Research ArticleNEUROSCIENCE

Target switch of centipede toxins for antagonistic switch

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Science Advances  07 Aug 2020:
Vol. 6, no. 32, eabb5734
DOI: 10.1126/sciadv.abb5734
  • Fig. 1 Short-term immobilization is related to KV channel subtypes.

    (A) Photograph of the S. subspinipes self-envenomation. These centipedes inject venom to each other during intraspecific interaction. Photo credit: Y.W., Northeast Forestry University. (B) Movement distance recorded per minute following injection of 10 μl of crude venom or saline (n = 5 centipedes for each condition). Red arrow, crude venom application. (C) Images of the S. subspinipes and the isolated DUM neuron. (D to F) Whole-cell DUM calcium (D), sodium (E), and potassium (F) currents challenged by crude venom (1 mg/ml), 20 μM verapamil, 25 μM Ni+, 1 μM TTX, and 100 mM TEA, respectively. (G) Phylogenetic tree of centipede KV channel subtypes. (H and I) Voltage-evoked whole-cell currents (H) and conductance-voltage relationships (I) of centipede KV channel subtypes.

  • Fig. 2 SsTx inhibits centipede Shal channel.

    (A) Comparison of venom sensitivity (1 mg/ml) of centipede KV channel subtypes, including Shaker, Shal, Shab, Slowpoke, and Eag channels. n = 5 for each bar. *P < 0.05. Photo credit: Y.W., Northeast Forestry University. (B) Representative whole-cell recoding of Shal currents challenged by crude venom (1 mg/ml). Before application of the crude venom, the cells were perfused with bath solution for 30 s. (C) Representative inhibitory effect of Shal in the presence of 1 μM SsTx (top). Overlapped absorbance peaks of venom components (gray) and purified SsTx (red) by a C18 reversed-phase high-performance liquid chromatography (RP-HPLC) column (bottom). The protein fractions were labeled by circles in red (active) or gray (inactive) when they were subsequently tested on the Shal currents. The number of protein fractions is indicated. The effect of these fractions (1 mg/ml) on the centipede’s Shal channel is shown in the Supplementary Materials.

  • Fig. 3 Enhanced SsTx-Shal interaction in centipede.

    (A) Representative inhibition of 300 nM SsTx in different K+ concentrations recorded from centipede Shal-expressing HEK293 cells. (B) IC50 of SsTx on Shal channel recorded from pipette solution with different K+ concentrations. *P < 0.05 (C) Concentration-response relationships of centipede Shal and channel mutant (E351A) fitted to a Hill equation (n = 5 per data point). (D) Comparison of IC50 values of wild-type (WT) Shal and its single-point mutants. n.s., not significant. *P < 0.05 (E) Analysis of the pairwise coupling between the SsTx K17 and Shal E351. Data points were fitted to a Hill equation. (F) Comparison of coupling energy between K17 of SsTx and E351 of centipede Shal (n = 3 to 5) with the 1.5-kT threshold for direct interaction indicated by a dashed line. (G) Distribution of E351 mapped to the centipede Shal model from top view. (H) Molecular docking of SsTx with the electrostatic potential distribution shown in color (scale bar) to centipede Shal (shown in gray). Two of the four copies of the toxins are shown in this modeled complex. (I) The salt bridge formed between SsTx K17 and Shal E351 was indicated in SsTx-Shal structural model. *P < 0.05.

  • Fig. 4 Target switching mechanism of centipede toxins.

    (A) Expression level of centipede Shal protein in the intestine, trachea, DUM neuron, heart tube, and muscle. (B) Representative photomicrographs of DUM or heart tube sections stained with hematoxylin and eosin (left) or Shal channel antibody (right). Scale bars, 200 μm (for the DUM) and 50 μm (for the heart tube). (C) Representative response of DUM neuron after application of 40-pA depolarizing current injected into the cell when challenged with 1 μM SsTx. (D) Comparison of spiking frequency in the presence of bath solution (n = 4) and 1 μM SsTx (n = 5). *P < 0.05 (E) Representative vascular contractility of centipede heart tube contraction when challenged with 1 μM SsTx and 10 μM phenylephrine (PE). (F) Representative whole-cell recordings of centipede Shaker (left) and single-point channel mutant (right) in the presence of crude venom (1 mg/ml) or 1 μM SsTx. (G) SsTx concentration-response relationships of centipede Shaker and channel mutants. Data points were fitted to a Hill equation. (H) Site 399 (centipede Shaker number) is highlighted in the amino acid sequence alignment of species-specific Shaker channels. (I) Comparison of inhibitory effects of 10 μM SsTx on centipede Shaker and its orthologs. *P < 0.05 (J) Schematic diagram summarizing the target-switching mechanism in centipede survival strategy of both intraspecific and interspecific interaction. Target specifications (Shal, Shaker, TRPV1, and KCNQ channels) underlying the envenomation of conspecifics and heterospecifics are shown. Representative point mutations of species-specific Shal and Shaker channels are differentiated in conspecifics and heterospecifics. *P < 0.05.

Supplementary Materials

  • Supplementary Materials

    Target switch of centipede toxins for antagonistic switch

    Shilong Yang, Yunfei Wang, Lu Wang, Peter Kamau, Hao Zhang, Anna Luo, Xiancui Lu, Ren Lai

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