Isolation and characterization of CvIV4: a pain inducing α-scorpion toxin

Abstract

Background: Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing α-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na(+) channel isoforms.

Principal findings: C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Na(v)1.7, a Na(+) channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Na(v)1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Na(v)1.2, Na(v)1.3 and Na(v)1.4. The effects of CvIV4 are similar to Old World α-toxins that target Na(v)1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Na(v)1.7 channels (D1586A and E1589Q, DIV S3-S4 linker) reduced but did not abolish the effects of CvIV4.

Conclusions: This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na(+) channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World α-toxins that target Na(v)1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution.

Figure 1. Mean (+1SE) duration of paw licking for Mus musculus injected with scorpion venom or water.

Samples of whole, soluble venom from three scorpion species induced more hind-paw licking in M. musculus than the water control during a ten-minute test period following the injection (F = 37.25; df = 1, 28; P<0.0001). However, Centruroides' venom induced more paw licking than V. spinigerus venom (F = 9.49; df = 1, 28; P = 0.0046). There was no statistical difference in the duration of paw licking induced by C. vittatus and C. exilicauda venom (F = 2.47; df = 1, 28; P = 0.1275).

Figure 2. Effect of C. vittatus venom and venom fractions on paw-licking behavior in Mus musculus.

A. High performance liquid chromatography (HPLC) profile of C. vittatus venom fractions. Whole, soluble venom from C. vittatus was separated into five fractions (peaks: P1, P2, P3, P4, P5). Each fraction was isolated and tested for pain using the paw-licking assay in M. musculus. B. Mean (+1SE) duration of paw licking for M. musculus injected with water, scorpion venom, or venom fractions. Paw licking was recorded for five minutes following the injection. P4 was the only fraction that was significantly more painful than water (P<0.0001) and P4 induced as much pain as whole venom (P = 0.9525). Histograms showing the same letter did not differ at the P<0.05 level of significance using Tukey's HSD test.

Figure 3. Effect of C. vittatus venom P4 subfractions on paw-licking behavior in Mus musculus.

A. High performance liquid chromatography (HPLC) profile of C. vittatus venom P4 subfractions. Fraction P4 was separated into four subfractions (P4-1, P4-2, P4-3, P4-4). B. Duration of hind-paw licking by M. musculus injected with C. vittatus P4 subfractions. Each sample was tested on two mice and each mouse was injected only once. Paw licking was recorded for five minutes following the injection. The paw licking values from both the first and second tests are shown in plot. Note, values for the first and second tests for P4-1 are identical and markers overlap.

Figure 4. Amino acid sequence of subfraction P4-4 (CvIV4) and C. vittatus venom gland cDNA that encodes toxin CvIV4.

A. Comparison of amino acid sequences representing CvIV4. The upper sequence represents the initial 40 amino acids of the purified peptide obtained from Edman degradation. Amino acid residues that could not be determined are shown as “X”. The lower sequence represents the translation of nucleotides from the cDNA encoding CvIV4 isolated from C. vittatus venom gland. Sequences representing the peptide and translated gene are identical with the exception of the five amino acid residues (X's) that could not be identified. B. Nucleotide sequence from venom gland cDNA that encodes toxin CvIV4. Nucleotide sequence from the 5′ and 3′ untranslated region (UTR) is shown as lower case letters. Sequence from the mature peptide is shown as upper case letters. Amino acid residues translated from nucleotide sequence are shown as upper case letters positioned below their corresponding codons. Amino acids designating the signal peptide are underlined and shown in bold.

Figure 5. Comparison of CvIV4 translated cDNA with Na⁺ channel toxin sequence from other scorpion species.

CvIV4 (underlined) is aligned with seven toxin sequences from other species. Alignment is based on cysteine residue position (shaded background) and toxins are arranged in order of descending percent identity with respect to CvIV4. Percent identify (%ID) is estimated from the number of amino acids shared by two toxins (NCBI Protein BLAST). While CvIV4 (JF938594) and CeII8 (P0CH40) are structurally similar (64% ID), they are functionally different as CvIV4 is classified as an α-toxin and CeII8 as a β-toxin (classification based on electrophysiological recordings from Na⁺ channel subtypes). CvIV4 shares over 50% of its amino acid residues with the remaining six toxins. Ts3 (P01496) and TsV (P46115) are classified as α-toxins based on electrophysiological studies of mammalian cells and tissues. Tst3 (P0C8X5), Pg8 (ACD35698), TbTx5 (P0C5K8) and LmNaTx10 (ACD35698) are classifed as α-toxins based solely on sequence similarity. Cv = Centruroides vittatus, Ce = Centruroides elegans, Tst = Tityus stigmurus, Ts = Tityus serrulatus, Pg = Parabuthus granulatus, Tb = Tityus bahaensis, Lm = Lychas mucronatus. GenBank accession numbers are shown in parentheses following the toxin name.

