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. 2015 Jun 17;10(6):e0128578.
doi: 10.1371/journal.pone.0128578. eCollection 2015.

The Scorpion Toxin Tf2 from Tityus fasciolatus Promotes Nav1.3 Opening

Thalita S Camargos  1 , Frank Bosmans  2 , Solange C Rego  1 , Caroline B F Mourão  1 , Elisabeth F Schwartz  1
Affiliations

The Scorpion Toxin Tf2 from Tityus fasciolatus Promotes Nav1.3 Opening

Thalita S Camargos et al. PLoS One. .

Abstract

We identified Tf2, the first β-scorpion toxin from the venom of the Brazilian scorpion Tityus fasciolatus. Tf2 is identical to Tb2-II found in Tityus bahiensis. We found that Tf2 selectively activates human (h)Nav1.3, a neuronal voltage-gated sodium (Nav) subtype implicated in epilepsy and nociception. Tf2 shifts hNav1.3 activation voltage to more negative values, thereby opening the channel at resting membrane potentials. Seven other tested mammalian Nav channels (Nav1.1-1.2; Nav1.4-1.8) expressed in Xenopus oocytes are insensitive upon application of 1 μM Tf2. Therefore, the identification of Tf2 represents a unique addition to the repertoire of animal toxins that can be used to investigate Nav channel function.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Purification and molecular mass determination of Tf2.
(A) Chromatography by RP-HPLC of 1 mg of T. fasciolatus crude venom. Fractionation was performed on a C18 analytical column, using a linear gradient from 0% solvent A (0.12% TFA in water) to 60% solvent B (0.10% TFA in acetonitrile) over 60 min at a flow rate of 1 mL/min, with detection at a wavelength of 216 and 280nm. The component eluted at 38.5% of acetonitrile corresponds to Tf2. (B-D) Three additional chromatographic protocols performed to obtain pure Tf2. (B) Linear gradient of B solvent, from 25 to 45% B in 40 minutes, at room temperature (22°C). (C) Linear gradient of B solvent, from 25 to 45% of B in 40 minutes, at 45°C. (D) Linear gradient of B solvent, from 30 to 40% of B in 40 minutes, at 45°C. Inset on (A) shows mass spectrometry analysis of Tf2 by micrOTOF-Q II, presenting the monoisotopic distribution of the +7 charged ion ([M+7H]7+ = 993.7050), which is equivalent to [M+H]+ 6949.9350 Da.
Fig 2
Fig 2. Tf2 sequence and alignment with scorpion Nav channel toxins.
(A) The nucleotide sequence of Tf2 was obtained by HiSeq (Illumina, USA). Signal peptide is underlined, mature peptide is highlighted in gray, and the amidation set point is marked in italic. (B) Multiple sequence alignment of Tf2 with other Nav channel toxins. Toxins are presented by their short names and UniProt KB codes. Capital letters denote amino acids. Lower-case letters denote: h, hydrophobic; s, small; b, big; p, polar; t, tiny; a, aromatic; l, aliphatic. Positive (+) and negative (-) amino acid residues that are part of the consensus sequence are also colored. Cys residues are shaded in black. aa means amino acid residues, and %Id is the percentage of sequence identity with Tf2.
Fig 3
Fig 3. Structural comparison between Tf2 and other Nav channel toxins.
(A) Structural alignment between Tf2 (in blue) and five other Nav channel scorpion toxins—Ts1, Ts2, CssII, CssIV, and AaHII (in gray). (B) Comparison of electrostatic potentials between the toxins Tf2, Ts1, Ts2, CssII, CssIV, and AaHII. The figure shows charge distribution along the toxin surface, divided into faces A and B. Shown in red are acidic residues whereas blue represents basic residues; in white, neutral regions are shown.
Fig 4
Fig 4. Effect of Tf2 on Nav channel isoforms expressed in X. laevis oocytes.
Shown on the left in each column is a representative trace of experimental Na+ currents obtained by depolarizing the membrane to a suitable voltage from a holding potential of -90mV, at -25mV for Nav 1.1–1.2, 1.4–1.7, at -40mV for Nav1.3, and at 20mV for Nav1.8. Shown on the right in each column is a deduced conductance (G)—voltage (V) relationship before (black) and after (red) the application of 1μM Tf2. This concentration only influences the activation of hNav1.3. Data is shown as mean ± SEM of n > 3.
Fig 5
Fig 5. Sequence alignment of the domain II paddle motif in 8 mammalian Nav channel isoforms.
Figure shows a sequence alignment of the domain II paddle motif as found in 8 mammalian Nav channel isoforms. As a reference, the number in italic indicates the coordinates of the first Gly residue in hNav1.1. Although the Ile in hNav1.3 (position 830 according to hNav1.3 coordinates, 840 according to hNav1.1 coordinates) differs from the Phe found in other neuronal isoforms, this residue is not present within the paddle motif and may not be accessible to Tf2. The Ser at position 842 (hNav1.3 coordinates—indicated in red) is unique among hNav1.1–1.3.
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