Limits...
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.

Show MeSH

Related in: MedlinePlus

Correlation of the recovery of ionic current and gating charge of WT and R4H at different potentials. (A) Semilogarithmic plot of ionic current (filled symbols) and gating charge (open symbols) recoveries obtained from different oocytes are superimposed for potentials of −140, −120, −100, and −80 mV. Values are mean ± SD of n > 3 cells. Superimposed triangles represent congruent data of ionic current (small, filled triangle) and gating charge recovery (large, open triangle) recorded sequentially in a single oocyte at −100 mV. (B) Simultaneous recording of ionic and gating currents around sodium reversal potential (ENa). Test pulses elicited from +5 to +15 mV in steps of 1 mV, holding potential −100 mV. Extracellular sodium concentration was reduced to 8.8 mM by choline replacement in Modified Barth's Solution. Ionic currents change polarity when crossing ENa, whereas gating currents follow the direction of the electric field. (C) Recordings of gating current and ionic current recovery at sodium reversal potential (ENa) obtained from a single oocyte, T = 8°C. Pulse-protocol: a 20 ms prepulse to 40 mV was followed by a recovery period of variable duration (1–60 ms) at −100 mV and a test pulse to −20 (ionic current) or 34 mV (ENa; gating current), test pulse duration 13 ms, holding potential −100 mV.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230646&req=5

Figure 7: Correlation of the recovery of ionic current and gating charge of WT and R4H at different potentials. (A) Semilogarithmic plot of ionic current (filled symbols) and gating charge (open symbols) recoveries obtained from different oocytes are superimposed for potentials of −140, −120, −100, and −80 mV. Values are mean ± SD of n > 3 cells. Superimposed triangles represent congruent data of ionic current (small, filled triangle) and gating charge recovery (large, open triangle) recorded sequentially in a single oocyte at −100 mV. (B) Simultaneous recording of ionic and gating currents around sodium reversal potential (ENa). Test pulses elicited from +5 to +15 mV in steps of 1 mV, holding potential −100 mV. Extracellular sodium concentration was reduced to 8.8 mM by choline replacement in Modified Barth's Solution. Ionic currents change polarity when crossing ENa, whereas gating currents follow the direction of the electric field. (C) Recordings of gating current and ionic current recovery at sodium reversal potential (ENa) obtained from a single oocyte, T = 8°C. Pulse-protocol: a 20 ms prepulse to 40 mV was followed by a recovery period of variable duration (1–60 ms) at −100 mV and a test pulse to −20 (ionic current) or 34 mV (ENa; gating current), test pulse duration 13 ms, holding potential −100 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)

Correlation of the recovery of ionic current and gating charge of WT and R4H at different potentials. (A) Semilogarithmic plot of ionic current (filled symbols) and gating charge (open symbols) recoveries obtained from different oocytes are superimposed for potentials of −140, −120, −100, and −80 mV. Values are mean ± SD of n > 3 cells. Superimposed triangles represent congruent data of ionic current (small, filled triangle) and gating charge recovery (large, open triangle) recorded sequentially in a single oocyte at −100 mV. (B) Simultaneous recording of ionic and gating currents around sodium reversal potential (ENa). Test pulses elicited from +5 to +15 mV in steps of 1 mV, holding potential −100 mV. Extracellular sodium concentration was reduced to 8.8 mM by choline replacement in Modified Barth's Solution. Ionic currents change polarity when crossing ENa, whereas gating currents follow the direction of the electric field. (C) Recordings of gating current and ionic current recovery at sodium reversal potential (ENa) obtained from a single oocyte, T = 8°C. Pulse-protocol: a 20 ms prepulse to 40 mV was followed by a recovery period of variable duration (1–60 ms) at −100 mV and a test pulse to −20 (ionic current) or 34 mV (ENa; gating current), test pulse duration 13 ms, holding potential −100 mV.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230646&req=5

Figure 7: Correlation of the recovery of ionic current and gating charge of WT and R4H at different potentials. (A) Semilogarithmic plot of ionic current (filled symbols) and gating charge (open symbols) recoveries obtained from different oocytes are superimposed for potentials of −140, −120, −100, and −80 mV. Values are mean ± SD of n > 3 cells. Superimposed triangles represent congruent data of ionic current (small, filled triangle) and gating charge recovery (large, open triangle) recorded sequentially in a single oocyte at −100 mV. (B) Simultaneous recording of ionic and gating currents around sodium reversal potential (ENa). Test pulses elicited from +5 to +15 mV in steps of 1 mV, holding potential −100 mV. Extracellular sodium concentration was reduced to 8.8 mM by choline replacement in Modified Barth's Solution. Ionic currents change polarity when crossing ENa, whereas gating currents follow the direction of the electric field. (C) Recordings of gating current and ionic current recovery at sodium reversal potential (ENa) obtained from a single oocyte, T = 8°C. Pulse-protocol: a 20 ms prepulse to 40 mV was followed by a recovery period of variable duration (1–60 ms) at −100 mV and a test pulse to −20 (ionic current) or 34 mV (ENa; gating current), test pulse duration 13 ms, holding potential −100 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