Research ArticleORGANISMAL BIOLOGY

A bimodal activation mechanism underlies scorpion toxin–induced pain

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Science Advances  02 Aug 2017:
Vol. 3, no. 8, e1700810
DOI: 10.1126/sciadv.1700810
  • Fig. 1 BmP01 directly activates TRPV1 channel.

    (A) Image of the scorpion M. martensii. (B) Peptide sequence and disulfide bonds of BmP01. (C) Solution structure of BmP01 [Protein Data Bank (PDB) ID: 1WM7]. Three disulfide bonds are colored in yellow. (D) Calcium imaging of hTRPV1-expressing HEK293 cells challenged sequentially with BmP01 (200 μM), capsaicin (10 μM), and ionomycin (1 mM). The scale bar represents 126 to 314 arbitrary units. (E) Fluorescence signals of hTRPV1-expressing HEK293T cells in response to BmP01, capsaicin, and ionomycin. **P < 0.001 (n = 3 to 5). (F) Representative single-channel current traces recorded in the absence or presence of 100 μM BmP01 (pH 7.0). (G) Representative whole-cell current response to BmP01 and agonists at saturating concentration [for hTRPV1, 10 μM capsaicin; for mTRPV3, 3 mM 2-aminoethoxydiphenyl borate (2APB)]. (H) Concentration-response relationship of BmP01 and hTRPV1 (n = 10) fitted to a Hill equation with the following parameters: EC50, 40.4 ± 12.3 μM; slope factor, 0.80 ± 0.05.

  • Fig. 2 Extracellular acidification strongly potentiates BmP01 in activating TRPV1 and inducing pain.

    (A) Determination of the acidity value of venom from M. martensii. (B) Comparison of the acidity value of different venoms. (C) Representative whole-cell currents from hTRPV1 at various BmP01 concentrations and pH levels. (D) Concentration-response relationships of BmP01 at various pH levels fitted to a Hill equation; n = 5 to 10. (E) Paw-licking behavior of mice following injection of either saline, BmP01 (pH 7.0 or 6.5), or capsaicin (pH 7.0 or 6.5). Ten microliters of each reagent was injected at the plantar surface of the left hind paw. NS, not significant. **P < 0.01.

  • Fig. 3 BmP01 targets the pre-S6 region of TRPV1.

    (A) Schematic representation of the chimeras between mTRPV1 (blue) and mTRPV3 (red), and mTRPV1 mutants containing a sequence replacement in the turret (yellow). (B) Comparison of the BmP01 response (shown as percentage of the capsaicin response). mTRPV1 was used as wild type (WT). **P < 0.001 (n = 3 to 5). (C) Concentration-response relationships of WT mTRPV1, V1/3S, and V1/3L fitted to a Hill equation. (D) Comparison of the BmP01 response of WT hTRPV1 and point mutants. **P < 0.001 (n = 3 to 5). (E) Representative whole-cell current recording from hTRPV1 treated with BmP01 and its point mutants (200 μM each) and 3 mM 2APB. (F) Concentration-response relationships for WT BmP01 and K23A fitted to a Hill equation (n = 5 to 10).

  • Fig. 4 Bimodal activation of TRPV1 by BmP01 and proton.

    (A) A salt bridge mediates BmP01-TRPV1 interaction. Left: Comparison of coupling energy between K23 of BmP01 and the pre-S6 channel residues (n = 4 to 7) with the 1.5-kT threshold for direct interaction indicated with a dashed line. Right: Structural model of the BmP01-TRPV1 complex, with the two proton-binding sites (E601 and E649) highlighted in red and orange, respectively. (B) Concentration-response relationships of E601A at varying pH levels fitted to a Hill equation (n = 5 to 10). Concentration-response relationships of WT hTRPV1 at the same pH levels (Fig. 2D) are superimposed as dashed lines for comparison. (C) Comparison of responses by WT hTRPV1 and its point mutants to 100 μM BmP01 at neutral acidity (pH 7.5) (n = 3 to 5). **P < 0.01. (D) Proton concentration-response relationships of WT mTRPV1, E601Q, and E649Q mutants fitted to a Hill equation (n = 4 to 7). (E) Cartoon summarizing the bimodal activation of TRPV1 by proton and BmP01. At pH 6.5, proton binding to the high-affinity site E601 potentiates TRPV1; under this situation, BmP01 binding to E649 leads to strong activation. At even lower pH levels, protons bind to both E601 and E649 to open the channel without BmP01.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1700810/DC1

    fig. S1. BmP01 activates TRPV1 by increasing the open probability.

    fig. S2. BmP01 inhibits Kv1.3 channel.

    fig. S3. Effects of BmP01 on TRPV1, TRPV3, and their chimeras.

    fig. S4. Effects of BmP01 on TRPV1 outer pore mutants.

    fig. S5. Low pH potentiates TRPV1 current response to toxins in the α-KTX8 toxin subfamily.

    fig. S6. Concentration-response curves used for the determination of coupling energy between BmP01 and TRPV1.

    table S1. Changes in charged fraction of the side chain of titratable residues in BmP01.

    table S2. BmP01 concentration-response parameters for TRPV1 WT and mutants.

    table S3. Proton concentration-response parameters for TRPV1 WT and mutants.

    table S4. BmP01 concentration-response parameters for Kv1.3 under different pH values.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. BmP01 activates TRPV1 by increasing the open probability.
    • fig. S2. BmP01 inhibits Kv1.3 channel.
    • fig. S3. Effects of BmP01 on TRPV1, TRPV3, and their chimeras.
    • fig. S4. Effects of BmP01 on TRPV1 outer pore mutants.
    • fig. S5. Low pH potentiates TRPV1 current response to toxins in the α-KTX8 toxin subfamily.
    • fig. S6. Concentration-response curves used for the determination of coupling energy between BmP01 and TRPV1.
    • table S1. Changes in charged fraction of the side chain of titratable residues in BmP01.
    • table S2. BmP01 concentration-response parameters for TRPV1 WT and mutants.
    • table S3. Proton concentration-response parameters for TRPV1 WT and mutants.
    • table S4. BmP01 concentration-response parameters for Kv1.3 under different pH values.

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