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

Voltage gated sodium channels (Na_v) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Na_vchannels 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 Na_vchannels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P₂decreases Na_v1.4 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)P₂binds stably to inactivated Na_vat 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)P₂prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P₂reduces 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 Na_vmodel, 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 Na_v-related diseases.