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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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Model of sodium channel fast inactivation controlled by S4D4. (A) Schematic illustration of our interpretations and conclusions displaying the relevant structures of the sodium channel and their movements at closed, open, and inactivated states as well as during recovery from inactivation. The positively charged amino acids of the S4 voltage sensors in domains 2–4 and the analyzed mutations in S4D4 are indicated. R symbolizes the putative receptor site in the S4–S5 linker of domain 4, which binds the docking region (D) of the inactivation loop connecting domains 3 and 4 (L3–4), leading to fast inactivation of the channel. Moreover, the position of L3–4 during inactivation causes the partial immobilization of the voltage sensors (most probably S4D4 and S4D3; see discussion), which is indicated here as blockade of m by B. (B) State diagram with lower level reflecting the voltage-dependent activation pathway from several closed (C) to the open state (O) and further to the open state (OR), which presents the receptor instantly followed by the voltage-independent binding of L3–4. The upper level reflects the transitions between several inactivated states producing the nonimmobilized gating current fraction. For recovery from fast inactivation, hyperpolarization causes the reverse movement of S4D4, which disrupts the connection of the inactivation loop to its receptor and simultaneously causes the partial immobilization of the voltage sensors, thereby permitting the return of the channels into the resting (closed) state.
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Figure 8: Model of sodium channel fast inactivation controlled by S4D4. (A) Schematic illustration of our interpretations and conclusions displaying the relevant structures of the sodium channel and their movements at closed, open, and inactivated states as well as during recovery from inactivation. The positively charged amino acids of the S4 voltage sensors in domains 2–4 and the analyzed mutations in S4D4 are indicated. R symbolizes the putative receptor site in the S4–S5 linker of domain 4, which binds the docking region (D) of the inactivation loop connecting domains 3 and 4 (L3–4), leading to fast inactivation of the channel. Moreover, the position of L3–4 during inactivation causes the partial immobilization of the voltage sensors (most probably S4D4 and S4D3; see discussion), which is indicated here as blockade of m by B. (B) State diagram with lower level reflecting the voltage-dependent activation pathway from several closed (C) to the open state (O) and further to the open state (OR), which presents the receptor instantly followed by the voltage-independent binding of L3–4. The upper level reflects the transitions between several inactivated states producing the nonimmobilized gating current fraction. For recovery from fast inactivation, hyperpolarization causes the reverse movement of S4D4, which disrupts the connection of the inactivation loop to its receptor and simultaneously causes the partial immobilization of the voltage sensors, thereby permitting the return of the channels into the resting (closed) state.

Mentions: According to the present understanding (Fig. 8 A), the S4–S5 linker in domain 4 represents part of the putative receptor that binds the docking region of the intracellular loop connecting domains 3 and 4 (Tang et al. 1996; Mitrovic et al. 1996; Filatov et al. 1998; McPhee et al. 1998). This loop contains a highly conserved triplet of three consecutive amino acids (IFM: isoleucine-phenylalanine-methionine) and is regarded as the physical inactivation gate (Vassilev et al. 1988; Stühmer et al. 1989; West et al. 1992; Eaholtz et al. 1994). In addition, gating current studies at the squid axon showed the partial immobilization of gating current and gave rise to the idea of the “foot in the door” effect, i.e., an obstacle in the restoration of some gating structures during recovery from inactivation (Armstrong and Bezanilla 1977). Our study is to contribute to the understanding of how these different structures may be functionally connected and in particular how the voltage sensor S4D4 is coupled to fast inactivation and gating charge immobilization. Besides fast inactivation, which proceeds over milliseconds during brief depolarizations (<100 ms), sodium channels can inactivate over a much longer time scale when depolarized for seconds or minutes, a phenomenon called slow inactivation. Previously, little has been known about the structural basis of slow inactivation, but recent experimental data suggest that S4D4 plays an important role also in slow inactivation (Abbruzzese et al. 1998; Mitrovic and Horn 1999). However, these studies have not analyzed the electrophysiologically silent transitions between different inactivated states because gating current measurements were not performed. In addition, Vedantham and Cannon 1998 have demonstrated that in voltage-gated sodium channels slow inactivation does not affect the movement of the fast inactivation gate. Because our approach was to correlate the movements of the S4D4 voltage sensor and the fast inactivation gate using ionic and gating current recordings, we performed our experiments under conditions that minimize the possible effects of slow inactivation.


