Selvanayagam Nirthanan

Irditoxin, a novel covalently linked heterodimeric three-finger toxin with high taxon-specific neurotoxicity

A novel heterodimeric three‐finger neurotoxin, irditoxin, was isolated from venom of the brown treesnake Boiga irregularis (Colubridae). Irditoxin subunit amino acid sequences were determined by Edman degradation and cDNA sequencing. The crystal structure revealed two subunits with a three‐finger protein fold, typical for “nonconventional” toxins such as denmotoxin, bucandin, and candoxin. This is the first colubrid three‐finger toxin dimer, covalently connected via an interchain disulfide bond. Irditoxin showed taxon‐specific lethality toward birds and lizards and was nontoxic toward mice. It produced a potent neuromuscular blockade at the avian neuromuscular junction (IC50=10 nM), comparable to α‐bungarotoxin, but was three orders of magnitude less effective at the mammalian neuromuscular junction. Covalently linked heterodimeric three‐finger toxins found in colubrid venoms constitute a new class of venom peptides, which may be a useful source of new neurobiology probes and therapeutic leads.—Pawlak, J., Mackessy, S. P., Sixberry, N. M., Stura, E. A, Le Du, M. H., Ménez, R., Foo, C. S., Ménez, A, Nirthanan, S., Kini, R. M. Irditoxin, a novel covalently linked heterodimeric three‐finger toxin with high taxon‐specific neurotoxicity. FASEB J. 23, 534–545 (2009)

Autonomic effects of some scorpion venoms and toxins.

1. The autonomic effects of venoms and toxins from several species of scorpions, including the Indian red scorpion Mesobuthus tamulus, the Chinese scorpion Buthus martensi Karsch and the Israeli scorpion Leiurus quinquestriatus quinquestriatus, all belonging to Buthidae, and the Asian black scorpions Heterometrus longimanus and Heterometrus spinifer, belonging to Scorpionidae, are reviewed.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of γ-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 μm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 μm) increased the submaximal response to acetylcholine (10 μm) with a 2.5-fold increase at 60 μm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [3H]tetracaine and [3H]phencyclidine to Torpedo nAChR-rich membranes (IC50 values of 0. 8 mm). nAChR-rich membranes were photolabeled with [3H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (δLeu-265) and M2-13 (αVal-255 and δVal-269), whereas labeling of αM2-10 (αSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in γM3 (γMet-299), a residue that contributes to the same pocket in the nAChR structure as αM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the α and γ subunits (labeling of αM2-10 and γMet-299) likely to be a site for positive allosteric modulation.

Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (King cobra)

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (αβγδ) and neuronal (α7, α3β2, and α4β2) nicotinic acetylcholine receptors (nAChRs) with highest affinity for α7-nAChRs. The high resolution (1.5 Å) crystal structure revealed haditoxin to be a homodimer, like κ-neurotoxins, which target neuronal α3β2- and α4β2-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain α-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain α-neurotoxins and κ-neurotoxins notwithstanding, haditoxin exhibited unique blockade of α7-nAChRs (IC50 180 nm), which is recognized by neither short-chain α-neurotoxins nor κ-neurotoxins. This is the first report of a dimeric short-chain α-neurotoxin interacting with neuronal α7-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.

Neuromuscular effects of candoxin, a novel toxin from the venom of the Malayan krait (Bungarus candidus)

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. 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 α‐neurotoxins, the neuromuscular blockade produced by candoxin was rapidly and completely reversed by washing or by the addition of the anticholinesterase neostigmine. 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 α‐neurotoxins that produce little or no fade. Electrophysiological experiments confirmed that candoxin produced a readily reversible blockade (IC50∼10 nM) of oocyte‐expressed muscle (αβγδ) nAChRs. Like α‐conotoxin MI, well known for its preferential binding to the α/δ interface of the muscle (αβγδ) nAChR, candoxin also demonstrated a biphasic concentration–response inhibition curve with a high‐ (IC50∼2.2 nM) and a low‐ (IC50∼98 nM) affinity component, suggesting that it may exhibit differential affinities for the two binding sites on the muscle (αβγδ) receptor. In contrast, curaremimetic α‐neurotoxins have been reported to antagonize both binding sites with equal affinity.

