A binding site for phosphoinositides described by multiscale simulations explains their modulation of voltage-gated sodium channels.

Voltage-gated sodium channels (Naáµ¥) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naáµ¥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naáµ¥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naáµ¥1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naáµ¥ at a conserved site within the DIV S4-S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naáµ¥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding 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 Naáµ¥ 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 Naáµ¥-related diseases.

New insights from modelling studies and molecular dynamics simulations of the DIS5-S6 extracellular linker of the skeletal muscle sodium channel NaV1.4.

In the CryoEM-structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59-residue DIS5-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.

Characterizing fenestration size in sodium channel subtypes and their accessibility to inhibitors.

Voltage-gated sodium channels (Nav) underlie the electrical activity of nerve and muscle cells. Humans have nine different subtypes of these channels, which are the target of small-molecule inhibitors commonly used to treat a range of conditions. Structural studies have identified four lateral fenestrations within the Nav pore module that have been shown to influence Nav pore blocker access during resting-state inhibition. However, the structural differences among the nine subtypes are still unclear. In particular, the dimensions of the four individual fenestrations across the Nav subtypes and their differential accessibility to pore blockers is yet to be characterized. To address this, we applied classical molecular dynamics simulations to study the recently published structures of Nav1.1, Nav1.2, Nav1.4, Nav1.5, and Nav1.7. Although there is significant variability in the bottleneck sizes of the Nav fenestrations, the subtypes follow a common pattern, with wider DI-II and DIII-IV fenestrations, a more restricted DII-III fenestration, and the most restricted DI-IV fenestration. We further identify the key bottleneck residues in each fenestration and show that the motions of aromatic residue sidechains govern the bottleneck radii. Well-tempered metadynamics simulations of Nav1.4 and Nav1.5 in the presence of the pore blocker lidocaine also support the DI-II fenestration being the most likely access route for drugs. Our computational results provide a foundation for future in vitro experiments examining the route of drug access to sodium channels. Understanding the fenestrations and their accessibility to drugs is critical for future analyses of diseases mutations across different sodium channel subtypes, with the potential to inform pharmacological development of resting-state inhibitors and subtype-selective drug design.