New insights from modelling studies and molecular dynamics simulations of the DI_S5‐S6 extracellular linker of the skeletal muscle sodium channel NaV1.4

Abstract In the CryoEM‐structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59‐residue DI S5‐S6 linker peptide was omitted due to absence of electron density. This peptide is intriguing – comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete hSkMNaV1.4 channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self‐assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N‐and C‐terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N‐linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to SkMNaV1.4 structure and function.