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)

Model of sodium channel fast inactivation controlled by S4D4. (A) Schematic illustration of our interpretations and conclusions displaying the relevant structures of the sodium channel and their movements at closed, open, and inactivated states as well as during recovery from inactivation. The positively charged amino acids of the S4 voltage sensors in domains 2–4 and the analyzed mutations in S4D4 are indicated. R symbolizes the putative receptor site in the S4–S5 linker of domain 4, which binds the docking region (D) of the inactivation loop connecting domains 3 and 4 (L3–4), leading to fast inactivation of the channel. Moreover, the position of L3–4 during inactivation causes the partial immobilization of the voltage sensors (most probably S4D4 and S4D3; see discussion), which is indicated here as blockade of m by B. (B) State diagram with lower level reflecting the voltage-dependent activation pathway from several closed (C) to the open state (O) and further to the open state (OR), which presents the receptor instantly followed by the voltage-independent binding of L3–4. The upper level reflects the transitions between several inactivated states producing the nonimmobilized gating current fraction. For recovery from fast inactivation, hyperpolarization causes the reverse movement of S4D4, which disrupts the connection of the inactivation loop to its receptor and simultaneously causes the partial immobilization of the voltage sensors, thereby permitting the return of the channels into the resting (closed) state.
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Related In: Results  -  Collection

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Figure 8: Model of sodium channel fast inactivation controlled by S4D4. (A) Schematic illustration of our interpretations and conclusions displaying the relevant structures of the sodium channel and their movements at closed, open, and inactivated states as well as during recovery from inactivation. The positively charged amino acids of the S4 voltage sensors in domains 2–4 and the analyzed mutations in S4D4 are indicated. R symbolizes the putative receptor site in the S4–S5 linker of domain 4, which binds the docking region (D) of the inactivation loop connecting domains 3 and 4 (L3–4), leading to fast inactivation of the channel. Moreover, the position of L3–4 during inactivation causes the partial immobilization of the voltage sensors (most probably S4D4 and S4D3; see discussion), which is indicated here as blockade of m by B. (B) State diagram with lower level reflecting the voltage-dependent activation pathway from several closed (C) to the open state (O) and further to the open state (OR), which presents the receptor instantly followed by the voltage-independent binding of L3–4. The upper level reflects the transitions between several inactivated states producing the nonimmobilized gating current fraction. For recovery from fast inactivation, hyperpolarization causes the reverse movement of S4D4, which disrupts the connection of the inactivation loop to its receptor and simultaneously causes the partial immobilization of the voltage sensors, thereby permitting the return of the channels into the resting (closed) state.
Mentions: According to the present understanding (Fig. 8 A), the S4–S5 linker in domain 4 represents part of the putative receptor that binds the docking region of the intracellular loop connecting domains 3 and 4 (Tang et al. 1996; Mitrovic et al. 1996; Filatov et al. 1998; McPhee et al. 1998). This loop contains a highly conserved triplet of three consecutive amino acids (IFM: isoleucine-phenylalanine-methionine) and is regarded as the physical inactivation gate (Vassilev et al. 1988; Stühmer et al. 1989; West et al. 1992; Eaholtz et al. 1994). In addition, gating current studies at the squid axon showed the partial immobilization of gating current and gave rise to the idea of the “foot in the door” effect, i.e., an obstacle in the restoration of some gating structures during recovery from inactivation (Armstrong and Bezanilla 1977). Our study is to contribute to the understanding of how these different structures may be functionally connected and in particular how the voltage sensor S4D4 is coupled to fast inactivation and gating charge immobilization. Besides fast inactivation, which proceeds over milliseconds during brief depolarizations (<100 ms), sodium channels can inactivate over a much longer time scale when depolarized for seconds or minutes, a phenomenon called slow inactivation. Previously, little has been known about the structural basis of slow inactivation, but recent experimental data suggest that S4D4 plays an important role also in slow inactivation (Abbruzzese et al. 1998; Mitrovic and Horn 1999). However, these studies have not analyzed the electrophysiologically silent transitions between different inactivated states because gating current measurements were not performed. In addition, Vedantham and Cannon 1998 have demonstrated that in voltage-gated sodium channels slow inactivation does not affect the movement of the fast inactivation gate. Because our approach was to correlate the movements of the S4D4 voltage sensor and the fast inactivation gate using ionic and gating current recordings, we performed our experiments under conditions that minimize the possible effects of slow inactivation.

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