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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 on the fluorescence changes that track the movement of the S4 segment of domain IV of mutant channels L1439C stained with TMRM. Traces were recorded as described in Fig. 6. (A) Sodium currents recorded at −20 mV before and after the treatment with 200 nM Ts3. (B) Fluorescence signals obtained at −20 mV before (black trace) and after (gray trace) the treatment with 200 nM of Ts3. The signals are shown as ΔF/F (%), where F is the fluorescence background. The arrow indicates the direction in which fluorescence increases. These experiments were performed at room temperature. (C) F-V curves obtained before (white symbols) and after (black symbols) the treatment with 200 nM of Ts3 (mean ± SEM, n = 3; paired experiments). Solid lines are the curves obtained by fitting the data with the function 3.
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fig8: Effects of Ts3 on the fluorescence changes that track the movement of the S4 segment of domain IV of mutant channels L1439C stained with TMRM. Traces were recorded as described in Fig. 6. (A) Sodium currents recorded at −20 mV before and after the treatment with 200 nM Ts3. (B) Fluorescence signals obtained at −20 mV before (black trace) and after (gray trace) the treatment with 200 nM of Ts3. The signals are shown as ΔF/F (%), where F is the fluorescence background. The arrow indicates the direction in which fluorescence increases. These experiments were performed at room temperature. (C) F-V curves obtained before (white symbols) and after (black symbols) the treatment with 200 nM of Ts3 (mean ± SEM, n = 3; paired experiments). Solid lines are the curves obtained by fitting the data with the function 3.

Mentions: To further verify this effect, we used a different mutant channel (L1439C). In this mutant, the binding affinity for the toxin was similar to that observed in wild-type channels, thus allowing a more direct comparison. The fluorescence of unmodified L1439C shows two components, indicating that the fluorophore samples two different environments (Chanda and Bezanilla, 2002). The early component causes an increase in fluorescence and late component quenches the fluorescence. This causes a characteristic “hooked” tails seen in fluorescence recordings from L1439C. Fig. 8 compares traces of sodium currents (A) and fluorescence signals (B) obtained at −20 mV before and after the treatment with Ts3. In the presence of Ts3, the fluorescence of L1439C showed an increase in fluorescence. We interpret this increase to suggest that fluorescence of the second quenching component is diminished considerably. This two-step movement interpretation also accounts for diminished size of the hook in the tail in toxin-modified channels. Ts3 shifted the F-V curves of L1439C channels by 20 mV in the positive direction (Fig. 8 C; see Table II for fitted parameters).


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 on the fluorescence changes that track the movement of the S4 segment of domain IV of mutant channels L1439C stained with TMRM. Traces were recorded as described in Fig. 6. (A) Sodium currents recorded at −20 mV before and after the treatment with 200 nM Ts3. (B) Fluorescence signals obtained at −20 mV before (black trace) and after (gray trace) the treatment with 200 nM of Ts3. The signals are shown as ΔF/F (%), where F is the fluorescence background. The arrow indicates the direction in which fluorescence increases. These experiments were performed at room temperature. (C) F-V curves obtained before (white symbols) and after (black symbols) the treatment with 200 nM of Ts3 (mean ± SEM, n = 3; paired experiments). Solid lines are the curves obtained by fitting the data with the function 3.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Effects of Ts3 on the fluorescence changes that track the movement of the S4 segment of domain IV of mutant channels L1439C stained with TMRM. Traces were recorded as described in Fig. 6. (A) Sodium currents recorded at −20 mV before and after the treatment with 200 nM Ts3. (B) Fluorescence signals obtained at −20 mV before (black trace) and after (gray trace) the treatment with 200 nM of Ts3. The signals are shown as ΔF/F (%), where F is the fluorescence background. The arrow indicates the direction in which fluorescence increases. These experiments were performed at room temperature. (C) F-V curves obtained before (white symbols) and after (black symbols) the treatment with 200 nM of Ts3 (mean ± SEM, n = 3; paired experiments). Solid lines are the curves obtained by fitting the data with the function 3.
Mentions: To further verify this effect, we used a different mutant channel (L1439C). In this mutant, the binding affinity for the toxin was similar to that observed in wild-type channels, thus allowing a more direct comparison. The fluorescence of unmodified L1439C shows two components, indicating that the fluorophore samples two different environments (Chanda and Bezanilla, 2002). The early component causes an increase in fluorescence and late component quenches the fluorescence. This causes a characteristic “hooked” tails seen in fluorescence recordings from L1439C. Fig. 8 compares traces of sodium currents (A) and fluorescence signals (B) obtained at −20 mV before and after the treatment with Ts3. In the presence of Ts3, the fluorescence of L1439C showed an increase in fluorescence. We interpret this increase to suggest that fluorescence of the second quenching component is diminished considerably. This two-step movement interpretation also accounts for diminished size of the hook in the tail in toxin-modified channels. Ts3 shifted the F-V curves of L1439C channels by 20 mV in the positive direction (Fig. 8 C; see Table II for fitted parameters).

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