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. 2017 Aug 11;292(32):13428-13440.
doi: 10.1074/jbc.M117.786509. Epub 2017 Jun 27.

Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel β4 subunit explain its role in cell-cell adhesion

Hideaki Shimizu  1   2   3 , Asako Tosaki  3 , Noboru Ohsawa  1   2 , Yoshiko Ishizuka-Katsura  1   2 , Shisako Shoji  1   2 , Haruko Miyazaki  3   4   5 , Fumitaka Oyama  3   6 , Takaho Terada  1   7 , Mikako Shirouzu  1   2 , Shun-Ichi Sekine  1   2 , Nobuyuki Nukina  3   4   5 , Shigeyuki Yokoyama  8   7
Affiliations

Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel β4 subunit explain its role in cell-cell adhesion

Hideaki Shimizu et al. J Biol Chem. .

Abstract

Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming α subunit and auxiliary β subunits, designated as β1/β1B-β4 (encoded by SCN1B-4B, respectively), which also function in cell-cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the β4 subunit, which contributes to its adhesive function. Here, using crystallographic and biochemical analyses, we show that the β4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys58) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30-Val35). Under reducing conditions, an N-terminally deleted β4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the β4 cis dimer contributes to the trans homophilic interaction of β4 in cell-cell adhesion. Furthermore, this mutant exhibited increased association with the α subunit, indicating that the cis dimerization of β4 affects α-β4 complex formation. These observations provide the structural basis for the parallel dimer formation of β4 in VGSCs and reveal its mechanism in cell-cell adhesion.

