Elucidating the Differential Impacts of Equivalent Gating-Charge Mutations in Voltage-Gated Sodium Channels

Voltage-gated sodium (Na _v ) channels are pivotal for cellular signaling and mutations in Na _v channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSD _I and VSD _II of the cardiac sodium channel Na _v 1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 µs molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our μs-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure- function relationships in Na _v channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.