The voltage-gated sodium channel pore exhibits conformational flexibility during slow inactivation

Slow inactivation in voltage-gated sodium channels (Na(V)s) directly regulates the excitability of neurons, cardiac myocytes, and skeletal muscles. Although Na(V) slow inactivation appears to be conserved across phylogenies—from bacteria to humans—the structural basis for this mechanism remains unclear. Here, using site-directed labeling and EPR spectroscopic measurements of membrane-reconstituted prokaryotic Na(V) homologues, we characterize the conformational dynamics of the selectivity filter region in the conductive and slow-inactivated states to determine the molecular events underlying Na(V) gating. Our findings reveal profound conformational flexibility of the pore in the slow-inactivated state. We find that the P1 and P2 pore helices undergo opposing movements with respect to the pore axis. These movements result in changes in volume of both the central and intersubunit cavities, which form pathways for lipophilic drugs that modulate slow inactivation. Our findings therefore provide novel insight into the molecular basis for state-dependent effects of lipophilic drugs on channel function.