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Isolation and Structure-Activity of µ-Conotoxin TIIIA, A Potent Inhibitor of Tetrodotoxin-Sensitive Voltage-Gated Sodium Channels

Institute for Molecular Bioscience, the University of Queensland, St. Lucia, Queensland, Australia (R.J.L., C.I.S., J.E., K.J.N., M.L., L.T., D.A.A., P.F.A.); School of Biomedical Sciences, the University of Queensland, St. Lucia, Queensland, Australia (R.J.L., J.E., D.J.A.); and Xenome Ltd., Indooroopilly, Queensland, Australia (K.N., R.D.).

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.

Address correspondence to: Richard J. Lewis, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia. E-mail: r.lewis{at}imb.uq.edu.au

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