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The present study describes the isolation and pharmacological characterisation of the neurotoxin N N-missulenatoxin-Mb1a (N N-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 e¡ects 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 N N-MSTX-Mb1a

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The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been hampered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal spider-venom peptide ω-HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability.