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.

N N-Atracotoxins (N N-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion K K-toxins. We have isolated and determined the amino acid sequence of a novel N N-ACTX, designated N N-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with N N-ACTXHv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other N N-ACTXs. However, N N-ACTX-Hv1b is approximately 15^30-fold less potent than other N N-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between N N-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.z 2000 Federation of European Biochemical Societies.

Unlike other site-3 neurotoxins, however, δ-ACTX bind with high affinity to both cockroach and mammalian sodium channels but low affinity to locust sodium channels. At present the pharmacophore of δ-ACTX is unknown but is believed to involve a number of basic residues distributed in a topologically similar manner to scorpion α-toxins and sea anemone toxins despite distinctly different protein scaffolds. As such, δ-ACTX provide us with specific tools with which to study sodium channel structure and function and determinants for phyla-and tissue-specific actions of neurotoxins interacting with site-3.

The venom of the male Australian duck-billed platypus contains a family of four polypeptides of appox. 5 kDa, which are referred to as defensin-like peptides (DLPs). They are unique in that their amino acid sequences have no significant similarities to those of any known peptides; however, the tertiary structure of one of them, DLP-1, has recently been shown to be similar to beta-defensin-12 and to the sodium neurotoxin peptide ShI (Stichodactyla helianthus neurotoxin I). Although DLPs are the major peptides in the platypus venom, little is known about their biological roles. In this study, we determined the three-dimensional structure of DLP-2 by NMR spectroscopy, with the aim of gaining insights into the natural function of the DLPs in platypus venom. The DLP-2 structure was found to incorporate a short helix that spans residues 9-12, and an antiparallel beta-sheet defined by residues 15-18 and 37-40. The overall fold and cysteine-pairing pattern of DLP-2 were found to be similar to those of DLP-1, and hence beta-defensin-12; however, the sequence similarities between the three molecules are relatively small. The distinct structural fold of the DLP-1, DLP-2, and beta-defensin-12 is based upon several key residues that include six cysteines. DLP-3 and DLP-4 are also likely to be folded similarly since they have high sequence similarity with DLP-2. The DLPs, and beta-defensin-12 may thus be grouped together into a class of polypeptide molecules which have a common or very similar global fold. The fact that the DLPs did not display antimicrobial, myotoxic, or cell-growth-promoting activities implies that the nature of the side chains in this group of peptides is likely to play an important role in defining the biological function(s).

1 Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 ml ml 71 failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 ml ml 71 ) produced potent e ects on transmitter release in both smooth and skeletal neuromuscular preparations. 2 In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 ml ml 71 ) caused rapid fasciculations and an increase in indirectly evoked twitches. 3 Male venom (0.02 and 0.05 ml ml 71 ) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no e ect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 ml ml 71 ) were attenuated by (+)-tubocurarine (100 mM) and by tetrodotoxin (TTX, 1 mM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml 71 ). 4 In the unstimulated rat vas deferens, male venom (0.05 ml ml 71 ) caused contractions which were inhibited by a combination of prazosin (0.3 mM) and P 2X -receptor desensitization (with a,bmethylene ATP 10 mM). In the rat stimulated vas deferens, male venom (0.05 ml ml 71 ) augmented indirectly evoked twitches. 5 Male venom (0.1 ml ml 71 ) causes a slowing of inactivation of TTX-sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons. 6 These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX-sensitive sodium channel gating. This action is similar to that of the d-atracotoxins from Australian funnel-web spiders.