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J. Biol. Chem., Vol. 282, Issue 42, 30699-30706, October 19, 2007

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Structure/Function Characterization of µ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels^*

Min-Min Zhang, Brad R. Green, Philip Catlin, Brian Fiedler, Layla Azam, Ashley Chadwick, Heinrich Terlau, Jeff R. McArthur^¶, Robert J. French^¶1, Josef Gulyas^||, Jean E. Rivier^||, Brian J. Smith^**2, Raymond S. Norton^**, Baldomero M. Olivera, Doju Yoshikami, and Grzegorz Bulaj³

From the Department of Biology, University of Utah, Salt Lake City, Utah 84112, Institute of Experimental and Clinical Pharmacology and Toxicology, Universitaetsklinikum Schleswig-Holstein, D-23538 Luebeck, Germany, ^¶Department of Physiology and Biophysics, University of Calgary and the Hotchkiss Brain Institute, Calgary, Alberta T2N 4N1, Canada, ^||The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037, ^**The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia, and Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. µ-Conotoxin KIIIA is representative of µ-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only 20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both Na_V1.2 and Na_V1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for Na_V1.2 and only partially for Na_V1.6. Other isoforms were blocked reversibly: Na_V1.3 (IC₅₀ 8 µM), Na_V1.5 (IC₅₀ 284 µM), and Na_V1.4 (IC₅₀ 80 nM). "Alanine-walk" and related analogs were synthesized and tested against both Na_V1.2 and Na_V1.4; replacement of Trp-8 resulted in reversible block of Na_V1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na_V1.4 than of Na_V1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na_V1.2 and that further engineering of µ-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

Received for publication, June 5, 2007 , and in revised form, August 6, 2007.

^* This work was supported in part by National Institutes of Health Program Project GM 48677. 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–S3 and supplemental Table S1.

¹ An Alberta Heritage Foundation for Medical Research Medical Scientist. Supported by operating grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, Nunavut, and Northwest Territories.

² Supported by the National Health and Medical Research Council.

³ To whom correspondence should be addressed. Tel.: 801-581-4629; Fax: 801-581-7087; E-mail: bulaj{at}pharm.utah.edu.

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