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The external pore loop interacts with S6 and S3-S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na(+) channel.

Yang YC, Hsieh JY, Kuo CC - J. Gen. Physiol. (2009)

Bottom Line: Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na(+) channel.Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action.We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na(+) channel.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Chang-Gung University, Tao-Yuan, Taiwan.

ABSTRACT
Carbamazepine, phenytoin, and lamotrigine are widely prescribed anticonvulsants in neurological clinics. These drugs bind to the same receptor site, probably with the diphenyl motif in their structure, to inhibit the Na(+) channel. However, the location of the drug receptor remains controversial. In this study, we demonstrate close proximity and potential interaction between an external aromatic residue (W1716 in the external pore loop) and an internal aromatic residue (F1764 in the pore-lining part of the sixth transmembrane segment, S6) of domain 4 (D4), both being closely related to anticonvulsant and/or local anesthetic binding to the Na(+) channel. Double-mutant cycle analysis reveals significant cooperativity between the two phenyl residues for anticonvulsant binding. Concomitant F1764C mutation evidently decreases the susceptibility of W1716C to external Cd(2+) and membrane-impermeable methanethiosulfonate reagents. Also, the W1716E/F1764R and G1715E/F1764R double mutations significantly alter the selectivity for Na(+) over K(+) and markedly shift the activation curve, respectively. W1716 and F1764 therefore very likely form a link connecting the outer and inner compartments of the Na(+) channel pore (in addition to the selectivity filter). Anticonvulsants and local anesthetics may well traverse this "S6 recess" without trespassing on the selectivity filter. Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na(+) channel. The effect of Y1618K mutation, however, is abolished by concomitant point mutations in the vicinity of Y1618, but not by those in the internally located inactivation machinery, supporting a direct local rather than a long-range allosteric action. Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action. We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na(+) channel.

