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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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Effects of Ts3 in the charge–voltage relationship. White symbols represent the Q-V curve obtained in control conditions (mean ± SEM, n = 3), and black symbols the curve obtained after the treatment with 200 nM of Ts3 (mean ± SEM, n = 3). The amount of charge recorded in each potential was normalized to the maximum charge recorded before the treatment with Ts3. Solid black lines are the curves obtained by fitting the data with a double Boltzmann function (function 2). Dotted gray lines are the curves obtained by fitting the data with a single Boltzmann function. Experiments were performed at 14°C.
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fig6: Effects of Ts3 in the charge–voltage relationship. White symbols represent the Q-V curve obtained in control conditions (mean ± SEM, n = 3), and black symbols the curve obtained after the treatment with 200 nM of Ts3 (mean ± SEM, n = 3). The amount of charge recorded in each potential was normalized to the maximum charge recorded before the treatment with Ts3. Solid black lines are the curves obtained by fitting the data with a double Boltzmann function (function 2). Dotted gray lines are the curves obtained by fitting the data with a single Boltzmann function. Experiments were performed at 14°C.

Mentions: Fig. 6 compares the Q-V relationships obtained before and after the treatment with Ts3. In the presence of Ts3 the total gating charge was reduced by 30%, as reported before with another site 3 toxin from the sea anemone (Sheets and Hanck, 1995). It is expected that at least part of this inhibited charge derives from the movement of the S4 segment of domain IV. The Q-V curves were fitted with either single or double Boltzmann functions (Eq. 2, making qb equal to zero or leaving it as a variable, respectively). The fitting with a double Boltzmann function consistently gave results with lesser deviation from the experimental points, as measured by the sum of squared residuals, thus suggesting the presence of a component that contributes with 30% of the charge and with a V1/2 of −70 mV. More interesting is the fact that Ts3 decreases mainly the charge of this component (Table I). In one experiment this component disappeared altogether in the presence of Ts3. This observation will benefit from more detailed investigation.


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)

Effects of Ts3 in the charge–voltage relationship. White symbols represent the Q-V curve obtained in control conditions (mean ± SEM, n = 3), and black symbols the curve obtained after the treatment with 200 nM of Ts3 (mean ± SEM, n = 3). The amount of charge recorded in each potential was normalized to the maximum charge recorded before the treatment with Ts3. Solid black lines are the curves obtained by fitting the data with a double Boltzmann function (function 2). Dotted gray lines are the curves obtained by fitting the data with a single Boltzmann function. 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

fig6: Effects of Ts3 in the charge–voltage relationship. White symbols represent the Q-V curve obtained in control conditions (mean ± SEM, n = 3), and black symbols the curve obtained after the treatment with 200 nM of Ts3 (mean ± SEM, n = 3). The amount of charge recorded in each potential was normalized to the maximum charge recorded before the treatment with Ts3. Solid black lines are the curves obtained by fitting the data with a double Boltzmann function (function 2). Dotted gray lines are the curves obtained by fitting the data with a single Boltzmann function. Experiments were performed at 14°C.
Mentions: Fig. 6 compares the Q-V relationships obtained before and after the treatment with Ts3. In the presence of Ts3 the total gating charge was reduced by 30%, as reported before with another site 3 toxin from the sea anemone (Sheets and Hanck, 1995). It is expected that at least part of this inhibited charge derives from the movement of the S4 segment of domain IV. The Q-V curves were fitted with either single or double Boltzmann functions (Eq. 2, making qb equal to zero or leaving it as a variable, respectively). The fitting with a double Boltzmann function consistently gave results with lesser deviation from the experimental points, as measured by the sum of squared residuals, thus suggesting the presence of a component that contributes with 30% of the charge and with a V1/2 of −70 mV. More interesting is the fact that Ts3 decreases mainly the charge of this component (Table I). In one experiment this component disappeared altogether in the presence of Ts3. This observation will benefit from more detailed investigation.

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