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...

5. Our studies confirmed that the rat anococcygeus muscle is an excellent nerve-smooth muscle preparation for investigating the effects of bioactive agents on noradrenergic and nitrergic transmission, as well as the direct agonist actions of these agents on post-synaptic ␣-adrenoceptors and M3 muscarinic cholinoceptors. Although many studies, including our own, have documented that scorpion venoms and toxins mediate their primary effects via a prejunctional mechanism that leads to the marked release of various autonomic neurotransmitters, our studies have shown that there are exceptions to this generally accepted phenomenon. In particular, we have provided firm evidence to show that the venoms from H. longimanus and H. spinifer do not have such a prejunctional site of action but, instead, the venoms mediate their autonomic effects through direct agonist actions on post-junctional muscarinic M3 cholinoceptors and ␣-adrenoceptors.

1 Candoxin (MW 7334.6), a novel toxin isolated from the venom of the Malayan krait Bungarus candidus, belongs to the poorly characterized subfamily of nonconventional three-finger toxins present in Elapid venoms. The current study details the pharmacological effects of candoxin at the neuromuscular junction. 2 Candoxin produces a novel pattern of neuromuscular blockade in isolated nerve-muscle preparations and the tibialis anterior muscle of anaesthetized rats. In contrast to the virtually irreversible postsynaptic neuromuscular blockade produced by curaremimetic a-neurotoxins, the neuromuscular blockade produced by candoxin was rapidly and completely reversed by washing or by the addition of the anticholinesterase neostigmine. 3 Candoxin also produced significant train-of-four fade during the onset of and recovery from neuromuscular blockade, both, in vitro and in vivo. The fade phenomenon has been attributed to a blockade of putative presynaptic nicotinic acetylcholine receptors (nAChRs) that mediate a positive feedback mechanism and maintain adequate transmitter release during rapid repetitive stimulation. In this respect, candoxin closely resembles the neuromuscular blocking effects of d-tubocurarine, and differs markedly from curaremimetic a-neurotoxins that produce little or no fade. 4 Electrophysiological experiments confirmed that candoxin produced a readily reversible blockade (IC 50 B10 nM) of oocyte-expressed muscle (abgd) nAChRs. Like a-conotoxin MI, well known for its preferential binding to the a/d interface of the muscle (abgd) nAChR, candoxin also demonstrated a biphasic concentration -response inhibition curve with a high-(IC 50 B2.2 nM) and a low-(IC 50 B98 nM) affinity component, suggesting that it may exhibit differential affinities for the two binding sites on the muscle (abgd) receptor. In contrast, curaremimetic a-neurotoxins have been reported to antagonize both binding sites with equal affinity.

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1. Nerve-evoked maximal twitches (T1, T2, T3, T4) of the rat isolated hemidiaphragm to train-of-four (TOF) stimulation (2 Hz X 2 s) were recorded continuously in the absence or presence of tubocurarine (1.5 mumol/l), succinylcholine (40 mumol/l) or alpha-bungarotoxin (1 mumol/l). The T1 and T4 response-time profiles for the three drugs were analysed with respect to amplitude depression and the TOF ratio (T4/T1) during the development of and recovery from neuromuscular blockade. 2. Tubocurarine produced T1 block accompanied by intense TOF fade; for the same degree of T1 block, the TOF ratio was lower during the recovery from blockade after washing out tubocurarine from the bath than during the onset of blockade. There was also a correspondingly slower recovery of the TOF ratio from 90% T1 block to control levels when compared with the time for complete T1 recovery. Fade and twitch tension depression were shown clearly to be separate responses, each with its own response-time profile. Fade is therefore not simply a consequence of postjunctional cholinoceptor blockade. 3. Succinylcholine produced T1 block with only moderate TOF fade; similar recovery rates from 90% T1 block to control levels were obtained for T1 and the TOF ratio. 4. alpha-Bungarotoxin produced irreversible and complete neuromuscular blockade during which TOF fade was virtually absent. 5. The results obtained in this study closely resemble those from other similar studies in animals and in humans and clearly demonstrate that the rat isolated hemidiaphragm is a suitable in vitro model for time course studies on TOF fade.