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Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization.

Kühn FJ, Greeff NG - J. Gen. Physiol. (1999)

Bottom Line: The double mutant R1635/1638H shows intermediate effects on inactivation.Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H.These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland.

ABSTRACT
The highly charged transmembrane segments in each of the four homologous domains (S4D1-S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.

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Effects of S4D4 point mutations on kinetics and on voltage dependence of macroscopic Na+ currents. (A) Comparison of normalized single current traces of WT (solid line) and mutant (dotted line) channels, elicited by a depolarizing pulse to −5 mV from a −100-mV holding potential. The settling times of the corresponding capacitance transients are almost identical (data not shown). (B) Semilogarithmic plots of single (mutants) or double (WT inactivation) exponential fits, as indicated, from representative current traces elicited by depolarizing pulses to −20, −5, and 20 mV; holding potential −100 mV. Single fits accord to the following equation. INa(t) = A * (1 − exp (−t/τm))3*exp(−t/τh) + P * (1 − exp(−t/τm))3. WT: (τm/τh (fast)/τh (slow) in ms) 0.48/0.70/4.20 (−20 mV); 0.28/0.70/5.10 (−5 mV); 0.17/0.51/3.10 (20 mV); R4H: (τm/τh in ms) 0.41/2.90 (−20 mV); 0.31/2.50 (−5 mV); 0.23/1.80 (20 mV); R4/5H: (τm/τh in ms) 0.45/4.30 (−20 mV); 0.24/3.80 (−5 mV); 0.15/2.90 (20 mV); R5H: (τm/τh in ms) 0.45/5.50 (−20 mV); 0.25/4.90 (−5 mV); 0.16/3.10 (20 mV).
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Figure 2: Effects of S4D4 point mutations on kinetics and on voltage dependence of macroscopic Na+ currents. (A) Comparison of normalized single current traces of WT (solid line) and mutant (dotted line) channels, elicited by a depolarizing pulse to −5 mV from a −100-mV holding potential. The settling times of the corresponding capacitance transients are almost identical (data not shown). (B) Semilogarithmic plots of single (mutants) or double (WT inactivation) exponential fits, as indicated, from representative current traces elicited by depolarizing pulses to −20, −5, and 20 mV; holding potential −100 mV. Single fits accord to the following equation. INa(t) = A * (1 − exp (−t/τm))3*exp(−t/τh) + P * (1 − exp(−t/τm))3. WT: (τm/τh (fast)/τh (slow) in ms) 0.48/0.70/4.20 (−20 mV); 0.28/0.70/5.10 (−5 mV); 0.17/0.51/3.10 (20 mV); R4H: (τm/τh in ms) 0.41/2.90 (−20 mV); 0.31/2.50 (−5 mV); 0.23/1.80 (20 mV); R4/5H: (τm/τh in ms) 0.45/4.30 (−20 mV); 0.24/3.80 (−5 mV); 0.15/2.90 (20 mV); R5H: (τm/τh in ms) 0.45/5.50 (−20 mV); 0.25/4.90 (−5 mV); 0.16/3.10 (20 mV).

Mentions: The kinetics of macroscopic sodium currents were analyzed by performing single or double exponential fits from normalized current traces at −20, −5, and 20 mV in order to determine the corresponding τh and τm values (Fig. 2). WT sodium current inactivation was well fit only by a double exponential because of the coexistence of slow and fast gating channels, as already mentioned. In contrast, the mutant sodium currents were well fit by a single exponential. The speed of our TEVC was fast enough to detect an acceleration of the activation kinetics for more depolarizing potentials (Fig. 2 B). The τm values of WT and mutant channels do not differ significantly, but the τh values are profoundly increased in the mutant channels according to the sequential order: WT (τh (fast)) < R4H < R4/5H < R5H ≅ WT (τh (slow)). Thus, the S4D4 mutants display a strong effect on inactivation rather than on activation kinetics. The unequal effects of S4D4 mutants on the gating properties of sodium channels were also observed by other groups (Chahine et al. 1994; Chen et al. 1996; Kontis et al. 1997). Between −20 mV and 20 mV, WT and mutant channels show a more pronounced voltage dependence of activation compared with inactivation represented by the slopes in Fig. 2 B; the voltage dependencies of activation and inactivation are similar in WT and mutant channels, respectively.


Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization.

