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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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Ts3 voltage-dependent displacement. (A) Pulse protocol used to remove the bound Ts3. A strong depolarizing pulse, varying from +60 to +180 mV during 20 ms, was applied just after a −20 mV test pulse. Between pulses the oocytes were held at −90 mV, and the protocol was repeated at least 15 times. (B) Traces (gray lines) obtained in control conditions, in the presence of Ts3 (Pulse #0) and after 14 successive depolarization to +120 mV, by applying the protocol described above (Pulse #14) in the absence of Ts3. Black lines shows the curves obtained by fitting the data with function 1 (see Materials and methods). (C) Voltage dependence of toxin removal, obtained by applying the pulse protocol described in A. The graph shows the data obtained from a representative experiment. The number of pulses needed to an e-fold displacement was calculated by fitting the slow component contribution decay with a single exponential function.
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fig2: Ts3 voltage-dependent displacement. (A) Pulse protocol used to remove the bound Ts3. A strong depolarizing pulse, varying from +60 to +180 mV during 20 ms, was applied just after a −20 mV test pulse. Between pulses the oocytes were held at −90 mV, and the protocol was repeated at least 15 times. (B) Traces (gray lines) obtained in control conditions, in the presence of Ts3 (Pulse #0) and after 14 successive depolarization to +120 mV, by applying the protocol described above (Pulse #14) in the absence of Ts3. Black lines shows the curves obtained by fitting the data with function 1 (see Materials and methods). (C) Voltage dependence of toxin removal, obtained by applying the pulse protocol described in A. The graph shows the data obtained from a representative experiment. The number of pulses needed to an e-fold displacement was calculated by fitting the slow component contribution decay with a single exponential function.

Mentions: Fig. 2 A shows the pulse protocol used to remove the toxin from muscle sodium channels.Fig. 2 B compares the sodium currents obtained by applying this protocol in control conditions, after the treatment with saturating Ts3, and after 14 depolarizing pulses to +120 mV following the washout of Ts3. The close similarity of the latter with the control record is accounted for by the displacement of Ts3 from the channel, since a further addition of the toxin restores its typical effect (unpublished data). To quantify the removal, the decay of each trace was fitted to a double exponential function (see Materials and methods). In control conditions the contribution of the fast component to the current decay was much larger than the contribution of the slow component (a = 93.7%; b = 6.3%; τf = 1.1 ms; τs = 16.8 ms).


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)

Ts3 voltage-dependent displacement. (A) Pulse protocol used to remove the bound Ts3. A strong depolarizing pulse, varying from +60 to +180 mV during 20 ms, was applied just after a −20 mV test pulse. Between pulses the oocytes were held at −90 mV, and the protocol was repeated at least 15 times. (B) Traces (gray lines) obtained in control conditions, in the presence of Ts3 (Pulse #0) and after 14 successive depolarization to +120 mV, by applying the protocol described above (Pulse #14) in the absence of Ts3. Black lines shows the curves obtained by fitting the data with function 1 (see Materials and methods). (C) Voltage dependence of toxin removal, obtained by applying the pulse protocol described in A. The graph shows the data obtained from a representative experiment. The number of pulses needed to an e-fold displacement was calculated by fitting the slow component contribution decay with a single exponential function.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Ts3 voltage-dependent displacement. (A) Pulse protocol used to remove the bound Ts3. A strong depolarizing pulse, varying from +60 to +180 mV during 20 ms, was applied just after a −20 mV test pulse. Between pulses the oocytes were held at −90 mV, and the protocol was repeated at least 15 times. (B) Traces (gray lines) obtained in control conditions, in the presence of Ts3 (Pulse #0) and after 14 successive depolarization to +120 mV, by applying the protocol described above (Pulse #14) in the absence of Ts3. Black lines shows the curves obtained by fitting the data with function 1 (see Materials and methods). (C) Voltage dependence of toxin removal, obtained by applying the pulse protocol described in A. The graph shows the data obtained from a representative experiment. The number of pulses needed to an e-fold displacement was calculated by fitting the slow component contribution decay with a single exponential function.
Mentions: Fig. 2 A shows the pulse protocol used to remove the toxin from muscle sodium channels.Fig. 2 B compares the sodium currents obtained by applying this protocol in control conditions, after the treatment with saturating Ts3, and after 14 depolarizing pulses to +120 mV following the washout of Ts3. The close similarity of the latter with the control record is accounted for by the displacement of Ts3 from the channel, since a further addition of the toxin restores its typical effect (unpublished data). To quantify the removal, the decay of each trace was fitted to a double exponential function (see Materials and methods). In control conditions the contribution of the fast component to the current decay was much larger than the contribution of the slow component (a = 93.7%; b = 6.3%; τf = 1.1 ms; τs = 16.8 ms).

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