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

Richard J. Lewis*, Christina I. Schroeder, Jenny Ekberg, Katherine J. Nielsen, Marion L. Loughnan, Linda Thomas, Denise A. Adams, Roger Drinkwater, David J. Adams, Paul F. Alewood

*Corresponding author for this work

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50 Citations (Scopus)

Abstract

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 (Nav1.4) 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 Nav1.2 and -1.4 over Nav1.3, -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.

Original languageEnglish
Pages (from-to)676-685
Number of pages10
JournalMolecular Pharmacology
Volume71
Issue number3
DOIs
Publication statusPublished - Mar 2007
Externally publishedYes

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    Lewis, R. J., Schroeder, C. I., Ekberg, J., Nielsen, K. J., Loughnan, M. L., Thomas, L., ... Alewood, P. F. (2007). Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels. Molecular Pharmacology, 71(3), 676-685. https://doi.org/10.1124/mol.106.028225