Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channels

Abstract

1. Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins. 2. We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660 nM and modify the resting potentials. 3. The effect on Na+ currents in rat cardiomyocytes was studied using the patch-clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78 nM, respectively. 4. On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8 microM), shift the steady-state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials. 5. The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins. 6. We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady-state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels.

Figure 1

Amino acid sequence determination of toxins. BgII: Direct sequencing by Edman degradation of reduced and alkylated toxin provided unequivocal identification of the 21 first amino acids, shown by underlining (-d->). Three additional fragments were obtained by HPLC separation of toxin BgII digested with Arg-C endopeptidase, which gave peptides with molecular masses of 1401.6, 1059.4 and 1259.4, respectively. The sequences were obtained by ionization, using MSIMS mass spectrometry, and correspond to sequence from positions 15 to 27, 28 to 36 and 37 to 48. The entire sequence corresponds exactly to that reported earlier (Loret et al., 1994). BgIII: Direct sequencing of reduced and alkylated toxin provided the 29 first amino acid residues, as indicated by the underlining (-d->). Three additional overlapping peptides were obtained by Arg-C endopeptidase digestion conducted as mentioned above for BgII, in which the only difference was found in the peptide corresponding to positions 15 to 27 (molecular mass 1402.6) where an aspartic acid substitutes an asparagine in position 16. The MSIMS spectrometry analysis confirmed the sequences shown, which also corresponds exactly to those earlier reported (Loret et al., 1994). The numbers on top of the sequences indicate the positions of the amino acids in the sequences.

Figure 2

Comparison of the amino acid sequences of BgII, BgIII with other sea anemone toxins. BgII and BgIII have been purified from the sea anemone Bunodosoma granulifera (Loret et al., 1994), ATXII from Anemonia sulcata (Wunderer et al., 1976), ApA and ApB from Anthopleura xanthogrammica (Norton, 1981; Reimer et al., 1985; Tanaka et al., 1977) and ShI from Stichodactyla helianthus (Kem et al., 1989; Wilcox et al., 1993). Identical amino acids are indicated with a black background, homologous amino acids are indicated with a gray background and ‘%id' stands for the percentage of identity in comparison to BgII. Dashes represent gaps.

Figure 3

Effects of Bunodosoma granulifera toxins on the resting and action potential characteristics of rat ventricular strips. Both BgII (A) and BgIII (B) were applied at a concentration of 10 μM. Within 1 min, both toxins markedly increased the action potential duration. After a longer time (5 min) the resting potential was decreased. Note that the increase in action potential duration and the decrease in resting potential were more marked with BgII.

Figure 4

Effect of BgII and BgIII toxins on Na⁺ currents in rat ventricular cardiomyocytes. (A,B) Time course of the effects of 10 μM BgII or 10 μM BgIII on I_Na of a rat ventricular cardiomyocyte. Points represent I_Na density values (pA/pF) at −40 mV. The holding potential was −100 mV. The thick horizontal line indicates the period during which the cell was superfused with the toxin. The inset on the top shows current traces recorded in control condition and during the action of toxins. Note in these cells, the marked increase in current amplitude and the slowing of inactivation. (C,D) Averaged normalized current-voltage relationship of I_Na established in six cells under control conditions and in the presence of 0.1 and 10 μM BgII (A) or 0.1 10 μM BgIII (B).

Figure 5

Effect of BgII and BgIII toxins on the cloned hH1 channel expressed in Xenopus oocytes. (A – C) Representative family of current traces evoked in an oocyte expressing hH1 channel by depolarizations ranging between −70 to+45 mV, using 5 mV increments, from a holding potential of −90 mV, in control conditions (A), in the presence of 1 μM BgII toxin (B) and in the presence of 60 μM BgIII toxin (C). Note that the slowing of inactivation is more marked with BgII than with BgIII and also the emergence of outward currents in the presence of both toxins. (D) Averaged normalized current-voltage relationship of hH1 in control conditions, in the presence of 5 μM of BgII toxin or 60 μM of BgIII toxin. (E) Averaged normalized steady-state inactivation in control condition, in the presence of 5 μM of BgII toxin or 60 μM of BgIII toxin. (F) Average normalized steady-state activation in the absence of toxins, in the presence of 5 μM of BgII toxin or 60 μM of BgIII toxin. In (D – F), data are mean±s.e.mean of n=20, n=6 and n=4 experiments, respectively.

Figure 6

Concentration dependence of the slowing of inactivation and of the modification of the reversal potential induced by BgII and BgIII toxins on hH1 Na⁺ channels. (A) Current traces were evoked by a step depolarization to 0 mV lasting 25 ms from a holding potential of −90 mV, in the absence (control) and in the presence of increasing concentrations of BgII toxin (as indicated), using the same oocyte. (B) Same protocol as in (A) but in the absence (control) and in the presence of increasing concentrations of BgIII toxin (as indicated), in the same oocyte. (C) Averaged time constant of inactivation (τ) plotted vs concentration of BgII toxin. Time constants of inactivation were calculated from a first order exponential fit of current traces evoked by a step depolarization to 0 mV from a holding potential of −90 mV. The EC₅₀ value determined by a sigmoidal fit is 382±140 nM. Data are the mean±s.e.mean of at least three experiments. (D) Averaged time constant inactivation (τ) plotted vs concentration of BgIII toxin. Time constants of inactivation were calculated as in (C). The EC₅₀ value as determined by a sigmoidal fit of τ is 7.8±1.2 μM. (E) Averaged reversal potential plotted vs concentration of BgII toxin. The EC₅₀ value determined by a sigmoidal fit is 340±70 nM. (F) Averaged reversal potential plotted vs concentration of BgIII toxin. The EC₅₀ value determined by a sigmoidal fit is 8.4±0.3 μM. Data are the mean±s.e.mean of at least three experiments at each concentration.