Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors

摘要

alpha delta-Bungarotoxins, a novel group of long-chain alpha-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of alpha-delta subunits. Three isoforms (alpha delta-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (alpha delta-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to alpha-bungarotoxin (alpha-BgTx), a classical blocker of muscle-type and neuronal alpha 7, alpha 8, and alpha 9 alpha 10 nAChRs. The toxicity of alpha delta-BgTx-1 (LD50 = 0.17-0.28 mu g/g mouse, i.p. injection) is essentially as high as that of alpha-BgTx. In the chick biventer cervicis nerve-muscle preparation, alpha delta-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by alpha-BgTx, acetylcholine response was fully reversible by washing. alpha delta-BgTxs, similar to alpha-BgTx, bind with high affinity to alpha 7 and muscle-type nAChRs. However, the major difference of alpha delta-BgTxs from alpha-BgTx and other naturally occurring alpha-neurotoxins is that alpha delta-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the alpha-delta site as compared with alpha-epsilon or alpha-gamma binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in alpha delta-BgTxs. Thus, alpha delta-BgTxs are new tools for studies on nAChRs.