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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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Gating current recordings in WT and mutant sodium channels. (A–D) Sodium ionic (INa) and gating (Ig) currents recorded from the whole cell membrane of Xenopus oocytes using a TEVC after blocking most (B and C) or all (D) of the ionic current with TTX. (A) Pulse protocol; the traces were elicited by 13 ms depolarizing test pulses to membrane potentials of −60 to 60 mV increasing in steps of 10 mV without (B) or with (C) a 20-ms prepulse to 0 mV from a holding potential of −100 mV, interval 1 ms at −100 mV, temperature 15°C. The OFF-gating current of the prepulse and the non-immobilized fraction of the test pulse ON-gating current (Ig,n) are indicated in C. Notice the elimination of INa and the partial immobilization (∼50%) of total gating current (Ig) due to the inactivating pulse. The corresponding capacitance current (Ic) reflects the actual clamp speed. (D) Total ON-gating currents of WT and mutant channels activated by step depolarizations in 20-mV increments from −80 to 80 mV from a holding potential of −100 mV, recorded in presence of 2 μM TTX, pulse duration 13 ms.
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Figure 3: Gating current recordings in WT and mutant sodium channels. (A–D) Sodium ionic (INa) and gating (Ig) currents recorded from the whole cell membrane of Xenopus oocytes using a TEVC after blocking most (B and C) or all (D) of the ionic current with TTX. (A) Pulse protocol; the traces were elicited by 13 ms depolarizing test pulses to membrane potentials of −60 to 60 mV increasing in steps of 10 mV without (B) or with (C) a 20-ms prepulse to 0 mV from a holding potential of −100 mV, interval 1 ms at −100 mV, temperature 15°C. The OFF-gating current of the prepulse and the non-immobilized fraction of the test pulse ON-gating current (Ig,n) are indicated in C. Notice the elimination of INa and the partial immobilization (∼50%) of total gating current (Ig) due to the inactivating pulse. The corresponding capacitance current (Ic) reflects the actual clamp speed. (D) Total ON-gating currents of WT and mutant channels activated by step depolarizations in 20-mV increments from −80 to 80 mV from a holding potential of −100 mV, recorded in presence of 2 μM TTX, pulse duration 13 ms.

Mentions: To gain further insights into the coupling of the S4D4 voltage sensor to the inactivation structure of the channel, we analyzed gating currents at the whole oocyte membrane either simultaneously with ionic currents in the same cell or in separate experiments. Compared with the cut-open oocyte method (Bezanilla et al. 1994; Stefani et al. 1994) or the macropatch technique (Conti and Stühmer 1989), the clamp speed of standard TEVCs is regarded as considerably slower. Therefore, the TEVC seemed to be unsuitable for characterizing the kinetics of fast mode sodium channels (Ruben et al. 1997). However, we succeeded in recording well-resolved sodium channel ionic and gating currents with an optimized TEVC by finding a compromise between maximum clamping speed and minimal signal distortion (see Material and Methods). Fig. 3 D shows representative ON-gating current traces of WT and mutant channels. A significant contamination of the gating current measurements with ionic current was excluded by recording in bath solution containing 2 μM TTX. Baseline distortions, e.g., visible in the R5H record, were due to small nonlinearities in leak subtraction that occur sporadically. Before integration of gating current traces, these artifacts were minimized by baseline correction. As the ON-gating current mainly represents the sum of the charge displacements of the S4 voltage sensors during activation, the similarity of the records supports the conclusion from our ionic current studies that the activation kinetics were not significantly disturbed by the mutations. In contrast, the association of a gating current component exclusively with the inactivation process is difficult and was not specifically analyzed in this study, but there exist some positive evidence (Greeff and Forster 1991; Sheets and Hanck 1995).


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)

Gating current recordings in WT and mutant sodium channels. (A–D) Sodium ionic (INa) and gating (Ig) currents recorded from the whole cell membrane of Xenopus oocytes using a TEVC after blocking most (B and C) or all (D) of the ionic current with TTX. (A) Pulse protocol; the traces were elicited by 13 ms depolarizing test pulses to membrane potentials of −60 to 60 mV increasing in steps of 10 mV without (B) or with (C) a 20-ms prepulse to 0 mV from a holding potential of −100 mV, interval 1 ms at −100 mV, temperature 15°C. The OFF-gating current of the prepulse and the non-immobilized fraction of the test pulse ON-gating current (Ig,n) are indicated in C. Notice the elimination of INa and the partial immobilization (∼50%) of total gating current (Ig) due to the inactivating pulse. The corresponding capacitance current (Ic) reflects the actual clamp speed. (D) Total ON-gating currents of WT and mutant channels activated by step depolarizations in 20-mV increments from −80 to 80 mV from a holding potential of −100 mV, recorded in presence of 2 μM TTX, pulse duration 13 ms.
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Related In: Results  -  Collection

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Figure 3: Gating current recordings in WT and mutant sodium channels. (A–D) Sodium ionic (INa) and gating (Ig) currents recorded from the whole cell membrane of Xenopus oocytes using a TEVC after blocking most (B and C) or all (D) of the ionic current with TTX. (A) Pulse protocol; the traces were elicited by 13 ms depolarizing test pulses to membrane potentials of −60 to 60 mV increasing in steps of 10 mV without (B) or with (C) a 20-ms prepulse to 0 mV from a holding potential of −100 mV, interval 1 ms at −100 mV, temperature 15°C. The OFF-gating current of the prepulse and the non-immobilized fraction of the test pulse ON-gating current (Ig,n) are indicated in C. Notice the elimination of INa and the partial immobilization (∼50%) of total gating current (Ig) due to the inactivating pulse. The corresponding capacitance current (Ic) reflects the actual clamp speed. (D) Total ON-gating currents of WT and mutant channels activated by step depolarizations in 20-mV increments from −80 to 80 mV from a holding potential of −100 mV, recorded in presence of 2 μM TTX, pulse duration 13 ms.
Mentions: To gain further insights into the coupling of the S4D4 voltage sensor to the inactivation structure of the channel, we analyzed gating currents at the whole oocyte membrane either simultaneously with ionic currents in the same cell or in separate experiments. Compared with the cut-open oocyte method (Bezanilla et al. 1994; Stefani et al. 1994) or the macropatch technique (Conti and Stühmer 1989), the clamp speed of standard TEVCs is regarded as considerably slower. Therefore, the TEVC seemed to be unsuitable for characterizing the kinetics of fast mode sodium channels (Ruben et al. 1997). However, we succeeded in recording well-resolved sodium channel ionic and gating currents with an optimized TEVC by finding a compromise between maximum clamping speed and minimal signal distortion (see Material and Methods). Fig. 3 D shows representative ON-gating current traces of WT and mutant channels. A significant contamination of the gating current measurements with ionic current was excluded by recording in bath solution containing 2 μM TTX. Baseline distortions, e.g., visible in the R5H record, were due to small nonlinearities in leak subtraction that occur sporadically. Before integration of gating current traces, these artifacts were minimized by baseline correction. As the ON-gating current mainly represents the sum of the charge displacements of the S4 voltage sensors during activation, the similarity of the records supports the conclusion from our ionic current studies that the activation kinetics were not significantly disturbed by the mutations. In contrast, the association of a gating current component exclusively with the inactivation process is difficult and was not specifically analyzed in this study, but there exist some positive evidence (Greeff and Forster 1991; Sheets and Hanck 1995).

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