κ₂-Bungarotoxin and κ₃-bungarotoxin: two new neuronal nicotinic receptor antagonists isolated from the venom of Bungarus multicinctus

Neuronal nicotinic acetylcholine receptors are recognized with high affinity by two snake venom κ-neurotoxins, κ-bungarotoxin and κ-flavitoxin. Native and radiolabeled κ-neurotoxins have been used to localize and quantitate neuronal nicotinic receptors in a variety of species We now report the identification of two new κ-neurotoxins, κ₂-Bungarotoxin and κ₃-bungarotoxin were purified from the venom of Bungarus multicinctus collected in the province of Guangdong, China. κ-Bungarotoxin has as yet not been found in this venom, although it is the only κ-neurotoxin to be isolated thus far from Taiwanese Bungarus multicinctus. The geographical separation of Guangdong and Taiwan might account for this evolutionary divergence within the species. Both of the new κ-neurotoxins are potent antagonists of nicotinic transmission in the chick ciliary ganglion. κ₃-Bungarotoxin, the least potent of the κ-neurotoxins, produces a complete blockade of nicotinic transmission in 60 min at 250 nM. Protection experiments using the short-acting nicotinic antagonists dihydro-β-erythroidine and (+)-tubocurarine demonstrate that κ₂-bungarotoxin blocks transmission by binding to the acetylcholine recognition sites of neuronal nicotinic receptors. The isoelectric point of κ₂-bungarotoxin (pI=8.9) is similar to that of κ-bungarotoxin and κ-flavitoxin, but κ₃-bungarotoxin is considerably more basic, basic, with pI >11. Partial amino acid sequences are reported for both κ₂-bungarotoxin. These sequences show a high degree of homology (∼80%) with other κ-neurotoxins, and allow the determination of the critical differences between the κ-neurotoxins and the structurally related α-neurotoxins. For example, all 4 κ-neurotoxins lack a tryptophanyl residue which is invariant and important for function in the α-neurotoxins. The κ-neurotoxins also differ from the α-neurotoxins by having an invariant prolinyl residue at a critical sequence position. Heterodimers were detected consisting of one subunit each of κ₂-bungarotoxin and κ₃-bungarotoxin. These heterodimers, which form between any combination of two κ-neurotoxins, appear to be physiologically active and confirm that a further distinction between κ-neurotoxins and α-neurotoxins is the strong tendency of the former to self-associate in solution. The present results help to establish the definition of 'κ-neurotoxins'. These snake toxins are now being used by a number of laboratories in physiological and biochemical experiments on neuronal nicotinic receptors from a variety of species. One note of caution to emerge from this study is that Bungarus multicinctus venom may contain any of 3 different κ-neurotoxins. These polypeptides are difficult or impossible to separate by low-pressure cation exchange columns, but can be distinguished by using cation exchange HPLC. This procedure, as well as partial amino acid sequencing, is essential in confirming the identity of purified κ-neurotoxins prior to their use as receptor probes.