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Synergetic action of domain II and IV underlies persistent current generation in Nav1.3 as revealed by a tarantula toxin.

Tang C, Zhou X, Zhang Y, Xiao Z, Hu Z, Zhang C, Huang Y, Chen B, Liu Z, Liang S - Sci Rep (2015)

Bottom Line: A pre-activated state binding model was proposed to explain the kinetics of toxin-channel interaction.Domain IV constructed the binding site for RTX-VII, while domain II might not participate in interacting with RTX-VII but could determine the efficacy of RTX-VII.Our results based on the use of RTX-VII as a probe suggest that domain II and IV cooperatively contribute to the generation of INaP in Nav1.3.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Hunan Normal University, Changsha, Hunan. 410081, China.

ABSTRACT
The persistent current (INaP) through voltage-gated sodium channels enhances neuronal excitability by causing prolonged depolarization of membranes. Nav1.3 intrinsically generates a small INaP, although the mechanism underlying its generation remains unclear. In this study, the involvement of the four domains of Nav1.3 in INaP generation was investigated using the tarantula toxin α-hexatoxin-MrVII (RTX-VII). RTX-VII activated Nav1.3 and induced a large INaP. A pre-activated state binding model was proposed to explain the kinetics of toxin-channel interaction. Of the four domains of Nav1.3, both domain II and IV might play important roles in the toxin-induced INaP. Domain IV constructed the binding site for RTX-VII, while domain II might not participate in interacting with RTX-VII but could determine the efficacy of RTX-VII. Our results based on the use of RTX-VII as a probe suggest that domain II and IV cooperatively contribute to the generation of INaP in Nav1.3.

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The substitution of the domain II and IV of Nav1.5 with those of Nav1.3 restores RTX-VII efficacy.(a) A 300-ms recording of the currents of Nav1.5 derived chimeric channels in the absence and presence of various concentration of RTX-VII (n = 6–9). The chimeric channels were constructed as follows: one or several domains (DI, DII, DIII or DIV) of Nav1.5 were substituted with the corresponding domain/s of Nav1.3 (see Supplementary Fig. S6). (b) Dose-response curves for RTX-VII enhancing the INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that did not or slightly restored toxin efficacy (steady-state INaP/INaT ratio at the saturated concentration of toxin) (n = 6–9). (c) Dose-response curves for RTX-VII enhancing INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that almost completely restored toxin potency and/or efficacy (n = 6–9). (d) Bars show the fold changes of the apparent EC50 of RTX-VII for each Nav1.5 derived chimeric channels compared with that for wt-Nav1.3 (n = 6–9). (e) Bars show the steady- state INaP/INaT ratio of wt-Nav1.3 and Nav1.5 derived chimeric channels in the presence of saturated concentrations of toxin. This values are 47.92 ± 5.43%, 46.37 ± 8.87%, 6.3 ± 1.82%, 10.60 ± 3.32%, 53.81 ± 5.56%, 12.68 ± 3.67%, 50.88 ± 8.33 and 20.41 ± 3.90% for Nav1.3, 1.5/1.3 DI-DII-DIII-DIV, 1.5/1.3 DIV, 1.5/1.3 DI-DIII-DIV, 1.5/1.3 DI-DII-DIV, 1.5/1.3 DI-DIV, 1.5/1.3 DII-DIV and 1.5/1.3 DI-DIII-DIV&DII-PD, respectively (***p < 0.001, N.S = not significant, when compared with wt-Nav1.3) (n = 6–9).
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f6: The substitution of the domain II and IV of Nav1.5 with those of Nav1.3 restores RTX-VII efficacy.(a) A 300-ms recording of the currents of Nav1.5 derived chimeric channels in the absence and presence of various concentration of RTX-VII (n = 6–9). The chimeric channels were constructed as follows: one or several domains (DI, DII, DIII or DIV) of Nav1.5 were substituted with the corresponding domain/s of Nav1.3 (see Supplementary Fig. S6). (b) Dose-response curves for RTX-VII enhancing the INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that did not or slightly restored toxin efficacy (steady-state INaP/INaT ratio at the saturated concentration of toxin) (n = 6–9). (c) Dose-response curves for RTX-VII enhancing INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that almost completely restored toxin potency and/or efficacy (n = 6–9). (d) Bars show the fold changes of the apparent EC50 of RTX-VII for each Nav1.5 derived chimeric channels compared with that for wt-Nav1.3 (n = 6–9). (e) Bars show the steady- state INaP/INaT ratio of wt-Nav1.3 and Nav1.5 derived chimeric channels in the presence of saturated concentrations of toxin. This values are 47.92 ± 5.43%, 46.37 ± 8.87%, 6.3 ± 1.82%, 10.60 ± 3.32%, 53.81 ± 5.56%, 12.68 ± 3.67%, 50.88 ± 8.33 and 20.41 ± 3.90% for Nav1.3, 1.5/1.3 DI-DII-DIII-DIV, 1.5/1.3 DIV, 1.5/1.3 DI-DIII-DIV, 1.5/1.3 DI-DII-DIV, 1.5/1.3 DI-DIV, 1.5/1.3 DII-DIV and 1.5/1.3 DI-DIII-DIV&DII-PD, respectively (***p < 0.001, N.S = not significant, when compared with wt-Nav1.3) (n = 6–9).

