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Role of domain 4 in sodium channel slow inactivation.

Mitrovic N, George AL, Horn R - J. Gen. Physiol. (2000)

Bottom Line: Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results.A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation.The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, PA 19107, USA.

ABSTRACT
Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2-20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.

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Lack of 5 Hz use dependence for I1455C and G1456C. A shows data for each mutant, either unmodified or modified by MTSES, using conditions and display identical to those in Fig. 2. The calibration bars represent 2 nA and 2 ms. B shows a helical wheel plot (100° angle between successive residues) between R2C and R5C, showing that significant use dependence is observed for modification along one face of the helix. *Use dependence only for MTSPeS. **Use dependence for both MTSES and MTSPeS.
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Figure 8: Lack of 5 Hz use dependence for I1455C and G1456C. A shows data for each mutant, either unmodified or modified by MTSES, using conditions and display identical to those in Fig. 2. The calibration bars represent 2 nA and 2 ms. B shows a helical wheel plot (100° angle between successive residues) between R2C and R5C, showing that significant use dependence is observed for modification along one face of the helix. *Use dependence only for MTSPeS. **Use dependence for both MTSES and MTSPeS.

Mentions: It is notable that cysteine mutants of the isoleucine and glycine residues between R3 and R4, namely, I1455C and G1456C, do not exhibit a hint of use dependence after modification by MTSES (Fig. 7 and Fig. 8 A). Fig. 8 B shows that this pattern is consistent with an α helical secondary structure in this region of D4/S4, as if one face of the helix, encompassing R3, R4, and A1453, can affect slow inactivation. These data dovetail nicely into our previous evidence, based on cysteine accessibility, that the region between R2 and R3 has a helical secondary structure (Yang et al. 1997).


Role of domain 4 in sodium channel slow inactivation.

Mitrovic N, George AL, Horn R - J. Gen. Physiol. (2000)

Lack of 5 Hz use dependence for I1455C and G1456C. A shows data for each mutant, either unmodified or modified by MTSES, using conditions and display identical to those in Fig. 2. The calibration bars represent 2 nA and 2 ms. B shows a helical wheel plot (100° angle between successive residues) between R2C and R5C, showing that significant use dependence is observed for modification along one face of the helix. *Use dependence only for MTSPeS. **Use dependence for both MTSES and MTSPeS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2232890&req=5

Figure 8: Lack of 5 Hz use dependence for I1455C and G1456C. A shows data for each mutant, either unmodified or modified by MTSES, using conditions and display identical to those in Fig. 2. The calibration bars represent 2 nA and 2 ms. B shows a helical wheel plot (100° angle between successive residues) between R2C and R5C, showing that significant use dependence is observed for modification along one face of the helix. *Use dependence only for MTSPeS. **Use dependence for both MTSES and MTSPeS.
Mentions: It is notable that cysteine mutants of the isoleucine and glycine residues between R3 and R4, namely, I1455C and G1456C, do not exhibit a hint of use dependence after modification by MTSES (Fig. 7 and Fig. 8 A). Fig. 8 B shows that this pattern is consistent with an α helical secondary structure in this region of D4/S4, as if one face of the helix, encompassing R3, R4, and A1453, can affect slow inactivation. These data dovetail nicely into our previous evidence, based on cysteine accessibility, that the region between R2 and R3 has a helical secondary structure (Yang et al. 1997).

Bottom Line: Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results.A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation.The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, PA 19107, USA.

ABSTRACT
Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2-20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.

Show MeSH
Related in: MedlinePlus