Received for publication June 30, 2006.
Revised November 15, 2006.
Accepted for publication November 16, 2006.

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

Abstract

µ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels (VGSCs) during prey capture. Using PCR techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new µ-conotoxin TIIIA. A ¹²⁵I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of C. 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-STX 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, which were critical for high affinity binding to TTX-sensitive Na⁺ channels. Surprisingly, [Glu15Ala]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC₅₀ 15 pM 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 oocytes and was without 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 analogues provide new biochemical probes as well insights into the structure-activity of µ-conotoxins.

Key words: Ion channel regulation, Sodium, Local anesthetics, Structure determinations, Structure-activity relationships and modeling, NMR, Nuclear Magnetic Resonance, Receptor binding studies, Neuropeptides, peptidases

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