Mentions: Considering the critical role of the DII and DIV of Nav1.3 in the RTX-VII-induced INaP, we assumed that reverse reconstruction of Nav1.3 DII and DIV into Nav1.5 might restore the efficacy of the toxin. A reversal chimeric strategy was used as follows: four domains of Nav1.3 were stepwise reconstructed into the scaffold of Nav1.5 (Supplementary Fig. S6). The nomenclature of a chimeric channel was defined as follows: for example, Nav1.5/1.3 DI was a chimeric channel in which the DI of Nav1.5 was substituted with that of Nav1.3. A total of 11 chimeric channels were constructed and their INaP generation by the toxin was compared. Again, INaP was measured at the time point of 295 ms (Figure 6a). The substitution of all four domains of Nav1.5 with those of Nav1.3 (Nav1.5/1.3 DI-II-III-IV) almost fully restored the efficacy of RTX-VII, thus eliminating the involvements of the intracellular loops of Nav1.3 in the toxin-induced INaP. Of the four single domain replaced chimeric channels, Nav1.5/1.3 DI, Nav1.5/1.3 DII and Nav1.5/1.3 DIII chimeras were resistant to RTX-VII, similar to wt-Nav1.5, whereas Nav1.5/1.3 DIV chimera was sensitive to RTX-VII. Furthermore, the toxin slowed the inactivation and induced a small steady- state INaP in this chimeric channel, indicating that Nav1.3 DIV is important but not sufficient for RTX-VII inducing large INaP. Of the two triple domain replaced chimeric channels, Nav1.5/1.3 DI-III-IV chimera did not fully restore toxin efficacy but Nav1.5/1.3 DI-II-IV chimera did, which indicates that the DII but not the DI and DIII of Nav1.3 is required for toxin inducing large INaP. Of the three double domain replaced chimeric channels, the reconstruction of the DI or DIII of Nav1.3 into the scaffold of Nav1.5/1.3 DIV chimera (Nav1.5/1.3 DIII-IV chimera or Nav1.5/1.3 DI-IV chimera) had a limited effect on restoring toxin efficacy, whereas the reconstruction of the DII of Nav1.3 into Nav1.5/1.3 DIV chimera (Nav1.5/1.3 DII-IV chimera) almost fully rescued toxin efficacy, suggesting the assembly of the DII and DIV of Nav1.3 should be sufficient for RTX-VII inducing large INaP. Additionally, the chimeric channel Nav1.5/1.3 DI-III-IV&DII PD, where only the DII-PD but not the whole DII of Nav1.3 was present, also attenuated the efficacy of RTX-VII compared with that of Nav1.5/1.3 DI-II-III-IV chimera, which strongly supports that the DII-VSD of Nav1.3 plays a vital role in toxin-induced INaP generation.


