The structural dynamics and molecular coupling in the slow inactivation of a prokaryotic voltage-gated sodium channel

Slow inactivation in voltage-gated sodium channels (Navs) is crucial for regulating cell excitability. Many studies indicate that the selectivity filter (SF) serves as the inactivation gate, but the structural mechanism and coupling between the distal primary and inactivation gates remain poorly understood. Our single-molecule FRET studies reveal that the NavAb SF transits among three conformational states, with depolarization enriching a high FRET state, potentially the slow inactivated conformation. We show L176 in the SF and T206 in the S6 helix as key residues that form a steric-hindrance coupler. Crystallographic analysis reveals that bulkier L176F overrides the opening tendency of an S6 C-terminal deletion (ΔC230) and closes the primary gate, while smaller T206A partially uncouples the slow inactivation gate. SmFRET data confirm that L176F restores the high FRET population depleted by ΔC230 or the open-pore blocker lidocaine. Our results indicate that ‘conformational talk’ through L176-T206 underlies slow inactivation in prokaryotic Navs. Slow inactivation of voltage-gated sodium channels is important for regulating cellular excitability. Here the authors probe the conformational dynamics in the selectivity filter using smFRET and reveal the critical residue pair that mediates conformational communication between the primary and slow inactivation gates