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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 macroscopic Na+ currents. (A) Na+ currents from WT-, R4H-, R4/5H-, and R5H-injected Xenopus oocytes elicited by step depolarizations from −80 to 80 mV, holding potential −100 mV. A single trace displaying an outward gating current followed by an inward ionic current near sodium reversal potential is indicated in R4/5H. (B) Corresponding current–voltage relationship of peak Na+ currents, normalized to the largest inward current. (C) Steady-state inactivation at 0 mV induced by a 100-ms prepulse at voltages between −120 and 20 mV, increments of 10 mV, holding potential −100 mV. The curves are fitted to a standard Boltzmann distribution with slopes and V0.5 given in the text. Values are mean ± SEM of n > 3 cells. (D) Semilogarithmic plot of time constant and voltage of recovery from fast inactivation. Pulse protocol: a 80-ms test pulse to 0 mV was preceded by a 100-ms pulse to 0 mV and variable recovery periods at four different holding potentials (−80, −100, −120, and −140 mV). Values are mean recovery time constants in milliseconds ± SEM of n = 1–7 cells; WT, 1.8 ± 0.6 (−140 mV), 3.9 ± 1.8 (−120 mV), 7.9 ± 1.5 (−100 mV), 24.8 ± 6.0 (−80 mV); R4H, 30.8 ± 9.6 (−140 mV), 89.4 ± 19.3 (−120 mV), 264 ± 59 (−100 mV), 347 ± 55.1 (−80 mV); R4/5H, 5.4 ± 0.5 (−140 mV), 9.1 ± 0.9 (−120 mV), 15.1 ± 0.8 (−100 mV), 26.7 ± 1.8 (−80 mV); R5H, 2.6 (−140mV), 3.7 ± 0.2 (−120 mV), 7.4 ± 0.6 (−100 mV), 15.4 ± 0.9 (−80 mV).
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Figure 1: Effects of S4D4 point mutations on macroscopic Na+ currents. (A) Na+ currents from WT-, R4H-, R4/5H-, and R5H-injected Xenopus oocytes elicited by step depolarizations from −80 to 80 mV, holding potential −100 mV. A single trace displaying an outward gating current followed by an inward ionic current near sodium reversal potential is indicated in R4/5H. (B) Corresponding current–voltage relationship of peak Na+ currents, normalized to the largest inward current. (C) Steady-state inactivation at 0 mV induced by a 100-ms prepulse at voltages between −120 and 20 mV, increments of 10 mV, holding potential −100 mV. The curves are fitted to a standard Boltzmann distribution with slopes and V0.5 given in the text. Values are mean ± SEM of n > 3 cells. (D) Semilogarithmic plot of time constant and voltage of recovery from fast inactivation. Pulse protocol: a 80-ms test pulse to 0 mV was preceded by a 100-ms pulse to 0 mV and variable recovery periods at four different holding potentials (−80, −100, −120, and −140 mV). Values are mean recovery time constants in milliseconds ± SEM of n = 1–7 cells; WT, 1.8 ± 0.6 (−140 mV), 3.9 ± 1.8 (−120 mV), 7.9 ± 1.5 (−100 mV), 24.8 ± 6.0 (−80 mV); R4H, 30.8 ± 9.6 (−140 mV), 89.4 ± 19.3 (−120 mV), 264 ± 59 (−100 mV), 347 ± 55.1 (−80 mV); R4/5H, 5.4 ± 0.5 (−140 mV), 9.1 ± 0.9 (−120 mV), 15.1 ± 0.8 (−100 mV), 26.7 ± 1.8 (−80 mV); R5H, 2.6 (−140mV), 3.7 ± 0.2 (−120 mV), 7.4 ± 0.6 (−100 mV), 15.4 ± 0.9 (−80 mV).

Mentions: Na+ currents obtained from Xenopus oocytes injected with either wild-type (WT) or mutant (R1635H; R1635/1638H; R1638H; subsequently named by the position in the S4 segment R4H; R4/5H; R5H) rBIIA sodium channel cRNA display characteristic patterns of voltage-dependent activation and inactivation (Fig. 1 A). For well-resolved gating current recordings in Xenopus oocytes, a very high expression of rBIIA sodium channels was necessary. For this purpose the genes of both WT and mutant sodium channels were expressed by use of a high expression vector (see Materials and Methods). Sodium peak currents of 10–40 μA, elicited between −10 and −20 mV in 88 mM external sodium, were obtained 2–4 d after injection of the corresponding cRNA. During this period, only ionic current measurements were performed, because the corresponding gating currents were still too small (<0.5 μA). Rs errors were <5 mV unless the currents exceeded ∼20–30 μA, because an optimized TEVC was used (see Materials and Methods). Between days 5 and 8, gating currents increased to peaks of 3–10 μA, whereas ionic currents started to decline after reaching maximum levels of up to −100 μA (Greeff et al. 1998). For recording of pure gating current traces, the corresponding ionic currents were suppressed by application of 2 μM TTX. An example for a simultaneous recording of ionic and gating current is given in Fig. 1 A (indicated by arrows). R4/5H shows an outward gating current of ∼2 μA near the sodium reversal potential (ENa). This gating current is merged with the outward ionic current at more depolarizing potentials. In some special experiments, performed without the application of TTX, gating currents were recorded at ENa essentially not disturbed by ionic currents (see Fig. 7 B).


