ABSTRACT
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 microl ml(-1) failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 microl ml(-1)) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations. In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 microl ml(-1)) caused rapid fasciculations and an increase in indirectly evoked twitches. Male venom (0.02 and 0.05 microl ml(-1)) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect 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 microl ml(-1)) were attenuated by (+)-tubocurarine (100 microM) and by tetrodotoxin (TTX, 1 microM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml(-1)). In the unstimulated rat vas deferens, male venom (0.05 microl ml(-1)) caused contractions which were inhibited by a combination of prazosin (0.3 microM) and P(2X)-receptor desensitization (with alpha,beta-methylene ATP 10 microM). In the rat stimulated vas deferens, male venom (0.05 microl ml(-1)) augmented indirectly evoked twitches. Male venom (0.1 microl ml(-1)) causes a slowing of inactivation of TTX-sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons. 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 delta-atracotoxins from Australian funnel-web spiders.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Ion Channel Gating/drug effects , Neurotoxins/pharmacology , Sodium Channels/drug effects , Spider Venoms/pharmacology , Acetylcholine/pharmacology , Adenosine Triphosphate/pharmacology , Adrenergic alpha-Antagonists/pharmacology , Animals , Chickens , Diaphragm/drug effects , Diaphragm/innervation , Dose-Response Relationship, Drug , Female , Ganglia, Spinal/drug effects , Ganglia, Spinal/physiology , Male , Membrane Potentials/drug effects , Mice , Mice, Inbred BALB C , Muscle Contraction/drug effects , Muscles/drug effects , Muscles/physiology , Nicotinic Antagonists/pharmacology , Phrenic Nerve/drug effects , Phrenic Nerve/physiology , Potassium Chloride/pharmacology , Prazosin/pharmacology , Rats , Rats, Sprague-Dawley , Tetrodotoxin/pharmacology , Tubocurarine/pharmacology , Vas Deferens/drug effects , Vas Deferens/physiology , Vasodilator Agents/pharmacologyABSTRACT
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).
Subject(s)
Proteins/chemistry , Venoms/chemistry , Amino Acid Sequence , Animals , Defensins , Magnetic Resonance Spectroscopy , Male , Models, Molecular , Molecular Sequence Data , Platypus , Protein Conformation , Protein Folding , Sequence Homology, Amino AcidABSTRACT
delta-Atracotoxins (delta-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. We have isolated and determined the amino acid sequence of a novel delta-ACTX, designated delta-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with delta-ACTX-Hv1a 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 delta-ACTXs. However, delta-ACTX-Hv1b is approximately 15-30-fold less potent than other delta-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between delta-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.
