BPS2026 – Elucidating the cross-talk between phosphorylation and disease-causing mutations in fast inactivation of voltage-gated sodium channels

Fast inactivation of Nav channels, which regulate electrical hyperexcitability in nerve and muscle cells, is mediated by the conserved IFM motif that allosterically occludes the central pore. Our recent work has elucidated the molecular mechanisms underlying numerous disease-associated mutations in the vicinity of the IFM-binding pocket. However, the functional and structural roles of several key residues remain unclear. At the same time, phosphorylation is increasingly recognized as a critical regulator of ion channel gating. Notably, some of the unresolved mutations are located near specific phosphorylation sites. Based on these observations, we hypothesize that cross-talk between phosphorylation and disease-causing mutations alters the impact of mutations on fast inactivation. To test such a hypothesis, we employed constant-velocity steered MD (cv-SMD) simulations to investigate how phosphorylation and adjacent mutations interact with each other and influence IFM binding for fast inactivation. The force-time profiles from multiple independent cv-SMDs allow us to estimate the free-energy barrier for this unbinding process and to assess the structural impacts of mutations and phosphorylation. The E1773K mutation markedly lowers the free-energy barrier and destabilizes IFM binding by disrupting a native interaction present in the WT. In contrast, phosphorylation at Y1495 in the E1773K background significantly increases the free-energy barrier, driven by the formation of a new stable salt bridge between K1773 and pY1495 that replaces the native hydrogen bonds, producing an opposite effect on fast inactivation. Together, these results highlight key salt bridges between phosphorylation and mutation that significantly stabilize the IFM binding, thereby improving the efficiency of fast inactivation. Our findings demonstrate that phosphorylation-dependent modulation of fast inactivation can alter or even reverse the functional impact of pathogenic mutations, providing mechanistic insight into how such variants contribute to disease phenotypes.