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A model illustrating the close proximity and interactions among the S3-4 linker (e.g., Y1618), the SS6 pore loop (e.g., W1716), and the internal pore-lining part of S6 (e.g., F1764) in domain 4 of the Na+ channel, making a pivotal apparatus for ion permeation, activation-inactivation coupling, and drug binding. (A) The transmembrane segments S3–S6 in domain 4 of the channel are plotted as rectangular cylinders and viewed from the pore. W1716 is adjacent to A1714 in sequence, and thus is most likely located externally to the selectivity filter in the SS6 pore loop. This position could reasonably interact with F1764 of S6 if there is a turn at the junction of SS6 loop and S6 helix. As a result, the S6 helix in the vicinity of F1764 forms a “recess” of the pore. The aromatic residues Y1618, W1716, and F1764 are plotted as yellow dots. Membrane voltage changes move the S4 segment, and thus move the S3-4, S4-5, and S5-6 linkers to contribute to the essential voltage-dependent physiological and pharmacological attributes of the channel. (B) An enlarged picture for the interacting aromatic residues in A. (C) A homology model for the S6 recess of domain 4 in Nav1.2 based on the crystallized structure of the KcsA K+ channel pore (done by an online server provided by the Swiss Institute of Bioinformatics: http://swissmodel.expasy.org). Because of the marked difference in the length of the pore loops between these two types of channels, the homology modeling is based on somewhat arbitrary sequence alignment of the key conservative residues of D4SS6 and S6 (marked by green and orange rectangles, respectively) in the Nav1.2 channel and a subunit in the KcsA channel, with a large portion of pore loop in Nav1.2 deleted (top panel; note the sequence numbering and the arrows indicating W1716 and F1764). In the molecular model (bottom panel), the S5, S6, and S5-S6 linker of domain 4 are shown as space fills and colored gray, orange, and dark green, respectively. A1714 is in light green to mark the possible location of the selectivity filter. The aromatic side chains of W1716 in SS6 and F1764 in S6 are shown in yellow. A carbamazepine molecule could be well docked to a receptor constituted by W1716 and F1764 in the recess region of this model with the Discovery Studio software (Accelyrs Inc.; not depicted). (D) The brown-colored areas illustrate the other part of the channel protein surrounding the aqueous pore region (light blue), which is made by the four SS5-SS6 loops from the four domains (illustrated as the four “walls” making the external part of the pore). W1716 on the SS5-SS6 loop and F1764 on S6 (dotted helix; both residues depicted as yellow phenyl groups) of domain 4 interact to form a recess, which is more readily depicted with an angle of view roughly perpendicular to that in A and B. The anticonvulsant drug (shown as a pink diphenyl motif) presumably binds to its receptor located at the S6 recess with dipole-induced dipole interactions among the phenyl groups of the drug (pink), W1716 and F1764 (both in yellow; the boxed picture). A hydrophobic drug molecule of suitable conformation could even go through the S6 recess and thus traverse the pore without trespassing on the selectivity filter, embodying the long-proposed “hydrophobic” pathway of local anesthetic action on the Na+ channel.
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fig11: A model illustrating the close proximity and interactions among the S3-4 linker (e.g., Y1618), the SS6 pore loop (e.g., W1716), and the internal pore-lining part of S6 (e.g., F1764) in domain 4 of the Na+ channel, making a pivotal apparatus for ion permeation, activation-inactivation coupling, and drug binding. (A) The transmembrane segments S3–S6 in domain 4 of the channel are plotted as rectangular cylinders and viewed from the pore. W1716 is adjacent to A1714 in sequence, and thus is most likely located externally to the selectivity filter in the SS6 pore loop. This position could reasonably interact with F1764 of S6 if there is a turn at the junction of SS6 loop and S6 helix. As a result, the S6 helix in the vicinity of F1764 forms a “recess” of the pore. The aromatic residues Y1618, W1716, and F1764 are plotted as yellow dots. Membrane voltage changes move the S4 segment, and thus move the S3-4, S4-5, and S5-6 linkers to contribute to the essential voltage-dependent physiological and pharmacological attributes of the channel. (B) An enlarged picture for the interacting aromatic residues in A. (C) A homology model for the S6 recess of domain 4 in Nav1.2 based on the crystallized structure of the KcsA K+ channel pore (done by an online server provided by the Swiss Institute of Bioinformatics: http://swissmodel.expasy.org). Because of the marked difference in the length of the pore loops between these two types of channels, the homology modeling is based on somewhat arbitrary sequence alignment of the key conservative residues of D4SS6 and S6 (marked by green and orange rectangles, respectively) in the Nav1.2 channel and a subunit in the KcsA channel, with a large portion of pore loop in Nav1.2 deleted (top panel; note the sequence numbering and the arrows indicating W1716 and F1764). In the molecular model (bottom panel), the S5, S6, and S5-S6 linker of domain 4 are shown as space fills and colored gray, orange, and dark green, respectively. A1714 is in light green to mark the possible location of the selectivity filter. The aromatic side chains of W1716 in SS6 and F1764 in S6 are shown in yellow. A carbamazepine molecule could be well docked to a receptor constituted by W1716 and F1764 in the recess region of this model with the Discovery Studio software (Accelyrs Inc.; not depicted). (D) The brown-colored areas illustrate the other part of the channel protein surrounding the aqueous pore region (light blue), which is made by the four SS5-SS6 loops from the four domains (illustrated as the four “walls” making the external part of the pore). W1716 on the SS5-SS6 loop and F1764 on S6 (dotted helix; both residues depicted as yellow phenyl groups) of domain 4 interact to form a recess, which is more readily depicted with an angle of view roughly perpendicular to that in A and B. The anticonvulsant drug (shown as a pink diphenyl motif) presumably binds to its receptor located at the S6 recess with dipole-induced dipole interactions among the phenyl groups of the drug (pink), W1716 and F1764 (both in yellow; the boxed picture). A hydrophobic drug molecule of suitable conformation could even go through the S6 recess and thus traverse the pore without trespassing on the selectivity filter, embodying the long-proposed “hydrophobic” pathway of local anesthetic action on the Na+ channel.

