery of pain treatments. Here, we hypothesized that Phα1β, a peptide from the venom of the armed spider Phoneutria nigriventer, produces analgesia by blocking the TRPA1 channel.

Experimental approach: Cultured rat dorsal root ganglion (DRG) neurons, human fetal lung fibroblasts (IMR90) or HEK293 cells expressing the human TRPA1 (hTRPA1-HEK293), human TRPV1 (hTRPV1-HEK293) or human TRPV4 channels (hTRPV4-HEK293), were used for calcium imaging and electrophysiology. Nociceptive responses induced by TRPA1, TRPV1 or TRPV4 agonists or by bortezomib were investigated in mice.

Key results: Phα1β selectively inhibited calcium responses and currents evoked by the TRPA1 agonist, allyl isothiocyanate (AITC), on hTRPA1-HEK293, IMR90 fibroblasts and DRG neurons. Phα1β did not affect calcium responses evoked by selective TRPV1 (capsaicin) or TRPV4 (GSK 1016790A) agonists on the various cell types. Intrathecal (i.t.) and intraplantar (i.pl.) administration of low doses of Phα1β (up to 300 pmol per paw) attenuated acute nociception and mechanical and cold hyperalgesia evoked by AITC (i.t. or i.pl.), without affecting responses produced by capsaicin or hypotonic solution. Notably, Phα1β abated the TRPA1-dependent neuropathic pain-like responses induced by bortezomib. In vitro and in vivo inhibition of TRPA1 by Phα1β was reproduced by a recombinant form of the peptide, CTK 01512-2.

Conclusions and implications: Phα1β and CTK 01512-2 selectively target TRPA1, but not other TRP channels. This specific action underlines the potential of Phα1β and CTK 01512-2 for pain treatment.

Figure 1

Phα1β and its recombinant form (CTK 01512–2) selectively inhibit the calcium response evoked by stimulation of human TRPA1 channels. (A) Typical traces of the inhibitory effect of pre‐exposure (10 min) to Phα1β (3 μM) and CTK 01512–2 (3 μM) on the calcium response evoked by the TRPA1 channel agonist, AITC (30 μM), in HEK293 cells transfected with the cDNA for human TRPA1 channels (hTRPA1‐HEK293). Concentration–response curve of the inhibitory effect of Phα1β and CTK 01512–2 on the calcium response evoked by AITC (AITC concentrations are 30 μM). Phα1β (3 μM) and CTK 01512–2 (3 μM) and the selective TRPA1 channel antagonist HC‐030031 (HC03, 30 μM) inhibit calcium response evoked in hTRPA1‐HEK293 cells by AITC. (B) Phα1β (3 μM) and CTK 01512–2 (3 μM) do not affect responses evoked by capsaicin (CPS, 0.1 μM) in HEK293 cells transfected with the cDNA for human TRPV1 channels (hTRPV1‐HEK293) and by GSK1016790A (GSK, 50 nM) in HEK293 cells transfected with the cDNA for human TRPV4 channels (hTRPV4‐HEK293). Values are mean ± SEM of n > 25 cells from at least three different experiments for each condition. ^§ P < 0.05, significantly different from vehicle (Veh); *P < 0.05, significantly different from AITC.

Figure 2

Phα1β and its recombinant form (CTK 01512–2) selectively inhibit calcium response or currents evoked by stimulation of TRPA1 channels. (A) Typical traces of the inhibitory effect of pre‐exposure (10 min) to the Phα1β (300 nM) and CTK 01512–2 (300 nM) on the calcium response evoked by the TRPA1 channel agonist, AITC (10 μM), in cultured IMR90 cells. Concentration–response curves of the inhibitory effect of Phα1β and CTK 01512–2 on the calcium response evoked by AITC in IMR90 cells (AITC concentrations are 1 μM). Phα1β (300 nM) and CTK 01512–2 (300 nM) and the selective TRPA1 channel antagonist HC‐030031 (HC03, 30 μM) inhibit calcium responses evoked in IMR90 cells by AITC. Phα1β (300 nM) and CTK 01512–2 (300 nM) do not affect responses evoked by the activating peptide of the human PAR2 receptor (hPAR2‐AP, 100 μM) in IMR90 cells. TRP, trypsin. (B) Typical traces of the inhibitory effect of pre‐exposure (10 min) to Phα1β (300 nM) and CTK 01512–2 (300 nM) on the calcium response evoked by AITC (10 μM), in cultured rat DRG (rDRG) neurons. Concentration–response curves of the inhibitory effect of Phα1β and CTK 01512–2 on the calcium response evoked by AITC in rDRG neurons (AITC concentrations are 10 μM). Phα1β (300 nM) and CTK 01512–2 (300 nM) and HC03 (30 μM) inhibit calcium response evoked in rDRG neurons by AITC. Phα1β (300 nM) and CTK 01512–2 (300 nM) do not affect responses evoked by capsaicin (CPS, 0.1 μM) in rDRG neurons. Values are mean ± SEM of n > 25 cells from at least three different experiments for each condition. ^§ P < 0.05, significantly different from vehicle (Veh); *P < 0.05, significantly different from AITC. (C) Typical traces and pooled data obtained by whole‐cell patch‐clamp recordings in cultured IMR90 exposed to the selective TRPA1 channel agonist AITC. The inward currents evoked at −60 mV by AITC (100 μM), but not those evoked by KCl (90 mM), are attenuated by the selective TRPA1 channel antagonist, HC‐030031 (HC03; 50 μM), Phα1β or CTK 01512–2 (3 μM). Values are the mean ± SEM of at least five independent experiments. §P < 0.05, significantly different from AITC 30 μM; **P < 0.05, significantly different from AITC 100 μM alone.

Figure 3

Phα1β and its recombinant form (CTK 01512–2) selectively block nocifensor responses evoked by reactive TRPA1 channel agonist. (A–C) Effect of increasing doses of i.pl. administration of Phα1β (30–300 pmol per paw) or CTK 01512–2 (300 pmol per paw) and the selective TRPA1 channel antagonist, HC‐030031 (HC, 300 nmol per paw) on the nociceptive response evoked by the i.pl. injection (10 μL, i.pl.) of AITC (10 nmol per paw) in C57BL/6 mice. (B–C) Time course and dose–response curve (0.25 h after AITC treatment) of Phα1β (30–300 pmol per paw) or CTK 01512–2 (300 pmol per paw) and HC‐030031 (HC, 300 nmol per paw) on mechanical (B) and cold (C) hyperalgesia evoked by the i.pl. injection of AITC (10 nmol per paw) in C57BL/6 mice. (D) Effect of i.pl. administration of Phα1β or CTK 01512–2 and the selective TRPV1 channel antagonist, capsazepine (CPZ, 1 nmol per paw) on the nociceptive response and mechanical hyperalgesia evoked by the injection (10 μL, i.pl.) of capsaicin (CPS, 0.1 nmol per paw) in C57BL/6 mice. (E) Effect of i.pl. administration of Phα1β or CTK 01512–2 and the selective TRPV4 channel antagonist, HC‐067047 (HC06, 300 nmol per paw) on the nociceptive response and mechanical hyperalgesia evoked by the injection (10 μL, i.pl.) of hypotonic solution (0.27% NaCl per paw) in C57BL/6 mice. Each column represents the mean of six mice, and vertical lines show the SEM. Statistical analysis was performed using one‐way or two‐way ANOVA followed by Student–Newman–Keuls or by Bonferroni's post hoc test respectively. *P < 0.05, significantly different from Veh; ^# P < 0.05, significantly different from BL values; ^§ P < 0.05, significantly different from Phα1β (300 pmol per paw).

Figure 4

Phα1β and its recombinant form (CTK 01512–2) block the hyperalgesia evoked by i.t. injection of AITC, a TRPA1 channel agonist. (A–B) Time course and dose–response curve of the effect of intrathecal (5 μL, i.t.) administration of AITC (0.01–3 nmol per site) on mechanical (A) and cold (B) nociception. (C–D) Time course and dose–response curve of i.t. administration of Phα1β (10–100 pmol per site), CTK 01512–2 (100 pmol per site) and the selective TRPA1 channel antagonist, HC‐030031 (HC, 30 nmol per site) on mechanical (C) and cold (D) hyperalgesia evoked by the injection (5 μL, i.t.) of AITC (1 nmol per site) in C57BL/6 mice. Dose–response curves were performed 0.5 h after AITC treatment. Each point and column represents the mean of six mice, and vertical lines show the SEM. Statistical analysis was performed using one‐way or two‐way ANOVA followed by Student–Newman–Keuls post hoc test or by Bonferroni's post hoc test respectively. *P < 0.05, significantly different from Veh; ^# P < 0.05, significantly different from BL values.

Figure 5

Phα1β and its recombinant form (CTK 01512–2) reduce TRPA1 channel‐dependent hyperalgesia in a model of neuropathic pain induced by BTZ. At day 7 after treatment with BTZ (1 mg·kg⁻¹, i.p.), mechanical hyperalgesia and cold hyperalgesia are increased (BL, basal level threshold at day 0 before BTZ). Time course and dose–response curve of i.t. administration of Phα1β (10–100 pmol per site), CTK 01512–2 (100 pmol per site) and the selective TRPA1 channel antagonist, HC‐030031 (HC, 30 nmol per site) on mechanical (A) and cold (B) hyperalgesia evoked by BTZ (1 mg·kg⁻¹, i.p.) at day 7 after treatment. Time course of the effect of the i.pl. administration of CTK 01512–2 (300 pmol per paw), ω‐conotoxin MVIIA (300 pmol per paw) and HC‐030031 (HC, 300 nmol per paw) on mechanical (C) and cold (D) hyperalgesia evoked by BTZ (1 mg·kg⁻¹, i.p.) at day 7 after treatment. Dose–response curves were performed 0.5 h after AITC treatment. Each point and column represents the mean of six mice, and vertical lines show the SEM. Statistical analysis was performed using one‐way or two‐way ANOVA followed by Student–Newman–Keuls post hoc test or by Bonferroni's post hoc test respectively. *P < 0.05, significantly different from BTZ/PBS; ^# P < 0.05, significantly different from BL values.