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Toxins (Basel)
2015 Jun 30;77:2494-513. doi: 10.3390/toxins7072494.
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Three Peptide Modulators of the Human Voltage-Gated Sodium Channel 1.7, an Important Analgesic Target, from the Venom of an Australian Tarantula.
Chow CY
,
Cristofori-Armstrong B
,
Undheim EA
,
King GF
,
Rash LD
.
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Voltage-gated sodium (NaV) channels are responsible for propagating action potentials in excitable cells. NaV1.7 plays a crucial role in the human pain signalling pathway and it is an important therapeutic target for treatment of chronic pain. Numerous spider venom peptides have been shown to modulate the activity of NaV channels and these peptides represent a rich source of research tools and therapeutic lead molecules. The aim of this study was to determine the diversity of NaV1.7-active peptides in the venom of an Australian Phlogius sp. tarantula and to characterise their potency and subtype selectivity. We isolated three novel peptides, μ-TRTX-Phlo1a, -Phlo1b and -Phlo2a, that inhibit human NaV1.7 (hNaV1.7). Phlo1a and Phlo1b are 35-residue peptides that differ by one amino acid and belong in NaSpTx family 2. The partial sequence of Phlo2a revealed extensive similarity with ProTx-II from NaSpTx family 3. Phlo1a and Phlo1b inhibit hNaV1.7 with IC50 values of 459 and 360 nM, respectively, with only minor inhibitory activity on rat NaV1.2 and hNaV1.5. Although similarly potent at hNaV1.7 (IC50 333 nM), Phlo2a was less selective, as it also potently inhibited rNaV1.2 and hNaV1.5. All three peptides cause a depolarising shift in the voltage-dependence of hNaV1.7 activation.
Figure 1. (A) Chromatogram resulting from fractionation of crude Phlogius sp. venom using C18 RP-HPLC. The numbers correspond to collected fractions, and active fractions are shaded grey; (B) Representative whole-cell current traces obtained from hNaV1.7 channels expressed in Xenopus oocytes. Current traces are shown in the absence and presence of F18, 19 and 23, and after ~3 min of peptide washout. Sodium currents were evoked using the voltage protocol shown above the central trace.
Figure 2. Chromatograms resulting from final purification of hNaV1.7-active peptides using C18 RP-HPLC. Absorbance was monitored at 214 and 280 nm. Inserts show MALDI-TOF mass spectra, with the monoisotopic M + H+ for each peptide indicated.
Figure 3. Positive-ion MALDI-ISD spectra of (A) F18, (B) F19, and (C) F23, obtained using 1,5-DAN matrix. The deduced peptide sequences are shown above the spectra.
Figure 4. Alignment of mature toxin sequences obtained by BLAST search of the partial sequences of F18 and F19 obtained from MALDI-TOF MS (highlighted in yellow) against a Phlogius sp. venom-gland transcriptome. A sequence logo for this alignment is shown, with conserved Cys residues that form the ICK motif highlighted in red or shaded grey. The theoretical M + H+ mass is shown for each oxidised peptide (assuming non-amidated C-termini). The M + H+ values in bold are those for the sequences of F18 and F19.
Figure 5. (A) MALDI-TOF mass spectra of tryptic digests of Phlo1a (upper panel) and Phlo1b (bottom panel). Amino acid positions (and number of missed cleavages) are indicated above the peak masses; (B) MS/MS analysis of the Phlo1a precursor ions 1805.20 and (C) 2215.32; (D) Comparison of the observed and theoretical M + H+ for the ions observed, their corresponding residue positions and fragment sequence.
Figure 6. (A) MALDI-TOF MS analysis of peptides fragments from tryptic digest of Phlo2a. Amino acid positions and the number of missed cleavage are indicated above the peak masses. (B) MS/MS analysis of tryptic peptides with m/z 1311.72. (C) Comparison of observed and theoretical M + H+ for tryptic fragments of alkylated Phlo2a obtained using MALDI-TOF MS.
Figure 7. MALDI-TOF mass spectra obtained at different times points (from 1 to 60 min) during CPY digestion of reduced/alkylated (A) Phlo1a and (B) Phlo1b.
Figure 8. (A) Summary of the experimental evidence for amino acid sequences of Phlo1a and Phlo1b, and partial sequence of Phlo2a, in comparison to predictions from the venom-gland transcriptome (confirmed sequence in bold); (B) Sequence alignment of Phlo1a and Phlo1b with other members of the NaSpTx2; (C) Sequence alignment of Phlo2a with other members of the NaSpTx3. Cysteine residues are shaded.
Figure 9. Effects of Phlogius peptides on hNaV1.7 expressed in oocytes. (A) Whole-cell current traces in absence (control) and presence of 0.1 or 1 μM peptide. Currents were evoked by a 50-ms step depolarisation to 0 mV from a holding potential of −80 mV every 10 s. (B) Time course for inhibition of hNaV1.7 by different peptide concentrations. Time controls show stable current amplitude in the absence of peptide. (C) Concentration-effect curves for inhibition of hNaV1.7 by Phlo1a, Phlo1b and Phlo2a (n = 5–7). Data are mean ± S.E.M. Hill coefficients are shown in parentheses.
Figure 10. Effect of Phlo1a (A), Phlo1b (B) and Phlo2a (C) on the I-V relationship for hNaV1.7. Oocytes were held at −80 mV, and sodium currents were elicited using 50-ms depolarising steps from −60 to +70 mV in 10 mV increments. I-V relationships were obtained in the absence (control, ●) and presence of each peptide at 300 nM (■) and 1 μM (▲). All currents were normalised to the maximum control peak current for each oocyte. Data are mean ± S.E.M. (n = 6). Insets in panels A–C show the voltage-dependence of inhibition. (D) Quantitation of the effect of each peptide on the V0.5 (in mV) of hNaV1.7.
Figure 11. Effects of Phlo1a and Phlo1b on (A) rNaV1.2 and (B) hNaV1.5 expressed in Xenopus oocytes. Currents were evoked by a 50-ms step depolarisation to 0 mV from a holding potential of −80 mV every 10 s. (C) Effect of Phlo2a on rNaV1.2 and hNaV1.5 currents in Xenopus oocytes. (D) Concentration-effect curves for inhibition of rNaV1.2 and hNaV1.5 currents by Phlo2a (n = 5). Data are presented as mean ± S.E.M and the Hill coefficients are shown in parentheses.
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