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

Beaudoin, C. A.; Hayes, J.; Liu, H.; Namadurai, S.; Deery, M. J.; August, E. A.; Krasner, S.; Hamaia, S. W.; Salvage, S. C.; Huang, C. L.- H.; Jackson, A. P.

doi:10.1101/2025.02.27.640513

bioRxiv · 10.1101/2025.02.27.640513

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

Beaudoin, C. A.·Hayes, J.·Liu, H.·Namadurai, S.·Deery, M. J.·August, E. A.·Krasner, S.·Hamaia, S. W.·Salvage, S. C.·Huang, C. L.- H.·Jackson, A. P.

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Abstract

Voltage-gated sodium (NaV) channel -subunits are modulated by associated {beta}-subunits that affect their localization, trafficking and gating behaviour. The {beta}-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 {beta}3-subunit can form cis trimers on the plasma membrane. The atomic resolution structure of a deglycosylated trimeric {beta}3-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 {beta}3-subunits. Here we use glycan profiling to confirm an extensive and heterogeneous pattern of {beta}3-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 {beta}3 Ig-domain are predicted to contain potential N-linked glycosylation sites. When the isolated, glycosylated full-length {beta}3-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 {beta}3 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 {beta}-subunits with corresponding -subunit sodium channels.

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Beaudoin, C. A., Hayes, J., Liu, H., Namadurai, S., Deery, M. J., August, E. A., Krasner, S., Hamaia, S. W., Salvage, S. C., Huang, C. L.- H., Jackson, A. P.. 2025-02-27. Structural implications of glycosylation on the voltage-gated sodium channel β3-subunit. https://doi.org/10.1101/2025.02.27.640513

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