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Evolutionarily conserved intracellular gate of voltage-dependent sodium channels.

Oelstrom K, Goldschen-Ohm MP, Holmgren M, Chanda B - Nat Commun (2014)

Bottom Line: The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved.These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels.Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neuroscience, University of Wisconsin, Madison, Wisconsin 53706, USA [2] Molecular Pharmacology Graduate Program, University of Wisconsin, Madison, Wisconsin 53706, USA.

ABSTRACT
Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

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Alignment of pore domains and homology models.(a) Aligned sequences of individual pore domains for rat Kv1.2, Shaker Kv, rat Nav1.4, rabbit Cav2.1 and the bacterial sodium channels NavAb, NavMs, NavRh and NaChBac (see Results). Red and blue highlighted residues in Nav1.4 DIV, Shaker Kv and Cav2.1 DI-III denote positions accessible to internal MTSET in both closed and open states (blue) or open states only (red). Residues highlighted in grey indicate positions where internal MTSET did not produce a functional change in the peak current response. Shaker Kv and Cav2.1 accessibility data are from previous experiments1012. Residues aligned with those contributing to the selectivity filter and intracellular pore gate in Nav1.4 are highlighted in yellow. Highly conserved residues are shown on a black background. Grey scale bar below the sequences shows the CORE index reliability score at each position (light grey=low reliability, black=high reliability) along with an indicator for highly conserved (:) and semi-conserved (.) positions27. (b) Homology models of the pore lining S6 segments from each domain in rat Nav1.4 channels based on prokaryotic structures in either the closed pore (NavAb, white) or open pore (NavMs, red) conformations. View is from the cytoplasm looking up through the channel pore. The residues comprising the pore gate are shown in stick representation in both their closed (white) and open (yellow) configurations. Images were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4; Schrödinger, LLC). (c) The solvent accessible surface at the gate region is shown in red for both the closed pore (left) and open pore (right) Nav1.4 models. View is the same as in b. (d) View of the gate region from within the plane of the membrane for closed (left) and open (right) Nav1.4 models. For clarity, only domains II and IV are shown. The residues contributing to the pore gate in the closed pore model are shown in stick representation and the solvent accessible surface is shown in red. For the open pore model, the solvent accessible surface is transparent so that the gate residues can be visualized.
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f6: Alignment of pore domains and homology models.(a) Aligned sequences of individual pore domains for rat Kv1.2, Shaker Kv, rat Nav1.4, rabbit Cav2.1 and the bacterial sodium channels NavAb, NavMs, NavRh and NaChBac (see Results). Red and blue highlighted residues in Nav1.4 DIV, Shaker Kv and Cav2.1 DI-III denote positions accessible to internal MTSET in both closed and open states (blue) or open states only (red). Residues highlighted in grey indicate positions where internal MTSET did not produce a functional change in the peak current response. Shaker Kv and Cav2.1 accessibility data are from previous experiments1012. Residues aligned with those contributing to the selectivity filter and intracellular pore gate in Nav1.4 are highlighted in yellow. Highly conserved residues are shown on a black background. Grey scale bar below the sequences shows the CORE index reliability score at each position (light grey=low reliability, black=high reliability) along with an indicator for highly conserved (:) and semi-conserved (.) positions27. (b) Homology models of the pore lining S6 segments from each domain in rat Nav1.4 channels based on prokaryotic structures in either the closed pore (NavAb, white) or open pore (NavMs, red) conformations. View is from the cytoplasm looking up through the channel pore. The residues comprising the pore gate are shown in stick representation in both their closed (white) and open (yellow) configurations. Images were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4; Schrödinger, LLC). (c) The solvent accessible surface at the gate region is shown in red for both the closed pore (left) and open pore (right) Nav1.4 models. View is the same as in b. (d) View of the gate region from within the plane of the membrane for closed (left) and open (right) Nav1.4 models. For clarity, only domains II and IV are shown. The residues contributing to the pore gate in the closed pore model are shown in stick representation and the solvent accessible surface is shown in red. For the open pore model, the solvent accessible surface is transparent so that the gate residues can be visualized.

Mentions: We aligned the pore sequences of the rat Kv1.2 and Shaker potassium channels with several bacterial voltage-gated sodium channels and the individual pore domains of rat Nav1.4 channels to assess the likelihood that (1) the location of the pore gate inferred from our accessibility studies represents a conserved feature among voltage-gated sodium channels and (2) our proposed gate in sodium channels is consistent with the gate inferred from similar accessibility studies in Shaker potassium and voltage-gated calcium channels (Fig. 6a). The multiple sequence alignment was performed using Expresso24, which utilizes the structural X-ray crystallographic data available for Kv1.2 (PDB 2A79)7, NavAb (PDB 4EKW)25, NavMs (PDB 4F4L)16 and NavRh (PDB 4DXW)26. The reliability of the alignment at each position was judged by the CORE index score as reported by T-Coffee27. This score was maximized in the regions corresponding to the selectivity filter and our proposed gate location in Nav1.4, but became progressively less reliable in regions associated with loops between helices or those extending into the cytoplasm as expected for less well-ordered structures.


