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Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels.

Campos FV, Chanda B, Beirão PS, Bezanilla F - J. Gen. Physiol. (2008)

Bottom Line: We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes.While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV.These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
Alpha-scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

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Effect of Ts3 on sodium currents from Xenopus oocytes expressing Nav1.4. (A) I-V curves obtained before (white circles) and after (black circles) the treatment with 200 nM of Ts3. Both curves were normalized by the maximal current obtained in the presence of toxin. Data shown as mean ± SEM (n = 4). Sodium currents were recorded with 20-ms pulses varying from −100 to +40 mV, which were followed and preceded by a 100-ms pulse to −100 mV. The holding potential was −90 mV. (B) Representative traces obtained in control conditions. (C) Representative traces obtained after the treatment with 200 nM of Ts3. (D) Superimposed recordings obtained at −20 mV before and after the treatment with Ts3. The traces were normalized by the peak value. Experiments were performed at 14°C.
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fig1: Effect of Ts3 on sodium currents from Xenopus oocytes expressing Nav1.4. (A) I-V curves obtained before (white circles) and after (black circles) the treatment with 200 nM of Ts3. Both curves were normalized by the maximal current obtained in the presence of toxin. Data shown as mean ± SEM (n = 4). Sodium currents were recorded with 20-ms pulses varying from −100 to +40 mV, which were followed and preceded by a 100-ms pulse to −100 mV. The holding potential was −90 mV. (B) Representative traces obtained in control conditions. (C) Representative traces obtained after the treatment with 200 nM of Ts3. (D) Superimposed recordings obtained at −20 mV before and after the treatment with Ts3. The traces were normalized by the peak value. Experiments were performed at 14°C.

Mentions: Fig. 1 shows sodium currents recorded at 14°C.Fig. 1 A compares the I-V curves obtained in control conditions and in the presence of 200 nM of Ts3. As reported previously (Campos et al., 2004), the presence of Ts3 increased the amplitude of the sodium current but did not change significantly the voltage dependence of the activation. Nav1.4 sodium current decay with a fast time course in control conditions (Fig. 1 B). In the presence of 200 nM of Ts3 the decay of the currents became slower and the peak current increased (Fig. 1, C and D). As the same effect was observed at a higher concentration of Ts3 (1 μM, not depicted) we considered 200 nM to be a saturating concentration, and this same amount of toxin was used for all the experiments unless otherwise stated. As we have shown before, Ts3 binds tightly to the channel and is removed only if high depolarizing pulses are applied, with higher voltages than that needed for full activation.


Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels.

Campos FV, Chanda B, Beirão PS, Bezanilla F - J. Gen. Physiol. (2008)

Effect of Ts3 on sodium currents from Xenopus oocytes expressing Nav1.4. (A) I-V curves obtained before (white circles) and after (black circles) the treatment with 200 nM of Ts3. Both curves were normalized by the maximal current obtained in the presence of toxin. Data shown as mean ± SEM (n = 4). Sodium currents were recorded with 20-ms pulses varying from −100 to +40 mV, which were followed and preceded by a 100-ms pulse to −100 mV. The holding potential was −90 mV. (B) Representative traces obtained in control conditions. (C) Representative traces obtained after the treatment with 200 nM of Ts3. (D) Superimposed recordings obtained at −20 mV before and after the treatment with Ts3. The traces were normalized by the peak value. Experiments were performed at 14°C.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483334&req=5

fig1: Effect of Ts3 on sodium currents from Xenopus oocytes expressing Nav1.4. (A) I-V curves obtained before (white circles) and after (black circles) the treatment with 200 nM of Ts3. Both curves were normalized by the maximal current obtained in the presence of toxin. Data shown as mean ± SEM (n = 4). Sodium currents were recorded with 20-ms pulses varying from −100 to +40 mV, which were followed and preceded by a 100-ms pulse to −100 mV. The holding potential was −90 mV. (B) Representative traces obtained in control conditions. (C) Representative traces obtained after the treatment with 200 nM of Ts3. (D) Superimposed recordings obtained at −20 mV before and after the treatment with Ts3. The traces were normalized by the peak value. Experiments were performed at 14°C.
Mentions: Fig. 1 shows sodium currents recorded at 14°C.Fig. 1 A compares the I-V curves obtained in control conditions and in the presence of 200 nM of Ts3. As reported previously (Campos et al., 2004), the presence of Ts3 increased the amplitude of the sodium current but did not change significantly the voltage dependence of the activation. Nav1.4 sodium current decay with a fast time course in control conditions (Fig. 1 B). In the presence of 200 nM of Ts3 the decay of the currents became slower and the peak current increased (Fig. 1, C and D). As the same effect was observed at a higher concentration of Ts3 (1 μM, not depicted) we considered 200 nM to be a saturating concentration, and this same amount of toxin was used for all the experiments unless otherwise stated. As we have shown before, Ts3 binds tightly to the channel and is removed only if high depolarizing pulses are applied, with higher voltages than that needed for full activation.

Bottom Line: We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes.While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV.These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
Alpha-scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.

Show MeSH
Related in: MedlinePlus