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 macroscopic Na+ currents. (A) Na+ currents from WT-, R4H-, R4/5H-, and R5H-injected Xenopus oocytes elicited by step depolarizations from −80 to 80 mV, holding potential −100 mV. A single trace displaying an outward gating current followed by an inward ionic current near sodium reversal potential is indicated in R4/5H. (B) Corresponding current–voltage relationship of peak Na+ currents, normalized to the largest inward current. (C) Steady-state inactivation at 0 mV induced by a 100-ms prepulse at voltages between −120 and 20 mV, increments of 10 mV, holding potential −100 mV. The curves are fitted to a standard Boltzmann distribution with slopes and V0.5 given in the text. Values are mean ± SEM of n > 3 cells. (D) Semilogarithmic plot of time constant and voltage of recovery from fast inactivation. Pulse protocol: a 80-ms test pulse to 0 mV was preceded by a 100-ms pulse to 0 mV and variable recovery periods at four different holding potentials (−80, −100, −120, and −140 mV). Values are mean recovery time constants in milliseconds ± SEM of n = 1–7 cells; WT, 1.8 ± 0.6 (−140 mV), 3.9 ± 1.8 (−120 mV), 7.9 ± 1.5 (−100 mV), 24.8 ± 6.0 (−80 mV); R4H, 30.8 ± 9.6 (−140 mV), 89.4 ± 19.3 (−120 mV), 264 ± 59 (−100 mV), 347 ± 55.1 (−80 mV); R4/5H, 5.4 ± 0.5 (−140 mV), 9.1 ± 0.9 (−120 mV), 15.1 ± 0.8 (−100 mV), 26.7 ± 1.8 (−80 mV); R5H, 2.6 (−140mV), 3.7 ± 0.2 (−120 mV), 7.4 ± 0.6 (−100 mV), 15.4 ± 0.9 (−80 mV).
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Related In: Results  -  Collection

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Figure 1: Effects of S4D4 point mutations on macroscopic Na+ currents. (A) Na+ currents from WT-, R4H-, R4/5H-, and R5H-injected Xenopus oocytes elicited by step depolarizations from −80 to 80 mV, holding potential −100 mV. A single trace displaying an outward gating current followed by an inward ionic current near sodium reversal potential is indicated in R4/5H. (B) Corresponding current–voltage relationship of peak Na+ currents, normalized to the largest inward current. (C) Steady-state inactivation at 0 mV induced by a 100-ms prepulse at voltages between −120 and 20 mV, increments of 10 mV, holding potential −100 mV. The curves are fitted to a standard Boltzmann distribution with slopes and V0.5 given in the text. Values are mean ± SEM of n > 3 cells. (D) Semilogarithmic plot of time constant and voltage of recovery from fast inactivation. Pulse protocol: a 80-ms test pulse to 0 mV was preceded by a 100-ms pulse to 0 mV and variable recovery periods at four different holding potentials (−80, −100, −120, and −140 mV). Values are mean recovery time constants in milliseconds ± SEM of n = 1–7 cells; WT, 1.8 ± 0.6 (−140 mV), 3.9 ± 1.8 (−120 mV), 7.9 ± 1.5 (−100 mV), 24.8 ± 6.0 (−80 mV); R4H, 30.8 ± 9.6 (−140 mV), 89.4 ± 19.3 (−120 mV), 264 ± 59 (−100 mV), 347 ± 55.1 (−80 mV); R4/5H, 5.4 ± 0.5 (−140 mV), 9.1 ± 0.9 (−120 mV), 15.1 ± 0.8 (−100 mV), 26.7 ± 1.8 (−80 mV); R5H, 2.6 (−140mV), 3.7 ± 0.2 (−120 mV), 7.4 ± 0.6 (−100 mV), 15.4 ± 0.9 (−80 mV).
Mentions: Na+ currents obtained from Xenopus oocytes injected with either wild-type (WT) or mutant (R1635H; R1635/1638H; R1638H; subsequently named by the position in the S4 segment R4H; R4/5H; R5H) rBIIA sodium channel cRNA display characteristic patterns of voltage-dependent activation and inactivation (Fig. 1 A). For well-resolved gating current recordings in Xenopus oocytes, a very high expression of rBIIA sodium channels was necessary. For this purpose the genes of both WT and mutant sodium channels were expressed by use of a high expression vector (see Materials and Methods). Sodium peak currents of 10–40 μA, elicited between −10 and −20 mV in 88 mM external sodium, were obtained 2–4 d after injection of the corresponding cRNA. During this period, only ionic current measurements were performed, because the corresponding gating currents were still too small (<0.5 μA). Rs errors were <5 mV unless the currents exceeded ∼20–30 μA, because an optimized TEVC was used (see Materials and Methods). Between days 5 and 8, gating currents increased to peaks of 3–10 μA, whereas ionic currents started to decline after reaching maximum levels of up to −100 μA (Greeff et al. 1998). For recording of pure gating current traces, the corresponding ionic currents were suppressed by application of 2 μM TTX. An example for a simultaneous recording of ionic and gating current is given in Fig. 1 A (indicated by arrows). R4/5H shows an outward gating current of ∼2 μA near the sodium reversal potential (ENa). This gating current is merged with the outward ionic current at more depolarizing potentials. In some special experiments, performed without the application of TTX, gating currents were recorded at ENa essentially not disturbed by ionic currents (see Fig. 7 B).

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