Kühn FJ, Greeff NG - J. Gen. Physiol. (1999)

Effects of S4D4 point mutations on kinetics and on voltage dependence of macroscopic Na+ currents. (A) Comparison of normalized single current traces of WT (solid line) and mutant (dotted line) channels, elicited by a depolarizing pulse to −5 mV from a −100-mV holding potential. The settling times of the corresponding capacitance transients are almost identical (data not shown). (B) Semilogarithmic plots of single (mutants) or double (WT inactivation) exponential fits, as indicated, from representative current traces elicited by depolarizing pulses to −20, −5, and 20 mV; holding potential −100 mV. Single fits accord to the following equation. INa(t) = A * (1 − exp (−t/τm))3*exp(−t/τh) + P * (1 − exp(−t/τm))3. WT: (τm/τh (fast)/τh (slow) in ms) 0.48/0.70/4.20 (−20 mV); 0.28/0.70/5.10 (−5 mV); 0.17/0.51/3.10 (20 mV); R4H: (τm/τh in ms) 0.41/2.90 (−20 mV); 0.31/2.50 (−5 mV); 0.23/1.80 (20 mV); R4/5H: (τm/τh in ms) 0.45/4.30 (−20 mV); 0.24/3.80 (−5 mV); 0.15/2.90 (20 mV); R5H: (τm/τh in ms) 0.45/5.50 (−20 mV); 0.25/4.90 (−5 mV); 0.16/3.10 (20 mV).
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Figure 2: Effects of S4D4 point mutations on kinetics and on voltage dependence of macroscopic Na+ currents. (A) Comparison of normalized single current traces of WT (solid line) and mutant (dotted line) channels, elicited by a depolarizing pulse to −5 mV from a −100-mV holding potential. The settling times of the corresponding capacitance transients are almost identical (data not shown). (B) Semilogarithmic plots of single (mutants) or double (WT inactivation) exponential fits, as indicated, from representative current traces elicited by depolarizing pulses to −20, −5, and 20 mV; holding potential −100 mV. Single fits accord to the following equation. INa(t) = A * (1 − exp (−t/τm))3*exp(−t/τh) + P * (1 − exp(−t/τm))3. WT: (τm/τh (fast)/τh (slow) in ms) 0.48/0.70/4.20 (−20 mV); 0.28/0.70/5.10 (−5 mV); 0.17/0.51/3.10 (20 mV); R4H: (τm/τh in ms) 0.41/2.90 (−20 mV); 0.31/2.50 (−5 mV); 0.23/1.80 (20 mV); R4/5H: (τm/τh in ms) 0.45/4.30 (−20 mV); 0.24/3.80 (−5 mV); 0.15/2.90 (20 mV); R5H: (τm/τh in ms) 0.45/5.50 (−20 mV); 0.25/4.90 (−5 mV); 0.16/3.10 (20 mV).
Mentions: The kinetics of macroscopic sodium currents were analyzed by performing single or double exponential fits from normalized current traces at −20, −5, and 20 mV in order to determine the corresponding τh and τm values (Fig. 2). WT sodium current inactivation was well fit only by a double exponential because of the coexistence of slow and fast gating channels, as already mentioned. In contrast, the mutant sodium currents were well fit by a single exponential. The speed of our TEVC was fast enough to detect an acceleration of the activation kinetics for more depolarizing potentials (Fig. 2 B). The τm values of WT and mutant channels do not differ significantly, but the τh values are profoundly increased in the mutant channels according to the sequential order: WT (τh (fast)) < R4H < R4/5H < R5H ≅ WT (τh (slow)). Thus, the S4D4 mutants display a strong effect on inactivation rather than on activation kinetics. The unequal effects of S4D4 mutants on the gating properties of sodium channels were also observed by other groups (Chahine et al. 1994; Chen et al. 1996; Kontis et al. 1997). Between −20 mV and 20 mV, WT and mutant channels show a more pronounced voltage dependence of activation compared with inactivation represented by the slopes in Fig. 2 B; the voltage dependencies of activation and inactivation are similar in WT and mutant channels, respectively.

Bottom Line: The double mutant R1635/1638H shows intermediate effects on inactivation.Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H.These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state.

View Article: PubMed Central - PubMed

Affiliation: Physiologisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland.

ABSTRACT
The highly charged transmembrane segments in each of the four homologous domains (S4D1-S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.

Show MeSH
Related in: MedlinePlus