Evolutionarily conserved intracellular gate of voltage-dependent sodium channels.

Oelstrom K, Goldschen-Ohm MP, Holmgren M, Chanda B - Nat Commun (2014)

Alignment of pore domains and homology models.(a) Aligned sequences of individual pore domains for rat Kv1.2, Shaker Kv, rat Nav1.4, rabbit Cav2.1 and the bacterial sodium channels NavAb, NavMs, NavRh and NaChBac (see Results). Red and blue highlighted residues in Nav1.4 DIV, Shaker Kv and Cav2.1 DI-III denote positions accessible to internal MTSET in both closed and open states (blue) or open states only (red). Residues highlighted in grey indicate positions where internal MTSET did not produce a functional change in the peak current response. Shaker Kv and Cav2.1 accessibility data are from previous experiments1012. Residues aligned with those contributing to the selectivity filter and intracellular pore gate in Nav1.4 are highlighted in yellow. Highly conserved residues are shown on a black background. Grey scale bar below the sequences shows the CORE index reliability score at each position (light grey=low reliability, black=high reliability) along with an indicator for highly conserved (:) and semi-conserved (.) positions27. (b) Homology models of the pore lining S6 segments from each domain in rat Nav1.4 channels based on prokaryotic structures in either the closed pore (NavAb, white) or open pore (NavMs, red) conformations. View is from the cytoplasm looking up through the channel pore. The residues comprising the pore gate are shown in stick representation in both their closed (white) and open (yellow) configurations. Images were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4; Schrödinger, LLC). (c) The solvent accessible surface at the gate region is shown in red for both the closed pore (left) and open pore (right) Nav1.4 models. View is the same as in b. (d) View of the gate region from within the plane of the membrane for closed (left) and open (right) Nav1.4 models. For clarity, only domains II and IV are shown. The residues contributing to the pore gate in the closed pore model are shown in stick representation and the solvent accessible surface is shown in red. For the open pore model, the solvent accessible surface is transparent so that the gate residues can be visualized.
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f6: Alignment of pore domains and homology models.(a) Aligned sequences of individual pore domains for rat Kv1.2, Shaker Kv, rat Nav1.4, rabbit Cav2.1 and the bacterial sodium channels NavAb, NavMs, NavRh and NaChBac (see Results). Red and blue highlighted residues in Nav1.4 DIV, Shaker Kv and Cav2.1 DI-III denote positions accessible to internal MTSET in both closed and open states (blue) or open states only (red). Residues highlighted in grey indicate positions where internal MTSET did not produce a functional change in the peak current response. Shaker Kv and Cav2.1 accessibility data are from previous experiments1012. Residues aligned with those contributing to the selectivity filter and intracellular pore gate in Nav1.4 are highlighted in yellow. Highly conserved residues are shown on a black background. Grey scale bar below the sequences shows the CORE index reliability score at each position (light grey=low reliability, black=high reliability) along with an indicator for highly conserved (:) and semi-conserved (.) positions27. (b) Homology models of the pore lining S6 segments from each domain in rat Nav1.4 channels based on prokaryotic structures in either the closed pore (NavAb, white) or open pore (NavMs, red) conformations. View is from the cytoplasm looking up through the channel pore. The residues comprising the pore gate are shown in stick representation in both their closed (white) and open (yellow) configurations. Images were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4; Schrödinger, LLC). (c) The solvent accessible surface at the gate region is shown in red for both the closed pore (left) and open pore (right) Nav1.4 models. View is the same as in b. (d) View of the gate region from within the plane of the membrane for closed (left) and open (right) Nav1.4 models. For clarity, only domains II and IV are shown. The residues contributing to the pore gate in the closed pore model are shown in stick representation and the solvent accessible surface is shown in red. For the open pore model, the solvent accessible surface is transparent so that the gate residues can be visualized.
Mentions: We aligned the pore sequences of the rat Kv1.2 and Shaker potassium channels with several bacterial voltage-gated sodium channels and the individual pore domains of rat Nav1.4 channels to assess the likelihood that (1) the location of the pore gate inferred from our accessibility studies represents a conserved feature among voltage-gated sodium channels and (2) our proposed gate in sodium channels is consistent with the gate inferred from similar accessibility studies in Shaker potassium and voltage-gated calcium channels (Fig. 6a). The multiple sequence alignment was performed using Expresso24, which utilizes the structural X-ray crystallographic data available for Kv1.2 (PDB 2A79)7, NavAb (PDB 4EKW)25, NavMs (PDB 4F4L)16 and NavRh (PDB 4DXW)26. The reliability of the alignment at each position was judged by the CORE index score as reported by T-Coffee27. This score was maximized in the regions corresponding to the selectivity filter and our proposed gate location in Nav1.4, but became progressively less reliable in regions associated with loops between helices or those extending into the cytoplasm as expected for less well-ordered structures.

Bottom Line: The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved.These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels.Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neuroscience, University of Wisconsin, Madison, Wisconsin 53706, USA [2] Molecular Pharmacology Graduate Program, University of Wisconsin, Madison, Wisconsin 53706, USA.

ABSTRACT
Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

Show MeSH
Related in: MedlinePlus