Molecular Dynamics and Neural Network Analysis Reveal Sequential Gating and Allosteric Communication in...

FMRFamide-activated sodium channels (FaNaCs) represent a unique class of neuropeptide-gated ion channels within the degenerin/epithelial sodium channel (DEG/ENaC) superfamily. While cryo-electron microscopy has revealed static binding architectures, the dynamic mechanisms underlying ligand recognition, allosteric signal transmission, and channel gating remain poorly understood. Here, we employed microsecond-scale molecular dynamics simulations coupled with neural relational inference analysis to elucidate the complete activation mechanism of FaNaC at atomic resolution. Our analysis revealed a sophisticated multistage activation process initiated by coordinated dynamics of FaNaC-specific insertions SI1 and SI2. Spontaneous FMRFamide-binding events suggested that SI1 functions as a dynamic gate that facilitates optimal ligand burial and stabilization, while SI2 appeared to serve as a conformational lid stabilizing the bound ligand through thermodynamically favorable induced-fit mechanisms. This ligand-induced conformational change, which involves the cooperative reorganization of the three peripheral loops (L1, L2, and L3) in the extracellular domain, propagates through the extracellular domain, particularly via a coordinated rigid-body motion of the β-ball/palm domain, leading to the reorganization of the central β-sheet in the extracellular vestibule and a subsequent conformational wave that compacts the intracellular vestibule. We further leveraged neural relational inference (NRI) to analyze residue-level allosteric networks, demonstrating that ligand binding enhances the network's connectivity and reorganizes allosteric communication pathways. These findings provide a high-resolution, dynamic view of FaNaC function, revealing a novel gating mechanism for the DEG/ENaC superfamily and laying the foundation for future studies into neuropeptide modulation.