The Janus-faced atracotoxins are specific blockers of invertebrate K(Ca) channels.

The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca(2+)-activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked K(Ca) channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nm, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+)-activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC(50) approximately 10 mum) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.

Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons.

The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Nav) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Nav channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons, P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Nav currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP, and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Nav channel gating, observed clinically in response to ciguatera poisoning.

Isolation of delta-missulenatoxin-Mb1a, the major vertebrate-active spider delta-toxin from the venom of Missulena bradleyi (Actinopodidae).

The present study describes the isolation and pharmacological characterisation of the neurotoxin delta-missulenatoxin-Mb1a (delta-MSTX-Mb1a) from the venom of the male Australian eastern mouse spider, Missulena bradleyi. This toxin was isolated using reverse-phase high-performance liquid chromatography and was subsequently shown to cause an increase in resting tension, muscle fasciculation and a decrease in indirect twitch tension in a chick biventer cervicis nerve-muscle bioassay. Interestingly, these effects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax robustus. Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that delta-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium current, a slowing of sodium current inactivation and a hyperpolarising shift in the voltage at half-maximal activation. In addition, delta-MSTX-Mb1a failed to affect TTX-resistant sodium currents. Subsequent Edman degradation revealed a 42-residue peptide with unusual N- and C-terminal cysteines and a cysteine triplet (Cys(14-16)). This toxin was highly homologous to a family of delta-atracotoxins (delta-ACTX) from Australian funnel-web spiders including conservation of all eight cysteine residues. In addition to actions on sodium channel gating and kinetics to delta-ACTX, delta-MSTX-Mb1a caused significant insect toxicity at doses up to 2000 pmol/g. Delta-MSTX-Mb1a therefore provides evidence of a highly conserved spider delta-toxin from a phylogenetically distinct spider family that has not undergone significant modification.