Biochemical and pharmacological characterization of the venom of the black scorpion Heterometrus spinifer.

The sting of the black scorpion Heterometrus spinifer, which can cause intense localized pain, has not been reported to produce lethal cardiovascular complications, which are well known to result from scorpion envenomation as a consequence of a massive release of catecholamines. Therefore, we have undertaken a biochemical and pharmacological characterization of the venom of H. spinifer. Pharmacologically, the venom (0.125 microL/mL) produced a marked, reversible contracture in the chick biventer cervicis muscle that was blocked by d-tubocurarine (2 microM) but not by tetrodotoxin (5 microM) and omega-conotoxin GVIA (3 microM). The anticholinesterase neostigmine (1 microM) potentiated the contracture by 5.3-fold. An ultra-filtrate fraction of MW < 3000 (F3K) of the venom produced a similar contracture in the biventer muscle, whereas the retentate of MW > 3000 did not. In the rat anococcygeus muscle, the venom produced a contractile response that was partially (37.4 +/- 1.6%) blocked by atropine (5 microM); phentolamine (5 microM) blocked the remaining response. Tetrodotoxin (5 microM) did not block the contractile response of the venom on the anococcygeus muscle. Electrospray ionization-mass spectrometry/mass spectrometry confirmed the presence of high concentrations of acetylcholine (79.8 +/- 1.7 microM) and norepinephrine (146.7 +/- 19.8 microM) in H. spinifer venom, which can fully account for the observed cholinergic and adrenergic effects. In contrast to scorpion venoms that selectively target neuronal ion channels in mediating transmitter release, our data show that H. spinifer venom does not possess such activity, which likely explains the apparent lack of lethality of black scorpion envenomation.

Functional site of bukatoxin, an α-type sodium channel neurotoxin from the Chinese scorpion (Buthus martensi Karsch) venom: probable role of the 52PDKVP56 loop

α‐Toxins from scorpion venoms prolong the action potential of excitable cells by blocking sodium channel inactivation. We have purified bukatoxin, an α‐toxin from scorpion (Buthus martensi Karsch) venom, to homogeneity. Bukatoxin produced marked relaxant responses in the carbachol‐precontracted rat anococcygeus muscle (ACM), which were mediated through the L‐arginine–nitric oxide synthase–nitric oxide pathway, consequent to a neuronal release of nitric oxide. Based on the presence of proline residues in the flanking segments of protein–protein interaction sites, we predicted the site between 52PP56 to be the potential interaction site of bukatoxin. A homology model of bukatoxin indicated the presence of this site on the surface. Buka11, a synthetic peptide designed based on this predicted site, produced a concentration‐dependent nitric oxide‐mediated relaxant response in ACM. Using alanine‐substituted peptides, we have shown the importance 53DKV55 flanked by proline residues in the functional site of bukatoxin.

Pharmacological characterization of mikatoxin, an α-neurotoxin isolated from the venom of the New-Guinean small-eyed snake Micropechis ikaheka

