Abstract
A new series of polypeptide presynaptic antagonists ("omega-agatoxins") was purified from venom of the funnel web spider Agelenopsis aperta. Physiological data indicate that all of these peptides are antagonists of voltage-sensitive calcium channels. Although all three omega-agatoxins (Aga) described here (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IIA) block insect neuromuscular transmission presynaptically, biochemical data permit their subclassification as Type I and Type II toxins. Type I toxins (omega-Aga-IA and -IB) are 7.5 kDa, have closely related amino acid sequences, and exhibit characteristic tryptophan-like UV absorbance spectra. Complete Edman sequencing of omega-Aga-IA reveals it to be a 66-amino acid polypeptide containing 9 cysteines and 5 tryptophan residues. omega-Aga-IIA, a Type II toxin, is 11 kDa, shows limited amino acid sequence similarity to the Type I toxins, and exhibits mixed tryptophan- and tyrosine-like absorbance. Nanomolar concentrations of omega-Aga-IIA inhibit the specific binding of 125I-labeled omega-conotoxin GVIA to chick synaptosomal membranes while omega-Aga-IA and -IB have no effect under identical conditions. The omega-agatoxins thus are defined as two subtypes of neuronal calcium channel toxins with different structural characteristics and calcium channel binding specificities.
Highlights
Neuromuscukw Block by w-Agutoxins-RPLC fractionation of crude A. upertu venom yielded a series of polypeptides (“wagatoxins”) which block neuromuscular transmission in housefly body wall muscle
Since the IP remains unchanged after EJP block by the w-agatoxins, toxin action cannot be explained by disruption of glutamate signaling at the postsynaptic membrane
The three classes of synaptic toxins occurring in A. aperta venom are defined as postsynaptic antagonists (a-agatoxins), presynaptic activators (p-agatoxins), and presynaptic antagonists (w-agatoxins)
Summary
Neuromuscukw Block by w-Agutoxins-RPLC fractionation of crude A. upertu venom yielded a series of polypeptides (“wagatoxins”) which block neuromuscular transmission in housefly body wall muscle Three of these peptides, w-AgaIA, w-Aga-IB, and w-Aga-IIA, were identified based on their ability to produce long lasting suppression of neurally evoked EJPs without affecting ionophoretic glutamate potentials (IPs; Fig. 1, A and B). Detailed electrophysiological analyses of neuromuscular antagonism by w-Aga-IA demonstrate its ability to suppress transmitter release by blocking voltage-activated calcium channels [4] This mode of action is clearly distinguishable from those of the Bath application of w-Aga-IA at 20 nM leads to a 90% reduction in EJP amplitude within lo-15 min, an effect which does not diminish following several hours of washing the preparation in toxin-free saline (Fig. 2). The long lasting block caused by w-agatoxins may reflect their slow dissociation from presynaptic binding sites
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