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. 2020 Aug 3;25(15):3551.
doi: 10.3390/molecules25153551.

Chemometric Models of Differential Amino Acids at the Navα and Navβ Interface of Mammalian Sodium Channel Isoforms

Fernando Villa-Diaz  1 , Susana Lopez-Nunez  1 , Jordan E Ruiz-Castelan  1 , Eduardo Marcos Salinas-Stefanon  2 , Thomas Scior  1
Affiliations

Chemometric Models of Differential Amino Acids at the Navα and Navβ Interface of Mammalian Sodium Channel Isoforms

Fernando Villa-Diaz et al. Molecules. .

Abstract

(1) Background: voltage-gated sodium channels (Navs) are integral membrane proteins that allow the sodium ion flux into the excitable cells and initiate the action potential. They comprise an α (Navα) subunit that forms the channel pore and are coupled to one or more auxiliary β (Navβ) subunits that modulate the gating to a variable extent. (2) Methods: after performing homology in silico modeling for all nine isoforms (Nav1.1α to Nav1.9α), the Navα and Navβ protein-protein interaction (PPI) was analyzed chemometrically based on the primary and secondary structures as well as topological or spatial mapping. (3) Results: our findings reveal a unique isoform-specific correspondence between certain segments of the extracellular loops of the Navα subunits. Precisely, loop S5 in domain I forms part of the PPI and assists Navβ1 or Navβ3 on all nine mammalian isoforms. The implied molecular movements resemble macroscopic springs, all of which explains published voltage sensor effects on sodium channel fast inactivation in gating. (4) Conclusions: currently, the specific functions exerted by the Navβ1 or Navβ3 subunits on the modulation of Navα gating remain unknown. Our work determined functional interaction in the extracellular domains on theoretical grounds and we propose a schematic model of the gating mechanism of fast channel sodium current inactivation by educated guessing.