Symptoms of envenomation by the New-Guinean small-eyed snake Micropechis ikaheka (Elapidae) include peripheral neurotoxicity and myotoxicity. We have now purified to homogeneity a long-chain neurotoxin, mikatoxin, from M. ikaheka venom by successive gel filtration and reverse-phase chromatography. Electrospray ionization mass spectrometry showed mikatoxin to be a homogenous peptide of MW 7775.6. Mikatoxin was devoid of any phospholipase A(2) activity associated with the crude venom and did not exhibit any intrinsic anticholinesterase activity. In the chick biventer cervicis muscle, it produced an irreversible, concentration-dependent block of responses to exogenously applied acetylcholine and carbachol as well as twitches evoked by nerve, but not by direct muscle stimulation. Moreover, mikatoxin, like alpha-bungarotoxin and erabutoxin-b, did not show significant fade response to train-of-four stimulation of the mouse phrenic nerve-hemi diaphragm muscle. It also failed to block ganglionic transmission in the guinea pig ileum and muscarinic responses in the rat anococcygeus muscle. Our study provides strong evidence for the presence of a neurotoxin (mikatoxin) in M. ikaheka venom that produces neuromuscular blockade in skeletal muscle attributable to selective and irreversible antagonism of postsynaptic nicotinic acetylcholine receptors of the neuromuscular junction and likely contributes to the peripheral neurotoxicity observed in M. ikaheka envenomation.

Crystallization and preliminary X-ray analysis of bucain, a novel toxin from the Malayan krait Bungarus candidus.

Bucain is a three-finger toxin, structurally homologous to snake-venom muscarinic toxins, from the venom of the Malayan krait Bungarus candidus. These proteins have molecular masses of approximately 6000-8000 Da and encompass the potent curaremimetic neurotoxins which confer lethality to Elapidae and Hydrophidae venoms. Bucain was crystallized in two crystal forms by the hanging-drop vapour-diffusion technique in 0.1 M sodium citrate pH 5.6, 15% PEG 4000 and 0.15 M ammonium acetate. Form I crystals belong to the monoclinic system space group C2, with unit-cell parameters a = 93.73, b = 49.02, c = 74.09 A, beta = 111.32 degrees, and diffract to a nominal resolution of 1.61 A. Form II crystals also belong to the space group C2, with unit-cell parameters a = 165.04, b = 49.44, c = 127.60 A, beta = 125.55 degrees, and diffract to a nominal resolution of 2.78 A. The self-rotation function indicates the presence of four and eight molecules in the crystallographic asymmetric unit of the form I and form II crystals, respectively. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated.

Characterization of the rat isolated retractor penis muscle as a model for the study of nitrergic transmission

INTRODUCTION The anococcygeus and retractor penis muscles are part of the erectile machinery in male rodents. The rat anococcygeus muscle is a widely used smooth muscle preparation for the study of the effects of test substances on adrenergic, nitrergic, and cholinergic transmission. There is, however, little information available on the process of autonomic transmission in the rat retractor penis muscle, although its autonomic innervation has generally been assumed to be similar to that of the anococcygeus muscle because of the contiguous nature of the two muscles. The present study investigated the involvement of nitrergic transmission in mediating relaxant responses of the rat retractor penis muscle to electrical field stimulation. METHODS The retractor penis muscle was isolated from Sprague-Dawley rats and mounted in Krebs solution. Phentolamine (5 microM) was added to the bath to block the adrenergic responses of the muscle, which was then precontracted with carbachol (10 microM). RESULTS Electrical field stimulation (20-30 V, 1 ms pulse width, at 0.5-20 Hz for 10 s) of the carbachol precontracted muscle elicited frequency-dependent relaxant responses (0.9-68%). Tetrodotoxin (1 microM), N(G)-nitro-L-arginine (L-NOARG) (50 microM), N(G)-nitro-L-arginine methylester (L-NAME) (100 microM), and haemoglobin (100 microM) inhibited these relaxant responses by 99.3%, 93.9%, 86.9%, and 77.5%, respectively. L-Arginine (250 microM) (but not its D-isomer) reversed the blockade produced by L-NOARG (72.7%) and L-NAME (81.5%). DISCUSSION Our results provide clear evidence that the inhibitory (relaxant) responses of the rat retractor penis muscle to electrical field stimulation are mediated by nitric oxide involving the L-arginine-nitric oxide synthase-nitric oxide pathway. The rat retractor penis muscle is a versatile preparation that can replace the cumbersome preparations from the pig, ox, and horse, hitherto used as pharmacological models for the study of the retractor penis muscle.