Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels

μ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new μ-conotoxin-TIIIA (TIIIA). A ¹²⁵I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other μ-conotoxins. TIIIA potently displaced [ ³H]saxitoxin and ¹²⁵I-TIIIA from rat brain (Na _v1.2) and skeletal muscle (Na_v1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na ⁺ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC₅₀, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Na_v1.2 and -1.4 over Na_v1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na ⁺ current. ¹H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for μ-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of μ-conotoxins.