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

Hideaki Shimizu,Takaho Terada,Mikako Shirouzu,Asako Tosaki,Shun-ichi Sekine,Yoshiko Ishizuka-Katsura,Fumitaka Oyama,Nobuyuki Nukina,Shigeyuki Yokoyama,Noboru Ohsawa,Shisako Shoji,Haruko Miyazaki

doi:10.1074/jbc.m117.786509

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.

Highlights

C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30-Val35)
The crystal packing included an anti-parallel contact between the ␤4ex molecules that was found to correspond to the trans homophilic interaction in cell– cell adhesion (Fig. 1, A and B)
Our structural analysis provided a detailed view of the parallel dimer of ␤4, involving the N–N interaction between the N-terminal segments of the two ␤4ex molecules and the S–S interaction formed by the intermolecular disulfide bond between the unpaired Cys residues in the parallel arrangement

Summary

Results

We determined the crystal structure of the mouse ␤4ex protein, containing all of the Cys residues, in the monoclinic crystal form at 1.7-Å resolution [8]. In this N–N interaction, the N-terminal segments form hydrogen bonds between their Glu and Ser with Ser and Thr, respectively, and join the ␤ sheets of the counterparts (Fig. 2, A–C, E, and F) As these intermolecular interactions are sophisticated and conserved in the human and mouse ␤4 structures (Fig. 2G), we hereafter designate the two ␤4ex molecules in the parallel arrangement as the parallel dimer. On the basis of these observations, we suggest that the ⌬N mutant on the cell surface could form a parallel dimer by S–S interaction under non-reducing conditions and become a monomeric form by the treatment with reducing agents, thereby decreasing the ability to mediate cell– cell adhesion. The crystal structures showed that the side chains of the N-terminal segment residues are located within the interface between the N–N interaction molecules and thereby unexposed on the parallel dimer surface (Fig. 6A). Cated that parallel dimer formation could decrease the association of ␤4 with Nav1.5

Discussion

Recombinant protein expression by Escherichia coli and crystallization

Analytical size exclusion chromatography

Cell culture and transfection

Western blotting

Cell aggregation assay

Immunofluorescence microscopy

Cell surface biotinylation

Statistical analysis