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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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Comparison of the recovery of ionic current and gating charge of WT and mutant sodium channels at different potentials. Sodium current recovery (left panel) and gating charge recovery (right panel) at recovery potentials of −80, −100, and −120 mV in WT-, R4H-, R4/5H-, and R5H-sodium channels. The data of each subdiagram were recorded from different oocytes and normalized as described in Fig. 5. Notice the different starting points of gating current recovery in R4H that strictly depend on the effective recovery potential (indicated by arrows). Corresponding sodium current (INa) and gating charge (Qg) recovery time constants obtained from single exponential fits are as follows (INa/Qg in ms): WT, 22.8/54.9 (−80 mV), 7.6/16.7 (−100 mV), 2.1/6.4 (−120 mV); R4H, 359/113 (−80 mV), 228/269 (−100 mV), 59.7/349 (−120 mV); R4/5H, 25.4/39.7 (−80 mV), 14.9/20.6 (−100 mV), 8.4/9.5 (−120 mV); R5H, 16.3/12.6 (−80 mV), 8.0/11.6 (−100 mV), 3.8/6.9 (−120 mV).
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Figure 6: Comparison of the recovery of ionic current and gating charge of WT and mutant sodium channels at different potentials. Sodium current recovery (left panel) and gating charge recovery (right panel) at recovery potentials of −80, −100, and −120 mV in WT-, R4H-, R4/5H-, and R5H-sodium channels. The data of each subdiagram were recorded from different oocytes and normalized as described in Fig. 5. Notice the different starting points of gating current recovery in R4H that strictly depend on the effective recovery potential (indicated by arrows). Corresponding sodium current (INa) and gating charge (Qg) recovery time constants obtained from single exponential fits are as follows (INa/Qg in ms): WT, 22.8/54.9 (−80 mV), 7.6/16.7 (−100 mV), 2.1/6.4 (−120 mV); R4H, 359/113 (−80 mV), 228/269 (−100 mV), 59.7/349 (−120 mV); R4/5H, 25.4/39.7 (−80 mV), 14.9/20.6 (−100 mV), 8.4/9.5 (−120 mV); R5H, 16.3/12.6 (−80 mV), 8.0/11.6 (−100 mV), 3.8/6.9 (−120 mV).

Mentions: The comparison of the recovery time courses of ionic and gating currents yields additional information about the kinetics and voltage dependence of fast inactivation in WT and mutant channels. Fig. 5 A illustrates recordings of WT ionic and gating current recoveries obtained from separate oocytes at different stages of expression. We used an alternating pulse protocol with and without prepulse for each recovery time in order to be able to normalize for the slow peak–current decay of the reference traces (without prepulse). This current decay results from the presence of a subpopulation of slow gating channels that predominantly appear in the absence of β1 coexpression (as discussed in the context of Fig. 1). During recovery, the fast gating channels recover first, followed by the slow gating channels. The gating currents that were recorded in the presence of 2 μM TTX show a more stable reference current compared with the ionic current, thus suggesting little decay. In view of the large differences of the recovery rates of WT and R4H obtained from ionic current data (see Fig. 1 D), we decided that it was more important to analyze the close correlation of ionic and gating current recovery concerning the time course and its voltage dependence, rather than attempting to discriminate the overlapping fast and slow gating channels. The gating current recovery shows a characteristic pattern: (a) the basic level where recovery starts is determined by the degree of immobilization that occurs when the duration and potential of the prepulse fully inactivate ionic current (Armstrong and Bezanilla 1977), and (b) the recovery of the gating current in its early time course shows a discontinuous change from a rapid rise to a slower one (Greeff 1986). We found that in WT and mutant channels ionic current and gating current recovery strongly correlate in the voltage range from −140 to −80 mV, concerning time course and voltage dependence (compare Fig. 1 D with Fig. 5Fig. 6Fig. 7). However, within a single phenotype the time constants of gating current recovery are significantly increased compared with the time constants of ionic current recovery. Similar results were obtained from studies of Shaker potassium channels where, notably at more depolarizing potentials, gating current recovery is considerably slower than is ionic current recovery (Roux et al. 1998). The observed mismatch in the corresponding recovery time constants of ionic and gating currents were obtained from data fitted with single exponential curves. Taking into account the expression of a mixture of fast and slow gating channels (Fig. 1 A and 5), we tried to fit double exponential curves, which, in some cases made the recovery time constants agree better. Nevertheless, we decided to fit our recovery data uniformly with single exponential curves since the gating charge recovery was difficult to fit by double exponential curves for two reasons: (a) the amplitude of the gating current recovery is rather small, and (b) the scattering of the data points obtained from gating charge recovery (Fig. 6 B) is more pronounced if compared with the ionic current recovery (Fig. 6 A).


