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J. Biol. Chem., Vol. 283, Issue 40, 27300-27313, October 3, 2008

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Tarantula Huwentoxin-IV Inhibits Neuronal Sodium Channels by Binding to Receptor Site 4 and Trapping the Domain II Voltage Sensor in the Closed Configuration^*

Yucheng Xiao, Jon-Paul Bingham^¶, Weiguo Zhu, Edward Moczydlowski^||, Songping Liang¹, and Theodore R. Cummins²

From the Department of Pharmacology and Toxicology, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202, ^¶Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii 96822, ^||Department of Biology, Clarkson University, Potsdam, New York 13699, and Life Sciences College, Hunan Normal University, Changsha, Hunan 410081, China

Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC₅₀ 26 nM). Following application of 1 µM HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>+100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by 300-fold, whereas the reverse mutations in rNav1.4 (N655D/Q657E) and the corresponding mutations in hNav1.5 (R812D/S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion β-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.

Received for publication, October 11, 2007 , and in revised form, June 11, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant NS054642 from the NINDS (to T. R. C. and J. P. B.). This work was also supported by the National Natural Science Foundation of China under Contract 30670640 (to S. L.) and the National 973 Project of China under Contract 2006CB708508 (to Y. X.). Part of this work was presented in abstract form at the Society for Neuroscience Annual Meeting, November 3-7, 2007 in San Diego. 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. 1-3 and Table 1.

¹ To whom correspondence may be addressed. E-mail: liangsp{at}hunnu.edu.cn.

² To whom correspondence may be addressed. E-mail: trcummin{at}iupui.edu.

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