Structure of Nav and modulation by phosphoinositides

(A) Nav channel topology featuring transmembrane helices (S1-S6), the selectivity filter (SF) and the DIII-IV linker (containing the IFM motif) located between DIII and DIV. (B) Nav1.4 structure (6agf) showing the four domain-swapped voltage sensor domains (VSDs I-IV), pore and DIII-IV linker on the intracellular side. (C) Summary of PI(4,5)P2 effects on transitions between Nav channel functional states (25) (D) Structure of the PI(4,5)P2 headgroup with the 4’- and 5’-phosphates indicated

Lipid fingerprint and binding of all PIP types to Nav1.4

(A) Nav1.4 embedded in a 360 Å x 360 Å model mammalian membrane containing 63 lipid species. (B) Lipid depletion enrichment index of lipids around Nav1.4 grouped into 12 headgroup classes (C) Nav1.4, shown from the intracellular (left) and membrane (right) sides, colored by PIP occupancy (darker purple = greater PIP occupancy) (D) Distribution of PIP binding occupancies (left) and occupancy of lipid species at 4 residues with the highest PIP occupancy.

Binding of different PIP species in enriched PIP simulations

(A) Enriched PIP simulation system, with Nav1.4 embedded in a POPC membrane (transparent gray) and 5% each of PIP1 (blue), PIP2 (purple) and PIP3 (pink) added to the cytoplasmic leaflet. (B) Representative snapshots from the five longest binding events from different replicates, showing the three different PIP species (PIP1 in blue, PIP2 in purple and PIP3 in pink) binding to VSD-IV and the DIII-IV linker. Nav1.4 is shown in white with interacting residues on the DIV S4-S5 linker and the DIII-IV linker colored in green and orange respectively. (C) A frequency distribution showing interaction times for each PIP species, defined as the length of a continuous period in which a PIP was within 0.7 nm of two VSD IV binding site residues. (D) Frequency plots showing number of positive residues interacting with bound PIP in the DIII-IV linker (vertical) and VSD-IV (horizontal). (E) Minimum distance between binding residues on Nav1.4 and bound PIPs lipid across simulation time for the five longest binding events, colored by distance and the type of PIP bound.

PI(4,5)P2 and PI(4)P binding to Nav1.4 stabilizes the DIII-IV linker in atomistic and flexible coarse-grained simulations

(A) Representative snapshots of PI(4,5)P2 bound from the VSD-I side (purple stick) and PI(4)P bound from the VSD-III side (cyan stick), with six basic residues forming the binding site located on the DIII-IV linker (orange VDW representation) and VSD-IV S4-S5 linker (shown in green VDW representation) visualized from the intracellular face of the protein (B) Proportion of frames where each of the binding site residues were identified to be within 4.5 Å with the different headgroup regions, P4, P5 and PO4, for PI(4,5)P2 (left) and PI(4)P (right) (C) Comparison of RMSF per carbon-alpha for simulations with and without bound PI(4,5)P2, showing residues on the S4-S5 linker and DIV linker with significant differences in mobility (p-value < 0.05) (D) Interaction network plots between the PIP headgroup and basic binding residues on DIII-IV linker (orange) and DIV S4-S5 linker (green), generated by ProLIF – showing the dominant interactions across simulations of PI(4,5)P2 and PI(4)P. (E) Density plots showing differences in the distributions of distance between IFM/IQM motif and its binding pocket in the presence and absence of PIPs for the Nav1.4 wild-type (left) and IFM->IQM mutant (right); with representative snapshots showing the two distinct conformations of the IQM motif in the mutant.

Comparison of the identified phosphoinositide binding site to Nav subtypes and other ion channels

(A) Binding poses of PI(4,5)P2 (in purple) and PI(4)P (in cyan) aligned with two other tetrameric channels structures Kv7.1 (6v01, in yellow) and Cav2.2 (7mix, in red) that were resolved with PI(4,5)P2 at their respective VSDs. (B) Sequence alignment of the S4 helix and S4-S5 linker of the four domains of Nav1.4, compared to VSD-II of Cav2.2 and one of the four identical VSDs of Kv7.1; residues colored by amino acid class; purple boxes indicate PI(4,5)P2 binding residues (identified with 5 Å of the headgroup). (C) Sequence alignment of the nine human Nav channel subtypes shows high sequence similarity in the S4 helix, S4-S5 linker and DIII-IV linker regions.

PIP binding to Nav1.7 with different VSD states in coarse-grained simulations

(A) Atomistic representation of the three different Nav1.7 structures simulated: (1) the inactivated state (blue, PDB ID: 6j8g) with the VSDs all in the activated, up state (2) the Nav1.7-NavPas chimera (pink, PDB ID: 6nt4) with the Nav1.7 VSD-IV in the deactivated, down state and a bound NavPas CTD (3) a Nav1.7 resting state model (orange, model generation detailed in Table. S2) with all four VSDs in the deactivated, down state. Panel insets show the different conformations of VSD-IV (left) and VSD-II (right) across different structures. The inactivation switch formed by the CTD and VSD-IV S4-S5 linker proposed by Clairfeuille et al. is shown (middle). (B) Combined occupancy of all PIP species (PIP1, PIP2, PIP3) at binding site residues in the three systems (C) Distribution of PIP binding durations at the identified site (D) Intracellular view of CTD covering the resting state VSD-IV. Representative snapshot of PIP binding at DIV S4-S5 linker in the Nav1.7-NavPas system. The CTD (dark pink) prevents PIP access to DIII-IV linker lysines, K1492 and K1495. (E) Combined PIP occupancy at the bottom three gating charges on VSD I-III in the inactivated (blue) and resting state model (orange) simulations. For VSD-IV, PIP occupancy in the Nav1.7-NavPas system (pink) is also shown. (F) Representative simulation snapshot showing PIP (purple) binding at the gating charges (orange) in the resting state model simulations.

Proposed mechanism of PIP effects on the sodium channel functional cycle