Neuronally selective μ-conotoxins from Conus striatus utilize an α-helical motif to target mammalian sodium channels

μ-Conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of μ-conotoxins SIIIA and SIIIB by ¹²⁵I- TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced ¹²⁵I-TIIIA from native rat brain Na_v1.2 (IC₅₀ values 10 and 5 nM, respectively) and muscle Na_v1.4 (IC₅₀ values 60 and 3 nM, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Na_v1.2 and Na _v1.4. An alanine scan of SIIIA-(2-20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2-20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2-20) had significantly enhanced neuronal affinity (IC₅₀ 0.65 nM), while the double mutant [D15A/H16R]SIIIA-(2-20) showed greatest Na_v1.2 versus 1.4 selectivity (136-fold). ¹H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and an α-helical motif across residues 11-16 that is not found in larger μ-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller μ-conotoxin pharmacophore.