Synergetic action of domain II and IV underlies persistent current generation in Nav1.3 as revealed by a tarantula toxin.

Tang C, Zhou X, Zhang Y, Xiao Z, Hu Z, Zhang C, Huang Y, Chen B, Liu Z, Liang S - Sci Rep (2015)

The substitution of the domain II and IV of Nav1.5 with those of Nav1.3 restores RTX-VII efficacy.(a) A 300-ms recording of the currents of Nav1.5 derived chimeric channels in the absence and presence of various concentration of RTX-VII (n = 6–9). The chimeric channels were constructed as follows: one or several domains (DI, DII, DIII or DIV) of Nav1.5 were substituted with the corresponding domain/s of Nav1.3 (see Supplementary Fig. S6). (b) Dose-response curves for RTX-VII enhancing the INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that did not or slightly restored toxin efficacy (steady-state INaP/INaT ratio at the saturated concentration of toxin) (n = 6–9). (c) Dose-response curves for RTX-VII enhancing INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that almost completely restored toxin potency and/or efficacy (n = 6–9). (d) Bars show the fold changes of the apparent EC50 of RTX-VII for each Nav1.5 derived chimeric channels compared with that for wt-Nav1.3 (n = 6–9). (e) Bars show the steady- state INaP/INaT ratio of wt-Nav1.3 and Nav1.5 derived chimeric channels in the presence of saturated concentrations of toxin. This values are 47.92 ± 5.43%, 46.37 ± 8.87%, 6.3 ± 1.82%, 10.60 ± 3.32%, 53.81 ± 5.56%, 12.68 ± 3.67%, 50.88 ± 8.33 and 20.41 ± 3.90% for Nav1.3, 1.5/1.3 DI-DII-DIII-DIV, 1.5/1.3 DIV, 1.5/1.3 DI-DIII-DIV, 1.5/1.3 DI-DII-DIV, 1.5/1.3 DI-DIV, 1.5/1.3 DII-DIV and 1.5/1.3 DI-DIII-DIV&DII-PD, respectively (***p < 0.001, N.S = not significant, when compared with wt-Nav1.3) (n = 6–9).
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f6: The substitution of the domain II and IV of Nav1.5 with those of Nav1.3 restores RTX-VII efficacy.(a) A 300-ms recording of the currents of Nav1.5 derived chimeric channels in the absence and presence of various concentration of RTX-VII (n = 6–9). The chimeric channels were constructed as follows: one or several domains (DI, DII, DIII or DIV) of Nav1.5 were substituted with the corresponding domain/s of Nav1.3 (see Supplementary Fig. S6). (b) Dose-response curves for RTX-VII enhancing the INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that did not or slightly restored toxin efficacy (steady-state INaP/INaT ratio at the saturated concentration of toxin) (n = 6–9). (c) Dose-response curves for RTX-VII enhancing INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that almost completely restored toxin potency and/or efficacy (n = 6–9). (d) Bars show the fold changes of the apparent EC50 of RTX-VII for each Nav1.5 derived chimeric channels compared with that for wt-Nav1.3 (n = 6–9). (e) Bars show the steady- state INaP/INaT ratio of wt-Nav1.3 and Nav1.5 derived chimeric channels in the presence of saturated concentrations of toxin. This values are 47.92 ± 5.43%, 46.37 ± 8.87%, 6.3 ± 1.82%, 10.60 ± 3.32%, 53.81 ± 5.56%, 12.68 ± 3.67%, 50.88 ± 8.33 and 20.41 ± 3.90% for Nav1.3, 1.5/1.3 DI-DII-DIII-DIV, 1.5/1.3 DIV, 1.5/1.3 DI-DIII-DIV, 1.5/1.3 DI-DII-DIV, 1.5/1.3 DI-DIV, 1.5/1.3 DII-DIV and 1.5/1.3 DI-DIII-DIV&DII-PD, respectively (***p < 0.001, N.S = not significant, when compared with wt-Nav1.3) (n = 6–9).
Mentions: Considering the critical role of the DII and DIV of Nav1.3 in the RTX-VII-induced INaP, we assumed that reverse reconstruction of Nav1.3 DII and DIV into Nav1.5 might restore the efficacy of the toxin. A reversal chimeric strategy was used as follows: four domains of Nav1.3 were stepwise reconstructed into the scaffold of Nav1.5 (Supplementary Fig. S6). The nomenclature of a chimeric channel was defined as follows: for example, Nav1.5/1.3 DI was a chimeric channel in which the DI of Nav1.5 was substituted with that of Nav1.3. A total of 11 chimeric channels were constructed and their INaP generation by the toxin was compared. Again, INaP was measured at the time point of 295 ms (Figure 6a). The substitution of all four domains of Nav1.5 with those of Nav1.3 (Nav1.5/1.3 DI-II-III-IV) almost fully restored the efficacy of RTX-VII, thus eliminating the involvements of the intracellular loops of Nav1.3 in the toxin-induced INaP. Of the four single domain replaced chimeric channels, Nav1.5/1.3 DI, Nav1.5/1.3 DII and Nav1.5/1.3 DIII chimeras were resistant to RTX-VII, similar to wt-Nav1.5, whereas Nav1.5/1.3 DIV chimera was sensitive to RTX-VII. Furthermore, the toxin slowed the inactivation and induced a small steady- state INaP in this chimeric channel, indicating that Nav1.3 DIV is important but not sufficient for RTX-VII inducing large INaP. Of the two triple domain replaced chimeric channels, Nav1.5/1.3 DI-III-IV chimera did not fully restore toxin efficacy but Nav1.5/1.3 DI-II-IV chimera did, which indicates that the DII but not the DI and DIII of Nav1.3 is required for toxin inducing large INaP. Of the three double domain replaced chimeric channels, the reconstruction of the DI or DIII of Nav1.3 into the scaffold of Nav1.5/1.3 DIV chimera (Nav1.5/1.3 DIII-IV chimera or Nav1.5/1.3 DI-IV chimera) had a limited effect on restoring toxin efficacy, whereas the reconstruction of the DII of Nav1.3 into Nav1.5/1.3 DIV chimera (Nav1.5/1.3 DII-IV chimera) almost fully rescued toxin efficacy, suggesting the assembly of the DII and DIV of Nav1.3 should be sufficient for RTX-VII inducing large INaP. Additionally, the chimeric channel Nav1.5/1.3 DI-III-IV&DII PD, where only the DII-PD but not the whole DII of Nav1.3 was present, also attenuated the efficacy of RTX-VII compared with that of Nav1.5/1.3 DI-II-III-IV chimera, which strongly supports that the DII-VSD of Nav1.3 plays a vital role in toxin-induced INaP generation.

