Kappa 2-bungarotoxin and kappa 3-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 kappa-neurotoxins, kappa-bungarotoxin and kappa-flavitoxin. Native and radiolabeled kappa-neurotoxins have been used to localize and quantitate neuronal nicotinic receptors in a variety of species. We now report the identification of two new kappa-neurotoxins. kappa 2-Bungarotoxin and kappa 3-bungarotoxin were purified from the venom of Bungarus multicinctus collected in the province of Guangdong, China. kappa-Bungarotoxin has as yet not been found in this venom, although it is the only kappa-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 kappa-neurotoxins are potent antagonists of nicotinic transmission in the chick ciliary ganglion. kappa 3-Bungarotoxin, the least potent of the kappa-neurotoxins, produces a complete blockage of nicotinic transmission in 60 min at 250 nM. Protection experiments using the short-acting nicotinic antagonists dihydro-beta-erythroidine and (+)-tubocurarine demonstrate that kappa 2-bungarotoxin blocks transmission by binding to the acetylcholine recognition sites of neuronal nicotinic receptors. The isoelectric point of kappa 2-bungarotoxin (pI = 8.9) is similar to that of kappa-bungarotoxin and kappa-flavitoxin, but kappa 3-bungarotoxin is considerably more basic, with pI greater than 11. Partial amino acid sequences are reported for both kappa 2-bungarotoxin and kappa 3-bungarotoxin. These sequences show a high degree of homology (approximately 80%) with other kappa-neurotoxins, and allow the determination of the critical differences between the kappa-neurotoxins and the structurally related alpha-neurotoxins. For example, all 4 kappa-neurotoxins lack a tryptophanyl residue which is invariant and important for function in the alpha-neurotoxins. The kappa-neurotoxins also differ from the alpha-neurotoxins by having an invariant prolinyl residue at a critical sequence position. Heterodimers were detected consisting of one subunit each of kappa 2-bungarotoxin and kappa 3-bungarotoxin. These heterodimers, which form between any combination of two kappa-neurotoxins, appear to be physiologically active and confirm that a further distinction between kappa-neurotoxins and alpha-neurotoxins is the strong tendency of the former to self-associate in solution. The present results help to establish the definition of 'kappa-neurotoxin'. 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.(ABSTRACT TRUNCATED AT 400 WORDS)