Mentions: Two aromatic residues, W1716 and F1764, have been reported to contribute to anticonvulsant binding to the Na+ channel (Ragsdale et al., 1994, 1996; Tsang et al., 2005; McNulty et al., 2007). In this study, we demonstrate that W1716 (in the external pore loop) is located in close proximity to F1764 (presumably lining the internal compartment of the pore of the S6 helix). First, in terms of the effect on carbamazepine binding affinity, the non-additive feature of W1716 and F1764 mutations suggests that the two residues do not independently contribute to the drug receptor. Moreover, the estimated interaction energy between W1716 and F1764 falls into the range expected for two closely spaced interacting aromatic side chains in a protein (Table I) (Serrano et al., 1991; Smith and Regan, 1995; Tatko and Waters, 2002; Hong et al., 2007). Second, F1764C reduces channel inactivation that is enhanced by W1716C and abolishes the accessibility of W1716C to the external MTSET, MTSES, and Cd2+. Third, the ion selectivity for Na+ over K+ of the W1716E mutant is abolished by introducing a countercharge at F1764 (i.e., F1764R). Fourth, double mutation of F1764R and G1715E (and also F1764R and L1719E) causes a large shift in the activation curve (but G1715E, L1719E, or F1764R single mutation does not have such an effect). These findings give rise to novel structural insight that the external pore-lining segment SS6 is located very close to the internal pore compartment of S6, enabling an alternative connection bypassing the selectivity filter between the external and internal compartments of the Na+ channel pore. This alternative connection can be reasonably conceived because there must be a turn between the SS6 (the carboxyl end) of the pore loop and the S6 helix (Fig. 11). In this regard, the S6 helix in the vicinity of F1764 may form a “recess” of the pore, so that the side chain at this F1764 position could present itself beside the SS6 pore loop to face the external part of the pore without trespassing the selectivity filter.


The external pore loop interacts with S6 and S3-S4 linker in domain 4 to assume an essential role in gating control and anticonvulsant action in the Na(+) channel.

Yang YC, Hsieh JY, Kuo CC - J. Gen. Physiol. (2009)