Bottom Line: A pre-activated state binding model was proposed to explain the kinetics of toxin-channel interaction.Domain IV constructed the binding site for RTX-VII, while domain II might not participate in interacting with RTX-VII but could determine the efficacy of RTX-VII.Our results based on the use of RTX-VII as a probe suggest that domain II and IV cooperatively contribute to the generation of INaP in Nav1.3.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Hunan Normal University, Changsha, Hunan. 410081, China.

ABSTRACT
The persistent current (INaP) through voltage-gated sodium channels enhances neuronal excitability by causing prolonged depolarization of membranes. Nav1.3 intrinsically generates a small INaP, although the mechanism underlying its generation remains unclear. In this study, the involvement of the four domains of Nav1.3 in INaP generation was investigated using the tarantula toxin α-hexatoxin-MrVII (RTX-VII). RTX-VII activated Nav1.3 and induced a large INaP. A pre-activated state binding model was proposed to explain the kinetics of toxin-channel interaction. Of the four domains of Nav1.3, both domain II and IV might play important roles in the toxin-induced INaP. Domain IV constructed the binding site for RTX-VII, while domain II might not participate in interacting with RTX-VII but could determine the efficacy of RTX-VII. Our results based on the use of RTX-VII as a probe suggest that domain II and IV cooperatively contribute to the generation of INaP in Nav1.3.

Show MeSH
Related in: MedlinePlus