Structural implications of glycosylation on the voltage-gated sodium channel β3-subunit

Abstract Voltage-gated sodium (Na V ) channel α-subunits are modulated by associated β-subunits that affect their localization, trafficking and gating behaviour. The β-subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules and the interactions between their extracellular Ig-domains may modify channel clustering. The full-length β3-subunit can form cis trimers on the plasma membrane. The atomic resolution structure of a deglycosylated trimeric β3-subunit Ig-domain has been solved by X-ray crystallography. However, it is not clear whether this particular trimeric Ig-domain structure is plausible for cell-expressed, glycosylated β3-subunits. Here we use glycan profiling to confirm an extensive and heterogeneous pattern of β3-subunit glycosylation, with the majority of glycans being bi- and tri-antennary structures with one or two terminal sialic acids. Two tryptic peptides of the β3 Ig-domain are predicted to contain potential N-linked glycosylation sites. When the isolated, glycosylated full-length β3-subunit was trypsin-digested and analysed by LC-MS/MS, only one of these peptides - containing an N-linked glycosylation site at residue N95 and located close to the trimer interface - was identified in its unmodified form, suggesting that residue N95 is under-glycosylated. All-atom molecular dynamics simulations of the glycosylated, membrane-bound full-length β3 trimer confirmed that glycans can be accommodated with the Ig-domain trimer and indeed, may contribute to protein-membrane and inter-protomer interactions within the full-length, membrane-embedded trimer. Further biochemical studies are warranted to explore the interactions between oligomeric β-subunits with corresponding α-subunit sodium channels.