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)

Comparison of the recovery of ionic current and gating charge of WT and mutant sodium channels at different potentials. Sodium current recovery (left panel) and gating charge recovery (right panel) at recovery potentials of −80, −100, and −120 mV in WT-, R4H-, R4/5H-, and R5H-sodium channels. The data of each subdiagram were recorded from different oocytes and normalized as described in Fig. 5. Notice the different starting points of gating current recovery in R4H that strictly depend on the effective recovery potential (indicated by arrows). Corresponding sodium current (INa) and gating charge (Qg) recovery time constants obtained from single exponential fits are as follows (INa/Qg in ms): WT, 22.8/54.9 (−80 mV), 7.6/16.7 (−100 mV), 2.1/6.4 (−120 mV); R4H, 359/113 (−80 mV), 228/269 (−100 mV), 59.7/349 (−120 mV); R4/5H, 25.4/39.7 (−80 mV), 14.9/20.6 (−100 mV), 8.4/9.5 (−120 mV); R5H, 16.3/12.6 (−80 mV), 8.0/11.6 (−100 mV), 3.8/6.9 (−120 mV).
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Figure 6: Comparison of the recovery of ionic current and gating charge of WT and mutant sodium channels at different potentials. Sodium current recovery (left panel) and gating charge recovery (right panel) at recovery potentials of −80, −100, and −120 mV in WT-, R4H-, R4/5H-, and R5H-sodium channels. The data of each subdiagram were recorded from different oocytes and normalized as described in Fig. 5. Notice the different starting points of gating current recovery in R4H that strictly depend on the effective recovery potential (indicated by arrows). Corresponding sodium current (INa) and gating charge (Qg) recovery time constants obtained from single exponential fits are as follows (INa/Qg in ms): WT, 22.8/54.9 (−80 mV), 7.6/16.7 (−100 mV), 2.1/6.4 (−120 mV); R4H, 359/113 (−80 mV), 228/269 (−100 mV), 59.7/349 (−120 mV); R4/5H, 25.4/39.7 (−80 mV), 14.9/20.6 (−100 mV), 8.4/9.5 (−120 mV); R5H, 16.3/12.6 (−80 mV), 8.0/11.6 (−100 mV), 3.8/6.9 (−120 mV).
Mentions: The comparison of the recovery time courses of ionic and gating currents yields additional information about the kinetics and voltage dependence of fast inactivation in WT and mutant channels. Fig. 5 A illustrates recordings of WT ionic and gating current recoveries obtained from separate oocytes at different stages of expression. We used an alternating pulse protocol with and without prepulse for each recovery time in order to be able to normalize for the slow peak–current decay of the reference traces (without prepulse). This current decay results from the presence of a subpopulation of slow gating channels that predominantly appear in the absence of β1 coexpression (as discussed in the context of Fig. 1). During recovery, the fast gating channels recover first, followed by the slow gating channels. The gating currents that were recorded in the presence of 2 μM TTX show a more stable reference current compared with the ionic current, thus suggesting little decay. In view of the large differences of the recovery rates of WT and R4H obtained from ionic current data (see Fig. 1 D), we decided that it was more important to analyze the close correlation of ionic and gating current recovery concerning the time course and its voltage dependence, rather than attempting to discriminate the overlapping fast and slow gating channels. The gating current recovery shows a characteristic pattern: (a) the basic level where recovery starts is determined by the degree of immobilization that occurs when the duration and potential of the prepulse fully inactivate ionic current (Armstrong and Bezanilla 1977), and (b) the recovery of the gating current in its early time course shows a discontinuous change from a rapid rise to a slower one (Greeff 1986). We found that in WT and mutant channels ionic current and gating current recovery strongly correlate in the voltage range from −140 to −80 mV, concerning time course and voltage dependence (compare Fig. 1 D with Fig. 5Fig. 6Fig. 7). However, within a single phenotype the time constants of gating current recovery are significantly increased compared with the time constants of ionic current recovery. Similar results were obtained from studies of Shaker potassium channels where, notably at more depolarizing potentials, gating current recovery is considerably slower than is ionic current recovery (Roux et al. 1998). The observed mismatch in the corresponding recovery time constants of ionic and gating currents were obtained from data fitted with single exponential curves. Taking into account the expression of a mixture of fast and slow gating channels (Fig. 1 A and 5), we tried to fit double exponential curves, which, in some cases made the recovery time constants agree better. Nevertheless, we decided to fit our recovery data uniformly with single exponential curves since the gating charge recovery was difficult to fit by double exponential curves for two reasons: (a) the amplitude of the gating current recovery is rather small, and (b) the scattering of the data points obtained from gating charge recovery (Fig. 6 B) is more pronounced if compared with the ionic current recovery (Fig. 6 A).

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