Figure 6. Effects of toxin CvIV4 on voltage-gated sodium channel isoforms.

A. CvIV4 (1 µM) slowed the fast inactivation of isoforms Na_v1.2, Na_v1.3, Na_v1.4 and Na_v1.7 expressed in human embryonic kidney cells (HEK). In contrast, CvIV4 had a minimal effect on Na_v1.5 (expressed in HEK) and no effect on neuronal TTX-R sodium current isolated from adult rat dorsal root ganglia (DRG) neurons (500 nM TTX was used to block TTX-S sodium current). All sodium current traces were elicited by depolarizing to −10 mV from a holding potential of −100 mV. B. Dose-response curves for CvIV4 slowing the fast inactivation of five sodium channel isoforms (Na_v1.2–1.7).

Figure 7. Effects of toxin CvIV4 on activation and inactivation of isoform Na_v1.7 expressed in Hek293 cells.

A. Depolarizing pulses in 5-mV increments were used to elicit current from Na_v1.7 expressed in HEK cells before (B) and after (C) the application of 1 µM CvIV4. D. Effects of 1 µM toxin on the current-voltage relationship of Na_v1.7. Currents not inactivated at 5 ms in the presence of 1 µM toxin were plotted as the percentage increase in the fraction of current at 5 ms (I_{5 ms}). E. Effects of CvIV4 on steady-state activation and inactivation of Na_v1.7. When data points were fitted with a Boltzmann equation, the V1/2 values were −90.2±1.2 and −94.1±1.6 mV before (filled circles) and after (open circles) toxin treatment, respectively. F. Effect of CvIV4 on rate of recovery from inactivation at −100 mV. Cells held at −100 mV were given a depolarizing prepulse to 0 mV for 20-ms followed by a step back to −100 mV with increasing duration to allow channels to recover from inactivation, followed by a 20-ms test depolarization of 0 mV to activate those channels that had recovered from inactivation. Data points presented as a fraction of the maximum recovered current were fitted with single exponential function to estimate the time constant. The time constants were 58.7±15.4 and 48.2±15.3 ms before (filled circles) and after (open circles) toxin treatment, respectively.

Figure 8. Effects of CvIV4 on activation and inactivation of Na_v1.2, Na_v1.3, Na_v1.4 and Na_v1.5.

50-ms depolarizing pulses were used to elicit Na⁺ current from channel isoforms expressed in HEK cells before and after the application of 1 µM CvIV4. Cells were held at −100 mV. Depolarizing potentials ranged from −100 to +120 mV in 5-mV increments. A. Currents not inactivated at 5 ms in the presence of 1 µM toxin were plotted as the percentage increase in the fraction of current at 5 ms (I_{5 ms}). B. Effects of CvIV4 on steady-state activation and inactivation of Na⁺ channel isoforms.

Figure 9. Comparison of CvIV4 with Old World α-toxins that modulate Na_v1.7.

CvIV4 is aligned with ODI, BmK MI, AahII and LqhIII, Old World α-toxins that slow the fast inactivation of Na_v1.7. The biological activity of CvIV4 is similar to these peptides, but its primary structure is not. Gaps were introduced to align cysteine residues (white font with black background). Hydrophobic residues (dark shaded background) that are critical for α-toxin structure and function are conserved in CvIV4 at positions 5, 14, 22, 36, 43 and 49 . CvIV4 shares a lysine (light shaded background) with AahII (position 60) that is critical for the biological activity of AahII . Additional amino acids that are identical among toxins are marked with an asterisk (*). GenBank accession numbers: CvIV4 (JF938594), ODI (P84646), BmK MI (P45697), AahII (P01484), LqhII (P59355). MAFFT version 6 used for sequence alignment (http://mafft.cbrc.jp/alignment/software/).

Figure 10. Effects of CvIV4 on mutant Na_v1.7 channels expressed in HEK293 cells.

A. Substitution of negatively charged amino acids in the Domain IV S3–S4 loop with neutral residues (D1586A, E1589Q) reduces the effects of 1 µM CvIV4 on Na_v1.7. B. Dose-response curves for CvIV4 on wildtype and mutant Na_v1.7 channels.