δ-Atracotoxins from Australian Funnel-web Spiders Compete with Scorpion α-Toxin Binding but Differentially Modulate Alkaloid Toxin Activation of Voltage-gated Sodium Channels

Michelle J Little,Cathy Zappia,Nicolas Gilles,Mark Connor,Margaret I Tyler,Marie-France Martin-Eauclaire,Dalia Gordon,Graham M Nicholson

doi:10.1074/jbc.273.42.27076

Abstract

delta-Atracotoxins from the venom of Australian funnel-web spiders are a unique group of peptide toxins that slow sodium current inactivation in a manner similar to scorpion alpha-toxins. To analyze their interaction with known sodium channel neurotoxin receptor sites, we studied their effect on [3H]batrachotoxin and 125I-Lqh II (where Lqh is alpha-toxin II from the venom of the scorpion Leiurus quinquestriatus hebraeus) binding and on alkaloid toxin-stimulated 22Na+ uptake in rat brain synaptosomes. delta-Atracotoxins significantly increased [3H]batrachotoxin binding yet decreased maximal batrachotoxin-activated 22Na+ uptake by 70-80%, the latter in marked contrast to the effect of scorpion alpha-toxins. Unlike the inhibition of batrachotoxin-activated 22Na+ uptake, delta-atracotoxins increased veratridine-stimulated 22Na+ uptake by converting veratridine from a partial to a full agonist, analogous to scorpion alpha-toxins. Hence, delta-atracotoxins are able to differentiate between the open state of the sodium channel stabilized by batrachotoxin and veratridine and suggest a distinct sub-conductance state stabilized by delta-atracotoxins. Despite these actions, low concentrations of delta-atracotoxins completely inhibited the binding of the scorpion alpha-toxin, 125I-Lqh II, indicating that they bind to similar, or partially overlapping, receptor sites. The apparent uncoupling between the increase in binding but inhibition of the effect of batrachotoxin induced by delta-atracotoxins suggests that the binding and action of certain alkaloid toxins may represent at least two distinguishable steps. These results further contribute to the understanding of the complex dynamic interactions between neurotoxin receptor site areas related to sodium channel gating.

Highlights

␦-Atracotoxins from the venom of Australian funnelweb spiders are a unique group of peptide toxins that slow sodium current inactivation in a manner similar to scorpion ␣-toxins
To analyze their interaction with known sodium channel neurotoxin receptor sites, we studied their effect on [3H]batrachotoxin and 125I-Lqh II binding and on alkaloid toxin-stimulated 22Na؉ uptake in rat brain synaptosomes. ␦-Atracotoxins significantly increased [3H]batrachotoxin binding yet decreased maximal batrachotoxinactivated 22Na؉ uptake by 70 – 80%, the latter in marked contrast to the effect of scorpion ␣-toxins
We report that ␦-atracotoxins interact with nanomolar affinities with neurotoxin receptor site 3 in rat brain synaptosomes but exhibit unusual allosteric interactions with the site 2 alkaloid toxin receptor

Summary

EXPERIMENTAL PROCEDURES

Purification of Toxins—Colonies of male A. robustus and female H. versuta spiders were “milked” by direct aspiration from the chelicerae of live spiders using glass pipettes. Synaptosomes were suspended in a solution consisting of (in mM) choline chloride 130, KCl 5.4, MgSO4 0.8, D-glucose 5.5, and HEPES-Tris 50 (pH 7.4, 37 °C) and were stored in liquid nitrogen until required for 22Naϩ uptake assays, or used within 3 h of preparation for [3H]BTX binding assays. Uptake was initiated by adding 150 ␮l of assay medium containing (in mM) choline chloride 128, KCl 5.4, MgSO4 0.8, glucose 5.5, HEPES-Tris (pH 7.4) 50, NaCl 2.66, ouabain 5, 1 mg/ml BSA, and 0.9 ␮Ci/ml carrier-free 22NaCl. After 5 s at 37 °C, uptake was terminated by the addition of 4 ml of ice-cold wash solution and rapid filtration through 0.45-␮m nitrocellulose membrane filters (Millipore, Sydney, Australia).

RESULTS

BTX Bmax

DISCUSSION