Structure of human NaV1.6 channel reveals Na ⁺ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin

The sodium channel Na _V 1.6 is widely expressed in neurons of the central and peripheral nervous systems, which plays a critical role in regulating neuronal excitability. Dysfunction of Na _V 1.6 has been linked to epileptic encephalopathy, intellectual disability and movement disorders. Here we present cryo-EM structures of human Na _V 1.6/β1/β2 alone and complexed with a guanidinium neurotoxin 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), revealing molecular mechanism of Na _V 1.6 inhibition by the blocker. In the apo-form structure, two potential Na ⁺ binding sites were revealed in the selectivity filter, suggesting a possible mechanism for Na ⁺ selectivity and conductance. In the 4,9-ah-TTX-bound structure, 4,9-ah-TTX binds to a pocket similar to the tetrodotoxin (TTX) binding site, which occupies the Na ⁺ binding sites and completely blocks the channel. Molecular dynamics simulation results show that subtle conformational differences in the selectivity filter affect the affinity of TTX analogues. Taken together, our results provide important insights into Na _V 1.6 structure, ion conductance, and inhibition.