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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 fluorescence changes that track the movement of the S4 segment of domains II and III, using the mutant channel S660C and L1115C stained with TMRM. Traces were obtained as described in Fig. 7. (A) F-V curves obtained from S660C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with the function 3. (B) F-V curves obtained from L1115C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with function 3.These experiments were performed at room temperature.
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fig10: Effects of Ts3 in the fluorescence changes that track the movement of the S4 segment of domains II and III, using the mutant channel S660C and L1115C stained with TMRM. Traces were obtained as described in Fig. 7. (A) F-V curves obtained from S660C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with the function 3. (B) F-V curves obtained from L1115C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with function 3.These experiments were performed at room temperature.

Mentions: To verify whether the movement of the other voltage sensors is affected by Ts3, we used mutants in the S4 segments of domain I (S216C), domain II (S660C), and domain III (L1115C). This approach is important because several evidences suggest that the four voltage sensors of the sodium channel move in a cooperative way in response to changes in the membrane potential (Chanda et al., 2004; Campos et al., 2007). Thus, by inhibiting the movement of the fourth voltage sensor, Ts3 could also affect the movement of the other voltage sensors. Fig. 9 shows the effects of Ts3 on S216C channels. Ts3 shifted the F-V curves of S216C channels by 20 mV in the positive direction (Fig. 9 A). Moreover, the slope of the curve became steeper after the treatment with the toxin (see Table II for fitted parameters). On the other hand, the fluorescence signal kinetics was not significantly affected by the toxin in any of the studied potentials (Fig. 9, B–D). The fluorescence signal recorded at each potential was normalized by the maximal fluorescence recorded in control conditions or in the presence of Ts3. To analyze the effects of the Ts3 in the fluorescence changes of the voltage sensor of domains II and III we used the S660C and L1115C mutants. Fig. 10 A compares the F-V curves obtained before and after the treatment with Ts3 for the mutant S660C. There was no shift on the curve in this case, but its steepness was slightly, yet significantly, increased (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 in the fluorescence changes that track the movement of the S4 segment of domains II and III, using the mutant channel S660C and L1115C stained with TMRM. Traces were obtained as described in Fig. 7. (A) F-V curves obtained from S660C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with the function 3. (B) F-V curves obtained from L1115C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with function 3.These experiments were performed at room temperature.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Effects of Ts3 in the fluorescence changes that track the movement of the S4 segment of domains II and III, using the mutant channel S660C and L1115C stained with TMRM. Traces were obtained as described in Fig. 7. (A) F-V curves obtained from S660C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with the function 3. (B) F-V curves obtained from L1115C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with function 3.These experiments were performed at room temperature.
Mentions: To verify whether the movement of the other voltage sensors is affected by Ts3, we used mutants in the S4 segments of domain I (S216C), domain II (S660C), and domain III (L1115C). This approach is important because several evidences suggest that the four voltage sensors of the sodium channel move in a cooperative way in response to changes in the membrane potential (Chanda et al., 2004; Campos et al., 2007). Thus, by inhibiting the movement of the fourth voltage sensor, Ts3 could also affect the movement of the other voltage sensors. Fig. 9 shows the effects of Ts3 on S216C channels. Ts3 shifted the F-V curves of S216C channels by 20 mV in the positive direction (Fig. 9 A). Moreover, the slope of the curve became steeper after the treatment with the toxin (see Table II for fitted parameters). On the other hand, the fluorescence signal kinetics was not significantly affected by the toxin in any of the studied potentials (Fig. 9, B–D). The fluorescence signal recorded at each potential was normalized by the maximal fluorescence recorded in control conditions or in the presence of Ts3. To analyze the effects of the Ts3 in the fluorescence changes of the voltage sensor of domains II and III we used the S660C and L1115C mutants. Fig. 10 A compares the F-V curves obtained before and after the treatment with Ts3 for the mutant S660C. There was no shift on the curve in this case, but its steepness was slightly, yet significantly, increased (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