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J. Biol. Chem., Vol. 283, Issue 31, 21621-21628, August 1, 2008

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Neuronally Selective µ-Conotoxins from Conus striatus Utilize an -Helical Motif to Target Mammalian Sodium Channels^*

Christina I. Schroeder¹, Jenny Ekberg, Katherine J. Nielsen^¶, Denise Adams, Marion L. Loughnan, Linda Thomas, David J. Adams, Paul F. Alewood, and Richard J. Lewis, An NHMRC Research Fellow^¶2

From the Institute for Molecular Bioscience and the School of Biomedical Sciences, The University of Queensland, Queensland 4072, Australia and ^¶Xenome Ltd, Indooroopilly, Queensland 4069, Australia

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-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.

Received for publication, April 14, 2008 , and in revised form, May 29, 2008.

^* This work was supported in part by a START grant from AusIndustry and by National Health and Medical Research Council Project (210307) and Program (351446) Grants. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Present address: UNSW Cancer Research Centre, University of New South Wales, Sydney NSW 2052, Australia.

² To whom correspondence should be addressed: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: 617-3346-2984; Fax: 617-3346-2101; E-mail: r.lewis{at}imb.uq.edu.au.

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