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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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Recovery from fast inactivation of macroscopic ionic and gating currents. (A) Recovery of WT gating current (Ig, in the presence of 2 μM TTX) and ionic current (INa, in the absence of TTX) obtained from different oocytes. Pulse protocol: a 100-ms prepulse to 0 mV from a holding potential of −100 mV was followed by a recovery period of variable duration (2–90 ms) at −100 mV. Test pulses have a duration of 80 ms (ionic current) or 13 ms (gating current) and responses are superimposed for all recovery periods. For the calculation of the recovery time course, the current traces with prepulse were routinely normalized to the current traces without prepulse in order to compensate the slight decrease of the current amplitude during pulse series. The series of increasing recovery times, starting from 2 ms, where INa is almost totally inactivated, was preceded by the longest recovery time where the plateau of recovery is observed (first and last pulses are indicated in INa recordings). (B) Gating current recovery of WT and mutant sodium channels at −100 mV. Pulse protocol was as described above. The time intervals for recovery were between 10 and 1,250 ms for R4H and between 2 and 90 ms for WT and the other mutant channels. The recovering gating charges were fitted to a single exponential with corresponding recovery time constants (τR) as indicated. Notice the different levels of the nonimmobilized gating current fraction at the onset of recovery, reflecting the different degrees of immobilization.
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Figure 5: Recovery from fast inactivation of macroscopic ionic and gating currents. (A) Recovery of WT gating current (Ig, in the presence of 2 μM TTX) and ionic current (INa, in the absence of TTX) obtained from different oocytes. Pulse protocol: a 100-ms prepulse to 0 mV from a holding potential of −100 mV was followed by a recovery period of variable duration (2–90 ms) at −100 mV. Test pulses have a duration of 80 ms (ionic current) or 13 ms (gating current) and responses are superimposed for all recovery periods. For the calculation of the recovery time course, the current traces with prepulse were routinely normalized to the current traces without prepulse in order to compensate the slight decrease of the current amplitude during pulse series. The series of increasing recovery times, starting from 2 ms, where INa is almost totally inactivated, was preceded by the longest recovery time where the plateau of recovery is observed (first and last pulses are indicated in INa recordings). (B) Gating current recovery of WT and mutant sodium channels at −100 mV. Pulse protocol was as described above. The time intervals for recovery were between 10 and 1,250 ms for R4H and between 2 and 90 ms for WT and the other mutant channels. The recovering gating charges were fitted to a single exponential with corresponding recovery time constants (τR) as indicated. Notice the different levels of the nonimmobilized gating current fraction at the onset of recovery, reflecting the different degrees of immobilization.

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)

Recovery from fast inactivation of macroscopic ionic and gating currents. (A) Recovery of WT gating current (Ig, in the presence of 2 μM TTX) and ionic current (INa, in the absence of TTX) obtained from different oocytes. Pulse protocol: a 100-ms prepulse to 0 mV from a holding potential of −100 mV was followed by a recovery period of variable duration (2–90 ms) at −100 mV. Test pulses have a duration of 80 ms (ionic current) or 13 ms (gating current) and responses are superimposed for all recovery periods. For the calculation of the recovery time course, the current traces with prepulse were routinely normalized to the current traces without prepulse in order to compensate the slight decrease of the current amplitude during pulse series. The series of increasing recovery times, starting from 2 ms, where INa is almost totally inactivated, was preceded by the longest recovery time where the plateau of recovery is observed (first and last pulses are indicated in INa recordings). (B) Gating current recovery of WT and mutant sodium channels at −100 mV. Pulse protocol was as described above. The time intervals for recovery were between 10 and 1,250 ms for R4H and between 2 and 90 ms for WT and the other mutant channels. The recovering gating charges were fitted to a single exponential with corresponding recovery time constants (τR) as indicated. Notice the different levels of the nonimmobilized gating current fraction at the onset of recovery, reflecting the different degrees of immobilization.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Recovery from fast inactivation of macroscopic ionic and gating currents. (A) Recovery of WT gating current (Ig, in the presence of 2 μM TTX) and ionic current (INa, in the absence of TTX) obtained from different oocytes. Pulse protocol: a 100-ms prepulse to 0 mV from a holding potential of −100 mV was followed by a recovery period of variable duration (2–90 ms) at −100 mV. Test pulses have a duration of 80 ms (ionic current) or 13 ms (gating current) and responses are superimposed for all recovery periods. For the calculation of the recovery time course, the current traces with prepulse were routinely normalized to the current traces without prepulse in order to compensate the slight decrease of the current amplitude during pulse series. The series of increasing recovery times, starting from 2 ms, where INa is almost totally inactivated, was preceded by the longest recovery time where the plateau of recovery is observed (first and last pulses are indicated in INa recordings). (B) Gating current recovery of WT and mutant sodium channels at −100 mV. Pulse protocol was as described above. The time intervals for recovery were between 10 and 1,250 ms for R4H and between 2 and 90 ms for WT and the other mutant channels. The recovering gating charges were fitted to a single exponential with corresponding recovery time constants (τR) as indicated. Notice the different levels of the nonimmobilized gating current fraction at the onset of recovery, reflecting the different degrees of immobilization.
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