Structure-based site-directed photo-crosslinking analyses of multimeric cell-adhesive interactions of voltage-gated sodium channel β subunits.

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

doi:10.1038/srep26618

Abstract

The β1, β2, and β4 subunits of voltage-gated sodium channels reportedly function as cell adhesion molecules. The present crystallographic analysis of the β4 extracellular domain revealed an antiparallel arrangement of the β4 molecules in the crystal lattice. The interface between the two antiparallel β4 molecules is asymmetric, and results in a multimeric assembly. Structure-based mutagenesis and site-directed photo-crosslinking analyses of the β4-mediated cell-cell adhesion revealed that the interface between the antiparallel β4 molecules corresponds to that in the trans homophilic interaction for the multimeric assembly of β4 in cell-cell adhesion. This trans interaction mode is also employed in the β1-mediated cell-cell adhesion. Moreover, the β1 gene mutations associated with generalized epilepsy with febrile seizures plus (GEFS+) impaired the β1-mediated cell-cell adhesion, which should underlie the GEFS+ pathogenesis. Thus, the structural basis for the β-subunit-mediated cell-cell adhesion has been established.

Highlights

The interfaces for the trans interactions of the βsubunits have remained elusive, in spite of their importance in cell-cell adhesion
The crystal structures of the β4 molecules lacking the N-terminal segment and the unpaired Cys residue did not provide any clues toward solving the mechanisms of cell-cell adhesion[14]
The crystal lattice successfully revealed the antiparallel arrangement of the β4 molecules containing the N-terminal segment and the unpaired Cys residue (Figs 1c and 2a)

Summary

Introduction

The interfaces for the trans interactions of the βsubunits have remained elusive, in spite of their importance in cell-cell adhesion. The C121W mutant was expressed on the cell surface and promoted the cell surface expression of the αsubunit, but exhibited impaired β1–β1 homophilic cell adhesion[12,13]. It is unclear why the C121W mutant lacks the adhesive activity, and whether the other GEFS+mutations besides C121W impair the β1–β1 homophilic cell adhesion. By structure-based mutagenesis and photo-crosslinking experiments combined with cell aggregation assays, we revealed that the identified interface is important for the trans homophilic interaction in the β4 -mediated cell-cell adhesion, in the native environment of living cells. Our findings establish the structural basis for the trans homophilic interaction of the βsubunits, in relation to their cellular functions and GEFS+pathogenesis

Methods

Results

Conclusion