A model illustrating the close proximity and interactions among the S3-4 linker (e.g., Y1618), the SS6 pore loop (e.g., W1716), and the internal pore-lining part of S6 (e.g., F1764) in domain 4 of the Na+ channel, making a pivotal apparatus for ion permeation, activation-inactivation coupling, and drug binding. (A) The transmembrane segments S3–S6 in domain 4 of the channel are plotted as rectangular cylinders and viewed from the pore. W1716 is adjacent to A1714 in sequence, and thus is most likely located externally to the selectivity filter in the SS6 pore loop. This position could reasonably interact with F1764 of S6 if there is a turn at the junction of SS6 loop and S6 helix. As a result, the S6 helix in the vicinity of F1764 forms a “recess” of the pore. The aromatic residues Y1618, W1716, and F1764 are plotted as yellow dots. Membrane voltage changes move the S4 segment, and thus move the S3-4, S4-5, and S5-6 linkers to contribute to the essential voltage-dependent physiological and pharmacological attributes of the channel. (B) An enlarged picture for the interacting aromatic residues in A. (C) A homology model for the S6 recess of domain 4 in Nav1.2 based on the crystallized structure of the KcsA K+ channel pore (done by an online server provided by the Swiss Institute of Bioinformatics: http://swissmodel.expasy.org). Because of the marked difference in the length of the pore loops between these two types of channels, the homology modeling is based on somewhat arbitrary sequence alignment of the key conservative residues of D4SS6 and S6 (marked by green and orange rectangles, respectively) in the Nav1.2 channel and a subunit in the KcsA channel, with a large portion of pore loop in Nav1.2 deleted (top panel; note the sequence numbering and the arrows indicating W1716 and F1764). In the molecular model (bottom panel), the S5, S6, and S5-S6 linker of domain 4 are shown as space fills and colored gray, orange, and dark green, respectively. A1714 is in light green to mark the possible location of the selectivity filter. The aromatic side chains of W1716 in SS6 and F1764 in S6 are shown in yellow. A carbamazepine molecule could be well docked to a receptor constituted by W1716 and F1764 in the recess region of this model with the Discovery Studio software (Accelyrs Inc.; not depicted). (D) The brown-colored areas illustrate the other part of the channel protein surrounding the aqueous pore region (light blue), which is made by the four SS5-SS6 loops from the four domains (illustrated as the four “walls” making the external part of the pore). W1716 on the SS5-SS6 loop and F1764 on S6 (dotted helix; both residues depicted as yellow phenyl groups) of domain 4 interact to form a recess, which is more readily depicted with an angle of view roughly perpendicular to that in A and B. The anticonvulsant drug (shown as a pink diphenyl motif) presumably binds to its receptor located at the S6 recess with dipole-induced dipole interactions among the phenyl groups of the drug (pink), W1716 and F1764 (both in yellow; the boxed picture). A hydrophobic drug molecule of suitable conformation could even go through the S6 recess and thus traverse the pore without trespassing on the selectivity filter, embodying the long-proposed “hydrophobic” pathway of local anesthetic action on the Na+ channel.
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fig11: A model illustrating the close proximity and interactions among the S3-4 linker (e.g., Y1618), the SS6 pore loop (e.g., W1716), and the internal pore-lining part of S6 (e.g., F1764) in domain 4 of the Na+ channel, making a pivotal apparatus for ion permeation, activation-inactivation coupling, and drug binding. (A) The transmembrane segments S3–S6 in domain 4 of the channel are plotted as rectangular cylinders and viewed from the pore. W1716 is adjacent to A1714 in sequence, and thus is most likely located externally to the selectivity filter in the SS6 pore loop. This position could reasonably interact with F1764 of S6 if there is a turn at the junction of SS6 loop and S6 helix. As a result, the S6 helix in the vicinity of F1764 forms a “recess” of the pore. The aromatic residues Y1618, W1716, and F1764 are plotted as yellow dots. Membrane voltage changes move the S4 segment, and thus move the S3-4, S4-5, and S5-6 linkers to contribute to the essential voltage-dependent physiological and pharmacological attributes of the channel. (B) An enlarged picture for the interacting aromatic residues in A. (C) A homology model for the S6 recess of domain 4 in Nav1.2 based on the crystallized structure of the KcsA K+ channel pore (done by an online server provided by the Swiss Institute of Bioinformatics: http://swissmodel.expasy.org). Because of the marked difference in the length of the pore loops between these two types of channels, the homology modeling is based on somewhat arbitrary sequence alignment of the key conservative residues of D4SS6 and S6 (marked by green and orange rectangles, respectively) in the Nav1.2 channel and a subunit in the KcsA channel, with a large portion of pore loop in Nav1.2 deleted (top panel; note the sequence numbering and the arrows indicating W1716 and F1764). In the molecular model (bottom panel), the S5, S6, and S5-S6 linker of domain 4 are shown as space fills and colored gray, orange, and dark green, respectively. A1714 is in light green to mark the possible location of the selectivity filter. The aromatic side chains of W1716 in SS6 and F1764 in S6 are shown in yellow. A carbamazepine molecule could be well docked to a receptor constituted by W1716 and F1764 in the recess region of this model with the Discovery Studio software (Accelyrs Inc.; not depicted). (D) The brown-colored areas illustrate the other part of the channel protein surrounding the aqueous pore region (light blue), which is made by the four SS5-SS6 loops from the four domains (illustrated as the four “walls” making the external part of the pore). W1716 on the SS5-SS6 loop and F1764 on S6 (dotted helix; both residues depicted as yellow phenyl groups) of domain 4 interact to form a recess, which is more readily depicted with an angle of view roughly perpendicular to that in A and B. The anticonvulsant drug (shown as a pink diphenyl motif) presumably binds to its receptor located at the S6 recess with dipole-induced dipole interactions among the phenyl groups of the drug (pink), W1716 and F1764 (both in yellow; the boxed picture). A hydrophobic drug molecule of suitable conformation could even go through the S6 recess and thus traverse the pore without trespassing on the selectivity filter, embodying the long-proposed “hydrophobic” pathway of local anesthetic action on the Na+ channel.
Mentions: Two aromatic residues, W1716 and F1764, have been reported to contribute to anticonvulsant binding to the Na+ channel (Ragsdale et al., 1994, 1996; Tsang et al., 2005; McNulty et al., 2007). In this study, we demonstrate that W1716 (in the external pore loop) is located in close proximity to F1764 (presumably lining the internal compartment of the pore of the S6 helix). First, in terms of the effect on carbamazepine binding affinity, the non-additive feature of W1716 and F1764 mutations suggests that the two residues do not independently contribute to the drug receptor. Moreover, the estimated interaction energy between W1716 and F1764 falls into the range expected for two closely spaced interacting aromatic side chains in a protein (Table I) (Serrano et al., 1991; Smith and Regan, 1995; Tatko and Waters, 2002; Hong et al., 2007). Second, F1764C reduces channel inactivation that is enhanced by W1716C and abolishes the accessibility of W1716C to the external MTSET, MTSES, and Cd2+. Third, the ion selectivity for Na+ over K+ of the W1716E mutant is abolished by introducing a countercharge at F1764 (i.e., F1764R). Fourth, double mutation of F1764R and G1715E (and also F1764R and L1719E) causes a large shift in the activation curve (but G1715E, L1719E, or F1764R single mutation does not have such an effect). These findings give rise to novel structural insight that the external pore-lining segment SS6 is located very close to the internal pore compartment of S6, enabling an alternative connection bypassing the selectivity filter between the external and internal compartments of the Na+ channel pore. This alternative connection can be reasonably conceived because there must be a turn between the SS6 (the carboxyl end) of the pore loop and the S6 helix (Fig. 11). In this regard, the S6 helix in the vicinity of F1764 may form a “recess” of the pore, so that the side chain at this F1764 position could present itself beside the SS6 pore loop to face the external part of the pore without trespassing the selectivity filter.

