Figure 1.
HPLC purification of mHWTX-IV.
The peaks marked by * contain mHWTX-IV. (A) Elution profile of Ornithoctonus huwena Wang venom by ion-exchange HPLC. (B) Isolation of mHWTX-IV by RP-HPLC on a C18 column in a gradient of 10–50% acetonitrile over 50 min. (C) Further purification of mHWTX-IV by a repetitive RP-HPLC with a gradient of 28–40% acetonitrile over 30 min.
Figure 2.
Mass spectrometry of mHWTX-IV and HWTX-IV.
(A) Molecular mass of mHWTX-IV detected by mass spectrometry, 4089.64 Da. (B) Molecular mass of HWTX-IV, 4107.94 Da. (C) Monoisotopic mass spectrum of a mixture of mHWTX-IV and HWTX-IV.
Figure 3.
MS/MS spectrum for sequence determination of the first fragment of mHWTX-IV (A) and of HWTX-IV (B) after digestion with trypsin.
The sequence was derived from the series of b-ions and y-ions.
Figure 4.
MS/MS spectrum for sequence determination of the N-terminal fragment of reduced and carboxamidomethylated mHWTX-IV.
Sequence was derived from the series of b-ions and y-ions.
Figure 5.
Effects of HWTX-IV and mHWTX-IV on sodium channel in rat DRG neurons.
All current traces were evoked by a 50-ms step depolarization to −10 mV from a holding potential of −80 mV at every second. The currents of TTX-S were significantly reduced by 1-µM HWTX-IV (A) and 1-μM mHWTX-IV (C); 10-µM HWTX-IV (B) and 10-μM mHWTX-IV (D) had no effect on TTX-R sodium currents. (E) shows the concentration dependent inhibition of TTX-S sodium currents on DRG neurons by HWTX-IV (B) and mHWTX-IV. Control for each panel means no toxin treatment. Every data point (mean ± S.E) was obtained from five separate experimental cells.
Figure 6.
Effects of HWTX-IV and mHWTX-IV on the kinetics of TTX-S sodium channels in rat DRG neurons.
Time course for block of TTX-S currents and reversal of block by mHWTX-IV (A) and HWTX-IV (B). Current-voltage (I–V) relationships of sodium currents before and after adding 100 nM mHWTX-IV (AC), HWTX-IV (BD). HWTX-IV and mHWTX-IV showed no obvious difference on the steady-state activation (CE) and inactivation (DF). Control for each panel means no toxin treatment. Every data point (mean ± S.E.) was obtained from five separate experimental cells. The data points for both activation and inactivation kinetics were well fitted with the Boltzmann equation.
Figure 7.
Sodium current recording after the application of mHWTX-IV detected by strong depolarization.
(A) DRG neurons were held at −80 mV and then with a 50 ms test pulse of −10 mV. A +200 mV 500 ms strong depolarization applied after cell back held at −80 mV. Finally, a −10 mV pulse used to test the currents. After the +200 mV strong depolarization, no current was induced. (B) Recovery of current from Nav1.7 following strong depolarization in the presence of 1 µM HWTX-IV or 1 µM mHWTX-IV. HEK293 cells were depolarized to +200, +150, +100 and +50 mV. Control for each panel means no toxin treatment. Every data point (mean ± S.E.) was obtained from five separate experimental cells.