Delta-atracotoxins from Australian funnel-web spiders compete with scorpion alpha-toxin binding on both rat brain and insect sodium channels.

Atracotoxins are novel peptide toxins from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. To analyse their interaction with known sodium channel neurotoxin receptor sites we determined their effect on scorpion toxin, batrachotoxin and saxitoxin binding. Nanomolar concentrations of delta-atracotoxin-Hv1 and delta-atracotoxin-Ar1 completely inhibited the binding of the scorpion alpha-toxin AaH II to rat brain synaptosomes as well as the binding of LqhalphaIT, a scorpion alpha-toxin highly active on insects, to cockroach neuronal membranes. Moreover, delta-atracotoxin-Hv1 cooperatively enhanced batrachotoxin binding to rat brain synaptosomes in an analogous fashion to scorpion alpha-toxins. Thus the delta-atracotoxins represent a new class of toxins which bind to both mammalian and insect sodium channels at sites similar to, or partially overlapping with, the receptor binding sites of scorpion alpha-toxins.

delta-Atracotoxins from australian funnel-web spiders compete with scorpion alpha-toxin binding but differentially modulate alkaloid toxin activation of voltage-gated sodium channels.

delta-Atracotoxins from the venom of Australian funnel-web spiders are a unique group of peptide toxins that slow sodium current inactivation in a manner similar to scorpion alpha-toxins. To analyze their interaction with known sodium channel neurotoxin receptor sites, we studied their effect on [3H]batrachotoxin and 125I-Lqh II (where Lqh is alpha-toxin II from the venom of the scorpion Leiurus quinquestriatus hebraeus) binding and on alkaloid toxin-stimulated 22Na+ uptake in rat brain synaptosomes. delta-Atracotoxins significantly increased [3H]batrachotoxin binding yet decreased maximal batrachotoxin-activated 22Na+ uptake by 70-80%, the latter in marked contrast to the effect of scorpion alpha-toxins. Unlike the inhibition of batrachotoxin-activated 22Na+ uptake, delta-atracotoxins increased veratridine-stimulated 22Na+ uptake by converting veratridine from a partial to a full agonist, analogous to scorpion alpha-toxins. Hence, delta-atracotoxins are able to differentiate between the open state of the sodium channel stabilized by batrachotoxin and veratridine and suggest a distinct sub-conductance state stabilized by delta-atracotoxins. Despite these actions, low concentrations of delta-atracotoxins completely inhibited the binding of the scorpion alpha-toxin, 125I-Lqh II, indicating that they bind to similar, or partially overlapping, receptor sites. The apparent uncoupling between the increase in binding but inhibition of the effect of batrachotoxin induced by delta-atracotoxins suggests that the binding and action of certain alkaloid toxins may represent at least two distinguishable steps. These results further contribute to the understanding of the complex dynamic interactions between neurotoxin receptor site areas related to sodium channel gating.

Characterisation of the effects of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, on sodium channel activation and inactivation.

The present study investigates the actions of robustoxin (atracotoxin-Ar1) purified from the venom of the male Sydney funnel-web spider Atrax robustus on sodium channel gating. Using whole-cell patch-clamp techniques the study assessed the actions of robustoxin on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents in rat dorsal root ganglion cells. Similar to the closely related funnel-web spider toxin versutoxin (delta-atracotoxin-Hv1) from Hadronyche versuta, robustoxin had no effect on TTX-R sodium currents but exerted potent effects on TTX-S sodium currents. The main action of robustoxin was a concentration-dependent slowing or removal of TTX-S sodium current inactivation. This steady-state current was maintained during long-lasting depolarisations at all test potentials. Robustoxin (30 nM) also caused a 13-mV hyperpolarising shift in the voltage midpoint of steady-state sodium channel inactivation (h infinity) leading to a reduced peak current at a holding potential of -80 mV. Moreover there was a steady-state or non-inactivating component present (18% of maximal sodium current) at prepulse potentials that normally inactivate all TTX-S sodium channels (more depolarised than -40 mV). In addition robustoxin produced a significant increase in the repriming kinetics of the sodium channel when channels returned to the resting state following activation. This increase in the rate of recovery of sodium current appears to explain the use-dependent effects on peak sodium current amplitude at high stimulation frequencies. Finally 30 nM robustoxin caused an 11-mV hyperpolarising shift in the voltage dependence of the channel but did not markedly modify tail current kinetics. These actions suggest that robustoxin inhibits conversion of the open state to the inactivated state of TTX-S sodium channels, thus allowing a fraction of the sodium current to remain at membrane potentials at which inactivation is normally complete. Given the recent reclassification of funnel-web spider toxins as atracotoxins, robustoxin should henceforth be known as delta-atracotoxin-Ar1 to reflect this main action on sodium channel inactivation. These present results further support the hypothesis that funnel-web spider toxins interact with neurotoxin receptor site 3 to slow channel inactivation in a manner similar to that of alpha-scorpion and sea anemone toxins.

Selective alteration of sodium channel gating by Australian funnel-web spider toxins.

The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of 22Na+ flux and an inhibition of batrachotoxin-activated 22Na+ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [3H]saxitoxin binding was unaffected. In addition, the partial activation of 22Na+ flux was not enhanced in the presence of alpha-scorpion toxin and further experiments suggest that VTX also enhances [3H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of alpha-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.

Residency in mental-health pharmacy with a research component.

A 12-month postdoctoral specialized residency program in mental-health pharmacy practice with a psychopharmacology research component is described. The resident spent approximately four hours a day in clinical practice and in research activities in the inpatient setting; 10 hours per week in clinical research activities in an ambulatory patient setting; and 10 hours per week in teaching and scholarly activities. The resident was a member of a psychopharmacology research team that also included a research psychiatrist, a nurse, and two psychiatric medical residents at an adult acute-care site. The resident prepared a review of the preclinical evaluations of each investigational drug and participated in the initial screening of study patients. The resident was responsible for patient consent procedures, coordinating clinical evaluations, and scheduling necessary laboratory tests. As a member of the clinical team, she was also responsible for obtaining medication histories, monitoring and reporting adverse drug reactions, maintaining accurate dosage records, and completing the appropriate rating scales in the clinical evaluation of patients. In the ambulatory setting, the resident participated in the outpatient management of study patients. The resident learned to assess patient's clinical progress, maintain progress notes, write medication orders and administer medications in acute situations, maintain individual treatment plans, follow approved drug research protocols, and provide a program of education for patients and families when necessary. In addition, the resident was involved in educational programs for pharmacy students. This training program enables the pharmacist to gain experience in drug research studies, clinical practice, and pharmaceutical education, while contributing to psychopharmacology research.