Bottom Line: Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na(+) channel.Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action.We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na(+) channel.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Chang-Gung University, Tao-Yuan, Taiwan.

ABSTRACT
Carbamazepine, phenytoin, and lamotrigine are widely prescribed anticonvulsants in neurological clinics. These drugs bind to the same receptor site, probably with the diphenyl motif in their structure, to inhibit the Na(+) channel. However, the location of the drug receptor remains controversial. In this study, we demonstrate close proximity and potential interaction between an external aromatic residue (W1716 in the external pore loop) and an internal aromatic residue (F1764 in the pore-lining part of the sixth transmembrane segment, S6) of domain 4 (D4), both being closely related to anticonvulsant and/or local anesthetic binding to the Na(+) channel. Double-mutant cycle analysis reveals significant cooperativity between the two phenyl residues for anticonvulsant binding. Concomitant F1764C mutation evidently decreases the susceptibility of W1716C to external Cd(2+) and membrane-impermeable methanethiosulfonate reagents. Also, the W1716E/F1764R and G1715E/F1764R double mutations significantly alter the selectivity for Na(+) over K(+) and markedly shift the activation curve, respectively. W1716 and F1764 therefore very likely form a link connecting the outer and inner compartments of the Na(+) channel pore (in addition to the selectivity filter). Anticonvulsants and local anesthetics may well traverse this "S6 recess" without trespassing on the selectivity filter. Furthermore, we found that Y1618K, a point mutation in the S3-4 linker (the extracellular extension of D4S4), significantly alters the consequences of carbamazepine binding to the Na(+) channel. The effect of Y1618K mutation, however, is abolished by concomitant point mutations in the vicinity of Y1618, but not by those in the internally located inactivation machinery, supporting a direct local rather than a long-range allosteric action. Moreover, Y1618 could interact with D4 pore residues W1716 and L1719 to have a profound effect on both channel gating and anticonvulsant action. We conclude that there are direct interactions among the external S3-4 linker, the external pore loop, and the internal S6 segment in D4, making the external pore loop a pivotal point critically coordinating ion permeation, gating, and anticonvulsant binding in the Na(+) channel.

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Related in: MedlinePlus