Keywords: extracellular loops; homology modeling; hot spot prediction; interactome; protein-protein interaction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of gating. The three schemes of eukaryotic Navs show the (a) closed; (b) open; and (c) inactivated gating states. (d) A typical membrane current of Rattus norvegicus of the Navα1.4 isoform responds to a depolarizing pulse reflecting the three main states of gating; IFM inactivation gate: brownish; S4 voltage sensors: sky blue.
Figure 1
Figure 1
Schematic representation of gating. The three schemes of eukaryotic Navs show the (a) closed; (b) open; and (c) inactivated gating states. (d) A typical membrane current of Rattus norvegicus of the Navα1.4 isoform responds to a depolarizing pulse reflecting the three main states of gating; IFM inactivation gate: brownish; S4 voltage sensors: sky blue.
Figure 2
Figure 2
Display of the 3D model for S6 DIV in eeNav1.4α/eeNavβ1 [48]. The box displays details about the interacting residues at the interface. Labels 2 to 8: PPI identification numbers (PPI-Ids) of computed polar interactions (Table S1, Supplementary Materials); the amino acids are labeled by one-letter-codes with their primary sequence residue numbers and interacting atoms, e.g. A24(N). Colors: extracellular membrane boundaries (dark red); intracellular membrane boundaries (navy blue); transmembrane and intracellular protein regions of Navα which do not participate in PPI (gray); Navβ1 subunit (cornflower blue); S5 DI: magenta; S1-S2 DIII: orange; S5 DIV: brown; S6 DIV: cyan; computed polar interactions: black dotted lines. Visualization achieved by Chimera Alpha 1.14.
Figure 3
Figure 3
Display of PPI models. Based on the 3D template in (a) hNav1.4α/hNavβ1 [49]; based on a homology model in (b) hNav1.4α/hNavβ3 [49,55] for S6 DIV. The box presents atomic details at the interface. Labels 1 to 8: Ids of computed polar interactions (Table S1, Supplementary Materials); the amino acids are labeled by one-letter-codes with their primary sequence residue numbers and interacting atoms in parentheses, e.g. bottommost D1515(O). Colors: extracellular membrane boundaries (dark red); intracellular membrane boundaries (navy blue); transmembrane and intracellular protein regions of Navα that do not participate in PPI (gray); Navβ1 subunit (cornflower blue); Navβ3 subunit (forest green); S5 DI: magenta; S1-S2 DIII: orange; S5 DIV: brown; S6 DIV: cyan); computed polar interactions: black dotted lines. Visualization achieved by Chimera Alpha 1.14 [61].
Figure 4
Figure 4
Structure alignments of the ectodomains (IgD) of β subunit templates. (a) eeNavβ1 with hNavβ1; (b) hNavβ1 with hNavβ3; (c) hNavβ1 with hNavβ2; (d) hNavβ1 with hNavβ4; eeNavβ1: sea green; hNavβ1: salmon; hNavβ3: forest green; hNavβ2: purple; hNavβ4: golden; labels 1 to 8: positions of MSA residues according to Table 2. All superpositions were achieved by Chimera Alpha 1.14 with MatchMaker [61].
Figure 5
Figure 5
S4 DIII voltage sensor of hNav1.4α [49] in close contact with hNavβ1 [49] or hNavβ3 [55]. (a) hNav1.4α interfaced with hNavβ1; (b) hNav1.4α interfaced with Navβ3; (c,e) the MEPS at the interface hNav1.4α/hNavβ1; (d,f) the MEPS at the interface between hNav1.4α and hNavβ3. The structures were prepared with Chimera add-on PDB2PQR [63] and MEPS calculated for PPI surfaces using the Adaptive Poisson-Boltzmann Solver (APBS) [64], a plug-in tool in Chimera Alpha 1.14 [61] and simulated under Chimera X [65]. S4 DIII voltage sensor: magenta; hNavβ1: cornflower blue; hNavβ3: green.
Figure 5
Figure 5
S4 DIII voltage sensor of hNav1.4α [49] in close contact with hNavβ1 [49] or hNavβ3 [55]. (a) hNav1.4α interfaced with hNavβ1; (b) hNav1.4α interfaced with Navβ3; (c,e) the MEPS at the interface hNav1.4α/hNavβ1; (d,f) the MEPS at the interface between hNav1.4α and hNavβ3. The structures were prepared with Chimera add-on PDB2PQR [63] and MEPS calculated for PPI surfaces using the Adaptive Poisson-Boltzmann Solver (APBS) [64], a plug-in tool in Chimera Alpha 1.14 [61] and simulated under Chimera X [65]. S4 DIII voltage sensor: magenta; hNavβ1: cornflower blue; hNavβ3: green.
Figure 6
Figure 6
Hypothetical modulation of fast inactivation of Navα gating by Navβ1 or Navβ3; (a) Navα in idle (closed) state in response to interacting Navβ1 or Navβ3; (b) Navα in open (activated) state in presence of Navβ1 or Navβ3 modulation; (c) fast inactivation modulated by Navβ1 or Navβ3; (d) fast inactivation triggered by the IFM inactivation gate; labels 1 to 8: Id of computed polar PPIs (Table S1); computed polar PPIs: navy blue arrows; return to its start position of S4 is forced by IF-ECLs by computed polar interactions with Navβ1 or Navβ3: green arrows; negative charges: red minus signs in parentheses; positive charges: navy blue plus signs in parentheses; Navα regions without PPI: dark and light gray; Navβ1 or Navβ3 subunit: light salmon; S5 DI: magenta; S1-S2 DIII: orange; S5 DIV: brown; S6 DIV: cyan; S4: sky blue, segments S1, S2, S3, S4, S5, and S6: light gray.
Figure 6
Figure 6
Hypothetical modulation of fast inactivation of Navα gating by Navβ1 or Navβ3; (a) Navα in idle (closed) state in response to interacting Navβ1 or Navβ3; (b) Navα in open (activated) state in presence of Navβ1 or Navβ3 modulation; (c) fast inactivation modulated by Navβ1 or Navβ3; (d) fast inactivation triggered by the IFM inactivation gate; labels 1 to 8: Id of computed polar PPIs (Table S1); computed polar PPIs: navy blue arrows; return to its start position of S4 is forced by IF-ECLs by computed polar interactions with Navβ1 or Navβ3: green arrows; negative charges: red minus signs in parentheses; positive charges: navy blue plus signs in parentheses; Navα regions without PPI: dark and light gray; Navβ1 or Navβ3 subunit: light salmon; S5 DI: magenta; S1-S2 DIII: orange; S5 DIV: brown; S6 DIV: cyan; S4: sky blue, segments S1, S2, S3, S4, S5, and S6: light gray.
Figure 7
Figure 7
Display of a trimeric hNavβ1 model. (a) Navβ1 trimer seen top-down (b) close-up view from above of the alleged hotspot (c) Aligned sequences from Mammalian species. Negatively charged residues are identical to Asp25 and Glu27 on Navβ1 from Homo sapiens; white box: analysis area; green dotted lines: computed repulsion of charges; sticks: negative charged residues; sticks and balls: residues that form a possible hydrophobic patch. Data generated by Chimera Alpha 1.14 [61].
Figure 7
Figure 7
Display of a trimeric hNavβ1 model. (a) Navβ1 trimer seen top-down (b) close-up view from above of the alleged hotspot (c) Aligned sequences from Mammalian species. Negatively charged residues are identical to Asp25 and Glu27 on Navβ1 from Homo sapiens; white box: analysis area; green dotted lines: computed repulsion of charges; sticks: negative charged residues; sticks and balls: residues that form a possible hydrophobic patch. Data generated by Chimera Alpha 1.14 [61].
Figure 8
Figure 8
Superposition of monomeric and trimeric 3D models of β proteins. (a) Experimentally determined homotrimeric hNavβ3 [55] (three colors: magenta, light blue, beige) in superposition with template hNav1.4/hNavβ1 (bluish/grey) [49] and (b) one IgD (out of three) subunit(s) of homotrimeric hNavβ3 (beige) in superposition with template hNav1.4α/hNavβ1. It can be seen—by eyesight—that in case a two out of the three subunits bump into the membrane. Extracellular membrane boundaries: dark red; intracellular membrane boundaries: navy blue; Navα subunit: gray; hNavβ1 subunit: cornflower blue; hNavβ3 chains A, B, and C: orange, cyan, and magenta, respectively.
Figure 9
Figure 9
Three-dimensional (3D) location of subunits Navβ1 and Navβ3. (a,b) Navα topology in complex with Navβ1 and Navβ3. Display of 3D models with solvent-excluded surface areas in panels (c,d). (c) Cryo-EM structure of hNavα1.4 in complex with Navβ1 [49] and (d) Cryo-EM structures of hNavα1.4 [49] in complex with crystal structure hNavβ3 [55] positioned according to structural analysis; S4: sky blue; in panels (c,d) the molecular surfaces are colored: Navβ1: cornflower blue; Navβ3: green forest; and grey color for Navα subunit surfaces. The same colors were applied to the panels (a,b) above. 3D models by Chimera X [65].
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