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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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Charge–voltage (Q/V) distributions in WT and mutant sodium channels. Open symbols are data obtained in the presence of a 20-ms inactivating prepulse to 0 mV (1-ms interval at −100 mV). Filled symbols are data without prepulse; test pulses were 13 ms long. The gating charge (Qg) represents the time integral of the corresponding gating current. The individual Q/V curves were fitted by a Boltzmann distribution as described in Table . The degree of immobilization at Qmax derived as mean value ± SD from Table  is indicated in each diagram.
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Figure 4: Charge–voltage (Q/V) distributions in WT and mutant sodium channels. Open symbols are data obtained in the presence of a 20-ms inactivating prepulse to 0 mV (1-ms interval at −100 mV). Filled symbols are data without prepulse; test pulses were 13 ms long. The gating charge (Qg) represents the time integral of the corresponding gating current. The individual Q/V curves were fitted by a Boltzmann distribution as described in Table . The degree of immobilization at Qmax derived as mean value ± SD from Table is indicated in each diagram.

Mentions: A summary of charge–voltage plots of WT and mutant sodium channels, generated in the presence or absence of an inactivating pulse and fitted to a standard Boltzmann distribution with slopes, half-activation potentials, and degrees of gating charge immobilization, is given in Fig. 4 and Table . R5H shows similar gating charge immobilization (48%) compared with WT (56%), whereas both R4H (34%) and R4/5H (34%) display strongly reduced gating charge immobilization. For R4H this observation reflects the fact that at more depolarizing potentials a substantial portion of the channels persists in the inactivated state (see discussion), as also indicated by the left-shift of the steady-state inactivation curve (Fig. 1 C). The slopes and half-activation potentials of WT and R5H only differ slightly from the corresponding values of R4H and R4/5H (Table ), which indicates the similar activation behavior of WT and mutant channels. The larger values of the immobilized gating charges at potentials more negative than −30 mV compared with the nonimmobilized total ON charges (Fig. 4) are most probably due to one of two possibilities. The first possibility is a contamination of the small total ON charges below −30 mV with residual ionic current in absence of an inactivating prepulse. This contamination could not be avoided at the very high expression levels and bath temperatures of 15°C despite of the presence of 2 μM TTX in the bath solution. The second possibility is an integration artifact resulting from a common baseline adjustment for the integration of gating current traces with different kinetics. However, our results were not distorted for that reason because the voltage ranges of main interest were not significantly affected.


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)

Charge–voltage (Q/V) distributions in WT and mutant sodium channels. Open symbols are data obtained in the presence of a 20-ms inactivating prepulse to 0 mV (1-ms interval at −100 mV). Filled symbols are data without prepulse; test pulses were 13 ms long. The gating charge (Qg) represents the time integral of the corresponding gating current. The individual Q/V curves were fitted by a Boltzmann distribution as described in Table . The degree of immobilization at Qmax derived as mean value ± SD from Table  is indicated in each diagram.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Charge–voltage (Q/V) distributions in WT and mutant sodium channels. Open symbols are data obtained in the presence of a 20-ms inactivating prepulse to 0 mV (1-ms interval at −100 mV). Filled symbols are data without prepulse; test pulses were 13 ms long. The gating charge (Qg) represents the time integral of the corresponding gating current. The individual Q/V curves were fitted by a Boltzmann distribution as described in Table . The degree of immobilization at Qmax derived as mean value ± SD from Table is indicated in each diagram.
Mentions: A summary of charge–voltage plots of WT and mutant sodium channels, generated in the presence or absence of an inactivating pulse and fitted to a standard Boltzmann distribution with slopes, half-activation potentials, and degrees of gating charge immobilization, is given in Fig. 4 and Table . R5H shows similar gating charge immobilization (48%) compared with WT (56%), whereas both R4H (34%) and R4/5H (34%) display strongly reduced gating charge immobilization. For R4H this observation reflects the fact that at more depolarizing potentials a substantial portion of the channels persists in the inactivated state (see discussion), as also indicated by the left-shift of the steady-state inactivation curve (Fig. 1 C). The slopes and half-activation potentials of WT and R5H only differ slightly from the corresponding values of R4H and R4/5H (Table ), which indicates the similar activation behavior of WT and mutant channels. The larger values of the immobilized gating charges at potentials more negative than −30 mV compared with the nonimmobilized total ON charges (Fig. 4) are most probably due to one of two possibilities. The first possibility is a contamination of the small total ON charges below −30 mV with residual ionic current in absence of an inactivating prepulse. This contamination could not be avoided at the very high expression levels and bath temperatures of 15°C despite of the presence of 2 μM TTX in the bath solution. The second possibility is an integration artifact resulting from a common baseline adjustment for the integration of gating current traces with different kinetics. However, our results were not distorted for that reason because the voltage ranges of main interest were not significantly affected.

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