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 Liang,Theodore R Cummins

doi:10.1074/jbc.m708447200

Abstract

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 (IC(50) approximately 26 nM). Following application of 1 microm 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 approximately 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 beta-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.

Highlights

Voltage-gated sodium channels (VGSCs)3 are important transmembrane proteins expressed in most excitable tissues
Vious work demonstrated that 1 ␮M HWTX-IV completely Effects of Subsaturating Concentrations of HWTX-IV on Actiinhibits TTX-sensitive sodium currents in adult rat dorsal root ganglia (DRG) neu- vation and Inactivation Properties of wild type (WT) VGSC Subtypes—We rons but has no effect on TTX-resistant currents [14]; asked whether HWTX-IV was acting as a simple inhibitor the effects of HWTX-IV on specific VGSC isoforms are or whether HWTX-IV might alter channel gating behavunclear
We intensively characterized the action of HWTX-IV, a 35-residue toxin from the tarantula O. huwena on five different VGSCs and showed that the toxin was able to inhibit neuronal subtypes Nav1.2, Nav1.3, and Nav1.7 but had little effect on skeletal muscle and cardiac isoforms Nav1.4 and

Summary

Introduction

Voltage-gated sodium channels (VGSCs)3 are important transmembrane proteins expressed in most excitable tissues. We tested whether HWTX-IV inhibition was altered by mutation of a pore residue in voltage-gated sodium channels that is critical for binding of toxins to neurotoxin site 1 such as TTX [27] as well as binding of another pore blocker, the ␮-conotoxin GIIIB, to Nav1.4 (data not shown).

Results

Conclusion