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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 intriguingcomprised 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.

Voltage gated sodium channels (Nav) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Nav channels undergo a transition from the resting, closed state to an open state which allows ion influx, before rapidly inactivating. Dysregulation of this functional cycle due to mutations leads to diseases including epilepsy, pain conditions and cardiac disorders, making Nav channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Nav1.4 activity by increasing the difficulty of channel opening, accelerating fast activation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Nav at a conserved site within the DIV S4-S5 linker, which couples the voltage sensing domain (VSD) to the pore. As the Nav C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesise that PI(4,5)P2 prolongs inactivation by competing to bind to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Nav model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Nav-related diseases.

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