Keywords: Navβ4; SCN4B; X-ray crystallography; cell adhesion; dimer; immunoglobulin-like domain; membrane protein; sodium channel.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Arrangements of the mouse/human β4 subunit extracellular domain molecules in the monoclinic, cubic, and hexagonal crystal forms. A, schematics of the cis and trans homophilic interactions of β4 and the α–β4 complex. The plasma membranes are colored gray. The cis homophilic interaction of β4 and the α–β4 complex occur on the same cell. Additionally, β4 can form a trans homophilic interaction between two different cells. B, arrangement of the mouse β4 molecule in the monoclinic crystal form (PDB code 5AYQ). The β4 molecules are colored yellow and cyan. Molecules with the same color are parallel to each other, and the yellow molecules are oriented anti-parallel to the cyan ones. Shown are a top view (top panel) and side view of the enclosed region (bottom panel). C and D, arrangements of the mouse β4 molecules in the cubic crystal form (C) and the human β4 molecules in the hexagonal crystal form (D). One of the pairs of β4 molecules interacting with each other in the parallel arrangement is enclosed. E, electron densities of the polyethylene glycol fragments. Shown is a 2Fo-Fc composite omit map (contoured at 1.5 σ) of the polyethylene glycol fragments (PEG1 and PEG2) in the hexagonal form.
Figure 2.
Figure 2.
Structures of the parallel dimer of β4. A–C, ribbon representations of the β4 parallel dimer in which one molecule is colored yellow and the other is red. Shown are a side view (top panel) and top view (bottom panel). Also shown are the human β4 hexagonal form (A), the mouse β4 cubic form (B), and the mouse β4 monoclinic form (C). D, 2Fo-Fc composite omit map (contoured at 1.5 σ) around the intermolecular disulfide bond between the Cys58 residues. E, the intermolecular hydrophobic interactions of the N-terminal segment (the N–N interaction). The surface-exposed hydrophobic residues are colored green. F, detailed views of the N–N interactions. The N-terminal segment of one molecule is colored cyan, and the other molecule is shown by a gray ribbon and a yellow stick model. Hydrogen bonds are shown with dotted red lines. G, amino acid sequences of the mouse and human β4s. The intermolecular interactions (shown in red) are conserved in the human and mouse β4s. The β strands are shown as arrows. Sequence alignment was performed using the Genetyx software (Genetyx Corp., Tokyo, Japan).
Figure 3.
Figure 3.
Chromatographic analysis of the parallel dimer formation mediated by the S–S interaction. A–D, elution profiles of the human β4ex protein using anion exchange chromatography under non-reducing conditions (A), the size exclusion chromatography (HiLoad 16/60 Superdex 75 column) for the first peak from the anion exchange chromatography under non-reducing conditions (B), the second peak under non-reducing conditions (C), and reducing conditions (D).
Figure 4.
Figure 4.
Chromatographic analysis of the parallel dimer formation mediated by the N–N interaction. Shown are size exclusion chromatography (Superdex 75 10/300 GL column) elution profiles of the human β4ex after 0–12 h of incubation at 20 °C (top panel) and SDS-PAGE analysis of the fractions eluted from size exclusion chromatography after 3 h of incubation (bottom panel). The incubation was performed under non-reducing conditions, and the chromatography and SDS-PAGE analysis were performed under reducing conditions.
Figure 5.
Figure 5.
Aggregation assay of ΔN-expressing cells. A, Western blot analysis of CHO cells stably expressing the WT and ΔN mutant of mouse β4. CHO cells were treated without (left panel) and with BS3 (right panel). Blots were probed with anti-β4. B, cell aggregation kinetics of CHO cells stably expressing the WT and ΔN mutant of β4 under non-reducing conditions. Aggregation is represented by the index Nt/N0, where Nt and N0 are the total number of particles at incubation times t and 0, respectively. Data are means ± S.E., n = 24, from three independent experiments. Mock (black), WT (red), and ΔN (blue). C, cell aggregation kinetics under reducing conditions. Data are means ± S.E., n = 45, from five independent experiments. *, p < 0.05; **, p < 0.01 (two-tailed unpaired Student's t test) compared with the WT. D, cell aggregation patterns of CHO cells stably expressing the WT and mutants of β4 under reducing conditions after a 120-min incubation. Shown are the WT (top panel) and ΔN (bottom panel). E, immunocytochemistry of CHO cells stably expressing the WT (top panel) and ΔN mutant (bottom panel) of β4. The β4 proteins were immunostained with anti-β4 and visualized with Alexa Fluor 647 (magenta), and nuclei were stained with Hoechst 33342 (cyan). F, cell surface biotinylation of CHO cells stably expressing the WT and ΔN mutant of β4. Shown are representative Western blots using anti-β4 (left panel, top) and anti-Na+/K+ ATPase (left panel, bottom) and quantification of the β4 bands normalized by the corresponding anti-Na+/K+ ATPase bands (right panel). Data are means ± S.D., n = 3, and individual data are shown in scatterplots.
Figure 6.
Figure 6.
Interface with the α subunit. A, the N-terminal residues required for modulation of the α subunits are mapped onto the β4 crystal structure. B, immunoprecipitation of HEK293 cells transiently transfected with Nav1.5-V5-His + WT (mouse), Nav1.5-V5-His + ΔN (mouse), and Nav1.5-V5-His alone using anti-β4 antiserum. Blots were probed with anti-V5. The graph shows the Nav1.5 intensity from Nav1.5 + ΔN–transfected cells relative to that from Nav1.5 + WT–transfected cells. Data are means ± S.D. from three independent experiments, and individual data are shown in scatterplots. C, Western blot analysis of the input samples for the co-immunoprecipitation study, probed with anti-β4. The graph shows the β4 intensity from Nav1.5 + ΔN–transfected cells relative to that from Nav1.5 + WT–transfected cells. Data are means ± S.D. from three independent experiments, and individual data are shown in scatterplots. D, mapping of the GEFS+ mutations on the β1 model structure. The model was generated by the superimposition of the monomer β1 model (built by the program MODELLER (34)) onto the mouse monoclinic β4 structure.
Figure 7.
Figure 7.
Schematics of β4. A, Open (top panel) and closed (bottom panel) conformations. The plasma membranes are colored gray. The β4 protein may accommodate the hydrophobic residues in its own hydrophobic pocket to form a monomer in a closed conformation. B, topology diagram of strand A/A′. C, the trans homophilic interactions of the parallel dimer and the monomer of β4 in cell–cell adhesion. The closed and open conformations of β4 are likely to exist in equilibrium, and the parallel dimer could be readily formed when two monomers in the open conformation meet each other. The monomeric β4 can associate with the α subunit. Both the α-β4 complex and parallel dimer could exhibit cell–cell adhesion.
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