Acetylcholine receptor binding characteristics of snake and cone snail venom postsynaptic neurotoxins: further studies with a non-radioactive assay.

The binding of postsynaptic neurotoxins from snake and marine cone snail (Conus sp.) venoms to nicotinic acetylcholine receptor (AchR) was investigated with an ELISA-based, non-radioactive assay. Three snake postsynaptic toxins from the long-chain group (Naja naja kaouthia cobratoxin, Naja oxiana neurotoxin I, Bungarus multicinctus alpha-bungarotoxin) and short-chain group (Naja naja atra cobrotoxin, Naja oxiana neurotoxin II, and Laticauda semifasciata erabutoxin b) were studied. Both types of snake postsynaptic toxins showed a dose-response with constant AchR (50 micrograms/ml) and varying toxin concentrations (50-0.035 micrograms/ml). The minimum detection limits of the assay for snake toxins ranged from 310 to 1240 ng/ml (40-160 pmole/ml), depending on the toxin. Unlike any of the short-chain toxins, long-chain toxins consistently bound less receptor and reached maximum absorbance levels with toxin concentrations of 10-50 micrograms/ml. Competition for AchR binding between cone snail postsynaptic neurotoxins (conotoxins GI, MI, SI) and alpha-bungarotoxin or cobrotoxin resulted in a dose-response. The postsynaptic conotoxins were uniformly better competitors for AchR binding with alpha-bungarotoxin than with cobrotoxin. Heat stability studies with neurotoxin I, erabutoxin b, or cobrotoxin revealed a loss in AchR binding activity with increasing temperature. alpha-Bungarotoxin heated at 90 degrees C had increased AchR binding activity by 105%, relative to 25 degrees C samples, but lost the majority of its binding activity after 100 degrees C. The enhanced binding of heated alpha-bungarotoxin to AchR was specific, as evidenced by a competitive dose-response with unheated alpha-bungarotoxin, but heated toxin lacked any biological activity in the mouse lethal assay. When conotoxins GI or MI were heated at 100 degrees C, there was no detectable loss in AchR binding activity, and only a slight decrease in mouse lethality.