Alpha-Elapitoxin-Aa2a, a long-chain snake alpha-neurotoxin with potent actions on muscle (alpha1)2betagammadelta nicotinic receptors, lacks the classical high affinity for neuronal alpha7 nicotinic receptors

Abstract

In contrast to all classical long-chain alpha-neurotoxins possessing the critical fifth disulfide bond, alpha-elapitoxin-Aa2a (alpha-EPTX-Aa2a), a novel long-chain alpha-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal alpha7-type nicotinic acetylcholine receptors (nAChRs). alpha-EPTX-Aa2a (8850 Da; 0.1-1 microM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. alpha-EPTX-Aa2a (1-10 nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA2 value of 8.311 ± 0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain alpha-neurotoxin, alpha-bungarotoxin. In contrast, alpha-EPTX-Aa2a produced complete, but weak, inhibition of 125I-alpha-bungarotoxin binding to rat hippocampal a7 nAChRs (pKI = 3.670), despite high sequence homology and similar mass to a wide range of long-chain alpha-neurotoxins. The mostly likely cause for the loss of alpha7 binding affinity is a leucine substitution, in loop II of alpha-EPTX-Aa2a, for the highly conserved Arg33 in long-chain alpha-neurotoxins. Arg33 has been shown to be critical for both neuronal and muscle activity. Despite this substitution, alpha-EPTX-Aa2a retains high affinity for muscle (alpha1)2betagammadelta nAChRs. This is probably as a result of an Arg29 residue, previously shown to be critical for muscle (alpha1)2betagammadelta nAChR affinity, and highly conserved across all short-chain, but not long-chain, alpha-neurotoxins. alpha-EPTX-Aa2a therefore represents a novel atypical long-chain alpha-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes.