Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel

Abstract

The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT) mice but not in TRPV1 knock-out (TRPV1 KO) mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG) neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, OPEN ACCESS Toxins 2015, 7 3672 BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1) BmP01 is one of the pain-inducing agents in scorpion venoms; and (2) BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation.

Keywords: BmP01; Kv channels; TRPV1; pain; peptide toxin; scorpion Mesobuthus martensii.

Figure 1

Purification of BmP01 from venom of the scorpion Mesobuthus martensii. (A) Venom of scorpion was fractionated using Sephadex G-50 gel filtration; (B) The peak eluting at 1000–1403 min (indicated by an arrow) was further fractionated on a C₁₈ RP-HPLC column. Ten fractions obtained were screened for pain behavioral study in mice. F1 fraction eluting at 38 min (indicated by an arrow) showed the pain inducing activity (inserted panel, n = 10); (C) F1 was fully purified on an analytical C₁₈ RP-HPLC column with a retention gradient of ~35% acetonitrile; (D) Molecular weight of the purified peptide was determined to be 3178.6 Da by MALDI-TOF analysis.

Figure 2

The sequence and structure of BmP01. (A) Sequence of cDNA encoding BmP01, the signal peptide (underlined) is of 28 amino acids in length, and the mature peptide consists of 29 amino acids, designated with white text on a blue background; (B) The 3D-structure of BmP01 is provided from Protein Data Bank (Protein Data Bank code 1WM7); (C) Alignment of purified BmP01 with three previously reported peptides from scorpion. They contain the same number of amino acids and disulfide bonds.

Figure 3

Effects of BmP01 on mouse Kv channels. (A) 1 mM BmP01 did not inhibit the mKv1.1 currents; (B) 10 μM of BmP01 inhibited the currents on mKv1.3; (C) the concentration–response relationship of BmP01 against mKv1.3 yielded an EC₅₀ of 269.15 ± 12.69 nM (n = 10); and (D) on-rate and off-rate of BmP01 interacting with mKv1.1.

Figure 4

Mean duration (±S.E.) of paw licking and electrophysiology on DRG neurons. (A) Different doses of BmP01 along with capsaicin and kaliotoxin were injected into WT mice. Kaliotoxin showed no significant pain behavior, whereas application of 500 µM BmP01 showed acute pain behavior similar to capsaicin; (B) Ten microliters (25 ng/μL) Crude venom injected into WT and TRPV1 KO mice showed the significant difference of the pain behavior between WT and TRPV1 KO mice; (C) Ten microlites saline (control), 500 μM of BmP01, kaliotoxin, capsaicin and 10 μL (25 ng/μL) crude venom were injected into the paw of WT mice. BmP01 and capsaicin induced pain in WT mice. Kaliotoxin was unable to induce pain whereas crude venom induces severe pain; (D) Ten microliters saline (control), 500 μM of BmP01, kaliotoxin, capsaicin and 10 μL (25 ng/μL) crude venom were injected into the paw of TRPV1 KO mice. Similar to capsaicin, BmP01 did not induce pain, while only crude venom induced pain; (E) Both BmP01 (300 μM) and capsaicin (10 μM) evoked the currents on DRG sensory neurons of WT mice. Contrastingly, in DRG neurons from TRPV1 KO mouse, BmP01 as well as capsaicin did not evoke currents; (F) Normalized currents evoked by BmP01 and capsaicin on DRG neurons of WT and TRPV1 KO mice. For both BmP01 and capsaicin, current traced is significantly lower in TRPV1 KO mice than in WT mice. * p < 0.01, n = 10.

Figure 5

BmP01 activated TRPV1 channel. (A) Application of BmP01 at the concentrations of 1 µM, 10 µM, 100 µM, 500 µM and 1 mM showed the increasing activity with higher concentration to activate TRPV1 currents. Along with Capsaicin, proton and 2APB, 500 µM of BmP01 completely activates the TRPV1 channel; (B) Concentration—response yielded an EC₅₀ of 131.8 ± 49.1 µM BmP01 (n = 10); (C) In HEK293T cells, negative for TRPV1 channel, BmP01 and capsaicin did not evoke the currents, whereas only proton induced some currents; (D) In contrast, BmP01 and capsaicin evoked the currents in HEK293T cells with over-expressed TRPV1 channels.