In contrast to most short and long chain curaremimetic neurotoxins that produce virtually irreversible neuromuscular blockade in isolated nerve-muscle preparations, candoxin, a novel three-finger toxin from the Malayan krait Bungarus candidus, produced postjunctional neuromuscular blockade that was readily and completely reversible. Nanomolar concentrations of candoxin (IC 50 ؍ ϳ10 nM) also blocked acetylcholineevoked currents in oocyte-expressed rat muscle (␣␥␦) nicotinic acetylcholine receptors in a reversible manner. In contrast, it produced a poorly reversible block (IC 50 ؍ ϳ50 nM) of rat neuronal ␣7 receptors, clearly showing diverse functional profiles for the two nicotinic receptor subsets. Interestingly, candoxin lacks the helix-like segment cyclized by the fifth disulfide bridge at the tip of the middle loop of long chain neurotoxins, reported to be critical for binding to ␣7 receptors. However, its solution NMR structure showed the presence of some functionally invariant residues involved in the interaction of both short and long chain neurotoxins to muscle (␣␥␦) and long chain neurotoxins to ␣7 receptors. Candoxin is therefore a novel toxin that shares a common scaffold with long chain ␣-neurotoxins but possibly utilizes additional functional determinants that assist in recognizing neuronal ␣7 receptors.Curaremimetic or ␣-neurotoxins from snake venoms are well known to bind with high affinity and selectivity and in most instances, almost irreversibly to Torpedo and muscle (␣␥␦) nicotinic acetylcholine receptors (nAChR), 1 thereby affecting synaptic neurotransmission and producing flaccid paralysis (1, 2). They belong to a family of proteins called "three-finger toxins," which adopt a flat, leaf-like shape formed by three adjacent loops that emerge from a small globular core, which is the location of the four conserved disulfide bridges (3-9). Other members of this family include -bungarotoxins, which recognize neuronal nicotinic receptors (10), muscarinic toxins with selectivity toward distinct types of muscarinic receptors (11), fasciculins that inhibit acetylcholinesterase (12), calciseptins that block the L-type calcium channels (13,14), cardiotoxins (cytotoxins) that exert their toxicity by forming pores in cell membranes (15), and dendroaspins, which are antagonists of various cell adhesion processes (16). Despite their common structural fold and comparable affinity for the Torpedo and muscle (␣␥␦) nAChRs, ␣-neurotoxins are classified as short chain neurotoxins (e.g. erabutoxin-b (Laticauda semifasciata)) that have 60 -62 residues and four conserved disulfide bonds and long chain neurotoxins (e.g. ␣-bungarotoxin (Bungarus multicinctus); ␣-cobratoxin (Naja kaouthia)) with 66 -75 residues and five disulfide bonds (3). The additional disulfide bridge in long chain ␣-neurotoxins, as well in the neuronal -bungarotoxin (B. multicinctus) is located in the middle loop (loop II) (3,8,9). This fifth bridge, which cyclizes a helix-like conformation at the tip of loop II, has been reported to be cru...
