Mode of action of atracotoxin at central and peripheral synapses of insects

Abstract

These studies characterized the paralytic and neurophysiological effects of an atracotoxin (ACTX), from the Australian funnel web spider, Hadronyche versuta, and compared it to the established P/Q-type calcium channel blocker, omega-agatoxin-IVA (omega-Aga-IVA). ACTX-induced paralysis was of a spastic form in housefly ( Musca domestica) larvae, but it was inactive on neuromuscular junction of housefly and tobacco budworm ( Heliothis virescens). On cockroach ( Periplaneta americana) cercal nerve-giant fiber synapse preparations, both toxins were effective blockers with potencies in the nanomolar range, but some spontaneous, high frequency trains of action potentials were observed with ACTX. In Drosophila melanogaster central nervous preparations, blockage of nerve firing occurred within 20 min when the nerve sheath was intact, demonstrating that the barrier could be breached by ACTX in vitro. There was a potent (pM) excitatory response to ACTX in this tissue, prior to the onset of block at higher concentrations. In contrast, omega-Aga-IVA was a pure blocker in both cockroach and Drosophila preparations. These studies demonstrate that central synaptic calcium channels underlie the action of ACTX. ACTX-dependent neuroexcitation has a number of possible mechanisms that warrant further study.

Scanning Mutagenesis of ω-Atracotoxin-Hv1a Reveals a Spatially Restricted Epitope That Confers Selective Activity against Insect Calcium Channels

“…That is, in the LD 50 assays, the activity of a toxin depends not only on its intrinsic affinity for the target channel but also on its in vivo stability and its ability to penetrate anatomical barriers. The latter point is crucial in that -ACTXHv1a targets the central nervous system of insects (4,5), whereas most other spider toxins act presynaptically at insect neuromuscular junctions. Thus, a mutant toxin might have reduced activity not because of a reduced affinity for the target channel, but rather because of increased susceptibility to proteolysis or more limited access to the central nervous system.…”

“…The phyletic specificity of these toxins appears to reside in their ability to block insect, but not vertebrate, voltage-gated calcium channels (2). Although nanomolar concentrations of -ACTX-Hv1a are sufficient to block high voltage-activated (HVA) calcium currents in the central nervous system of the fruit fly Drosophila melanogaster and the American cockroach Periplaneta americana (2,5), 1 M toxin does not block HVA calcium currents in vertebrate neurons (2) and the toxin is harmless when injected subcutaneously into newborn mice (1).…”

“…These toxins comprise 36 -37 residues with six strictly conserved cysteine residues that are paired to form three disulfide bridges (4). The best studied family member, -ACTX-Hv1a, is one of the most potent insecticidal peptide toxins discovered so far (4,5); it has proved lethal to all insect orders that have been tested, including coleopterans, dictyopterans, hemipterans, orthopterans, and refractory lepidopteran pests such as the tobacco budworm Heliothis virescens and the cotton bollworm Helicoverpa armigera (1,2,5). The phyletic specificity of these toxins appears to reside in their ability to block insect, but not vertebrate, voltage-gated calcium channels (2).…”

Biological Activity of Insecticidal Toxins: Structural Basis, Site-Directed Mutagenesis and Perspectives

“…However, this analysis indicates that 1T0Z (from the Asian scorpion Buthus martensi Karsch) and 1I25 (from the Chinese bird spider O. huwena) may have anti-coleopteran properties due to the fact that they are in the same branch as 1WWN and 1TI5, respectively ( Figure 1). Studies have shown that insecticidal toxins purified from arthropod venoms exert their effects via specific interactions with ion channels and receptors in the central or peripheral nervous system (De Lima et al, 2007;Bloomquist, 2003;Johnson et al, 1998;Fletcher et al, 1997). B. martensi Karsch venom has four peptides related to the excitatory insect toxin family and 10 related to the depressant insect toxin (Goudet et al, 2002).…”

Peptide toxins that selectively target insect NaVand CaVchannels

“…124,126 In affected insects, ω-ACTX-Hv1a induces a slow-onset, irreversible spastic paralysis that precedes flaccid paralysis and death. 124,130 D. melanogaster that were engineered to express an inducible ω-ACTX-Hv1a transgene often developed a phenotype reminiscent of a hypomorphic allele of the dmca1D gene which encodes the Drosophila Ca V 1 channel. 131 This led to the suggestion that insect Ca V 1 channels are the primary target of ω-ACTX-Hv1a 131 , since similar phenotypes have not been reported for hypomorphic alleles of dmca1A.…”