Structural and functional mechanisms underlying activation gate dynamics and IFM motif accessibility in human Na_v1.5

Voltage-gated sodium channels are vital for regulating excitability in muscle and nerve cells, and their dysregulation is linked to a range of diseases. However, therapeutic targeting of Na_v channels remains challenging due to a limited understanding of their gating mechanisms. Here, we present a cryo-EM structure of human Na_v1.5 in an intermediate open state, stabilized by interactions between the N-terminal domain and the S6_I segment. This structure reveals a possible Na⁺ binding site adjacent to the conserved inactivation (IFM) motif. Molecular dynamics simulations demonstrate that monovalent cations stably occupy this site, while electrophysiological recordings demonstrate that ion binding modulates IFM motif docking and fast inactivation kinetics. Our findings reveal that IFM accessibility is dynamically regulated in this intermediate state, refining the canonical door-wedge model of fast inactivation. Collectively, our study provides a revised structural framework for Na_v1.5 gating mechanisms, suggesting an alternative pathway for ion accessibility that may inform better mechanistic and therapeutic strategies for treating Na_v1.5-related cardiac arrhythmias.