Electrophysiological analysis of the neurotoxic action of a funnel-web spider toxin, δ-atracotoxin-Hv1a, on insect voltage-gated Na⁺ channels

The effects of δ-ACTX-Hv1a, purified from the venom of the funnel-web spider Hadronyche versuta, were studied on the isolated giant axon and dorsal unpaired median (DUM) neurones of the cockroach Periplaneta americana under current- and voltage-clamp conditions using the double oil-gap technique for single axons and the patch-clamp technique for neurones. In parallel, the effects of the toxin were investigated on the excitability of rat dorsal root ganglion (DRG) neurones. In both DRG and DUM neurones, δ-ACTX-Hv1a induced spontaneous repetitive firing accompanied by plateau potentials. However, in the case of DUM neurones, plateau action potentials were facilitated when the membrane was artificially hyperpolarized. In cockroach giant axons, δ-ACTX-Hv1a also produced plateau action potentials, but only when the membrane was pre-treated with 3-4 diaminopyridine. Under voltage-clamp conditions, δ-ACTX-Hv1a specifically affected voltage-gated Na+ channels in both axons and DUM neurones. Both the current/voltage and conductance/voltage curves of the δ-ACTX-Hv1a-modified inward current were shifted 10 mV to the left of control curves. In the presence of δ-ACTX-Hv1a, steady-state Na+ channel inactivation became incomplete, causing the appearance of a non-inactivating component at potentials more positive than -40mV. The amplitude of this non-inactivating component was dependent on the holding potential. From this study, it is concluded that, in insect neurones, δ-ACTX-Hv1a mainly affects Na+ channel inactivation by a mechanism that differs slightly from that of scorpion α-toxins

Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore