Synthesis, Solution Structure, and Phylum Selectivity of a Spider δ-Toxin That Slows Inactivation of Specific Voltage-gated Sodium Channel Subtypes

Nahoko Yamaji,Michelle J Little,Hideki Nishio,Bert Billen,Elba Villegas,Yuji Nishiuchi,Jan Tytgat,Graham M Nicholson,Gerardo Corzo

doi:10.1074/jbc.m109.030841

Abstract

Magi 4, now renamed delta-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to delta-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (Na(V)) channels but, unlike delta-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in Na(V) channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect Na(V) channel subtypes showing that delta-hexatoxin-Mg1a selectively slows channel inactivation of mammalian Na(V)1.1, Na(V)1.3, and Na(V)1.6 but more importantly shows higher affinity for insect Na(V)1 (para) channels. Consequently, delta-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded delta-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion alpha-toxins and delta-hexatoxins are distributed in a topologically similar manner in delta-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of delta-hexatoxin-Mg1a for certain mammalian and insect Na(V) channel subtypes. As such, delta-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.

Highlights

Toxins interacting with these neurotoxin receptor sites have been instrumental in the study of NaV channel topology, function, and pharmacology [6]
The CD spectra of synthetic and native ␦-HXTX-Mg1a superimposed indicating that their secondary structures were similar. Both native and synthetic ␦-HXTX-Mg1a were able to displace the binding of 125I-Lqh␣IT from insect NaV channels in cockroach synaptosomes with similar IC50 values
The toxin-induced increase in peak INa seen with DmNaV1 and to a lesser extent in DUM neurons, rNaV1.1, rNaV1.3, and NaV1.6, was in general well correlated with the magnitude of the sustained current at the end of the depolarizing test pulse

Summary

Introduction

At least seven distinct toxin-binding sites have been identified by radioligand binding and electrophysiological studies on vertebrate and insect NaV channels [4, 5] Toxins interacting with these neurotoxin receptor sites have been instrumental in the study of NaV channel topology, function, and pharmacology [6]. A wide range of scorpion ␣-toxins, sea anemone toxins, and spider ␦-hexatoxins (formerly ␦-atracotoxins [7]) compete for binding to receptor site-3 on the extracellular surface of NaV channels These polypeptide toxins all inhibit the fast inactivation of NaV channels to prolong Naϩ currents (INa), despite huge diversity in primary and tertiary structures [8, 9]. For all of these toxins, the precise pattern of NaV channel subtype selectivity is either unknown or at best is incomplete

Objectives

Results

Conclusion