Critical role of extracellular loops in differential modulations of TTX-sensitive and TTX-resistant Na channels.

The cardiac voltage-gated sodium channel Na1.5 is resistant to tetrodotoxin (TTXr). Here, we report a cryo-electron microscopy (cryo-EM) structure of wild-type human Na1.5, coexpressed with the β1 auxiliary subunit and treated with high-concentration TTX, at 3.4 Å resolution. Structural comparison reveals the molecular determinants for the distinct responses to TTX as well as β subunits between TTXr and TTX-sensitive (TTXs) Na channels. A conserved cation-π interaction between the guanidinium group of TTX and Tyr or Phe on the P2 helix in TTXs Na channels is lost in all TTXr subtypes owing to the replacement by Cys/Ser at the corresponding locus, explaining their differential TTX sensitivities. The β1 subunit is invisible in the EM map. Comparison of Na1.5 with Na1.7 and Na1.8, which are, respectively, TTXs and TTXr, identifies four sites on the extracellular loops (ECLs) that may account for their different β1-binding abilities. When the corresponding residues in TTXs Na1.7 are replaced with those from Na1.5, the modulatory effects of β1 on channel activation and inactivation are diminished. Consistently, β1 is absent in the 3D EM reconstruction of this Na1.7 mutant. Together with our previous structure-guided discovery that TTXr channels lack a Cys on the ECL for disulfide bond formation with β2 or β4, the structure-function relationship studies underscore the importance of the ECLs in the mechanistic distinctions between TTXs and TTXr Na channels. The ECLs may be further explored for the development of subtype-specific drugs.