Abstract
Rapid and effectual inactivation in voltage-gated sodium channels is required for canonical action-potential firing. This "fast" inactivation arises from swift and reversible protein conformational changes that utilize transmembrane segments and the cytoplasmic linker between channel domains III and IV. Until recently, fast inactivation had been accepted to rely on a "ball-and-chain" mechanism whereby a hydrophobic triplet of DIII-IV amino acids (IFM) impairs conductance by binding to a site in central pore of the channel made available by channel opening. New structures of sodium channels have upended this model. Specifically, cryo-electron microscopic structures of eukaryotic sodium channels depict a peripheral binding site for the IFM motif, outside of the pore, opening the possibility of a yet unidentified allosteric mechanism of fast-inactivation gating. We set out to study fast inactivation by photo-trapping human sodium channels in various functional states under voltage control. This was achieved by genetically encoding the crosslinking unnatural amino acid benzophenone phenylalanine at various sites within the DIII-IV linker in the cardiac sodium channel Na(V)1.5. These data show dynamic state- and positional-dependent trapping of the transient conformations associated with fast inactivation, each yielding different phenotypes and rates of trapping. These data reveal distinct conformational changes that underlie fast inactivation and point to a dynamic environment around the IFM locus.