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

The cardiac voltage-gated sodium channel Nav1.5 is resistant to tetrodotoxin (TTXr). Here, we report a cryo-electron microscopy (cryo-EM) structure of wild-type human Nav1.5, 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) Nav channels. A conserved cation-π interaction between the guanidinium group of TTX and Tyr or Phe on the P2I helix in TTXs Nav 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 Nav1.5 with Nav1.7 and Nav1.8, 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 Nav1.7 are replaced with those from Nav1.5, the modulatory effects of β1 on channel activation and inactivation are diminished. Consistently, β1 is absent in the 3D EM reconstruction of this Nav1.7 mutant. Together with our previous structure-guided discovery that TTXr channels lack a Cys on the ECLII 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 Nav channels. The ECLs may be further explored for the development of subtype-specific drugs.