Acetylcholine neurotoxins

Radioiodinated derivatives have been prepared to define more closely the target site of a-conotoxins on the acetylcholine receptor (R. Myers, unpublished data). In membrane preparations from Torpedo electroplax, photoactivatable azidosalicylate derivatives of a-conotoxin GIA preferentially label the p and 7 subunits of the acetylcholine receptor. However, when the photoactivatable derivative is cross-linked to detergent solubilized acetylcholine receptor (AChR), only the 7 subunit is labeled. Since snake a-neurotoxins mainly bind to the a subunits of AChR and a-conotoxins compete directly with a-bungarotoxin, the cross-linking results above are both intriguing and problematic. 

Neurotoxins present in sea snake venoms are summarized. All sea snake venoms are extremely toxic, with low LD5Q values. Most sea snake neurotoxins consist of only 60-62 amino acid residues with 4 disulOde bonds, while some consist of 70 amino acids with 5 disulfide bonds. The origin of toxicity is due to the attachment of 2 neurotoxin molecules to 2 a subunits of an acetylcholine receptor that is composed of a2 6 subunits. The complete structure of several of the sea snake neurotoxins have been worked out. Through chemical modification studies the invariant tryptophan and tyrosine residues of post-synaptic neurotoxins were shown to be of a critical nature to the toxicity function of the molecule. Lysine and arginine are also believed to be important. Other marine vertebrate venoms are not well known. 

In order to understand the exact mechanism of the neurotoxic action, it is important to know the secondary structure of the neurotoxins as well. It is now known that postsynaptic neurotoxins attach to the a-subunits of acetylcholine receptor (AChR). 

When a nerve-muscle preparation is stimulated in the presence of a sea snake neurotoxin, there is no twitch. However, when the muscle itself is stimulated directly in the presence of a neurotoxin, the muscle contracts. This means that neurotoxin does not inhibit the muscle itself. Moreover, postsynaptic neurotoxin does not inhibit the release of acetylcholine from the nerve ending. Therefore, the site of snake toxin inhibition must be in the postsynaptic site 20). Later it was shown that a neurotoxin strongly binds to the acetylcholine receptor (AChR). 

The AChR is composed of five subunits, ql2Pi - A neurotoxin attaches to the a subunit. Since there are 2 mol of the a subunits, 2 mol of neurotoxins attach to 1 mol of AChR. A neurotransmitter, acetylcholine (ACh), also attaches to the a subunit. When the ACh attaches to the AChR, the AChR changes conformation, opening up the transmembrane pore so that cations (Na" ", K ) can pass through. By this mechanism the depolarization wave from a nerve is now conveyed to a muscle. The difference between neurotoxin and ACh is that the former s attachment does not open the transmembrane pore. As a consequence, the nerve impulse from a nerve cannot be transmitted through the postsynaptic site (27). 

Delayed-action paralytic neurotoxins that block the release of acetylcholine causing a symmetric, descending flaccid paralysis of motor and autonomic nerves. Paralysis always begins with the cranial nerves. Toxins are obtained from an anaerobic bacteria (Clostridium botulinum). Toxin A is a white powder or crystalline solid that is readily soluble in water. It is stable for up to 7 days as an aqueous solution. All toxins are destroyed by heat and decompose when exposed to air for more than 12 h. 

Mixture of neurotoxins that block the acetylcholine receptors. The /3-bungarotoxin is a pre-synaptic neural toxin, a-bungarotoxin is a postsynaptic neural toxin, and K-bungarotoxin is specific to the neuronal receptors in ganglions. They are obtained from the venom of the banded krait (Bungarus multicinctus). 

Delayed-action neurotoxin that blocks the release of acetylcholine that is a crystalline solid obtained from bacteria (Clostridium tetani). Dried material is stable for years when stored between 39 and 45°F otherwise, it is relatively unstable and very sensitive to heat. 

All botulin neurotoxins act in a similar way. They only differ in the amino-acid sequence of some protein parts (Prabakaran et al., 2001). Botulism symptoms are provoked both by oral ingestion and parenteral injection. Botulin toxin is not inactivated by enzymes present in the gastrointestinal tracts. Foodborne BoNT penetrates the intestinal barrier, presumably due to transcytosis. It is then transported to neuromuscular junctions within the bloodstream and blocks the secretion of the neurotransmitter acetylcholine. This results in muscle limpness and palsy caused by selective hydrolysis of soluble A-ethylmalemide-sensitive factor activating (SNARE) proteins which participate in fusion of synaptic vesicles with presynaptic plasma membrane. SNARE proteins include vesicle-associated membrane protein (VAMP), synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP-25). Their degradation is responsible for neuromuscular palsy due to blocks in acetylcholine transmission from synaptic terminals. In humans, palsy caused by BoNT/A lasts four to six months. 

Damle, V.N. and Karlin, A., Affinity labeling of one of two a-neurotoxin binding sites in acetylcholine receptor from Torpedo californica, Biochemistry, 17, 2039, 1978. 

Leutje, C. W., Wada, K., Rogers, W., Abramson, S.N., Tsuji, K., Heinemann, S., and Patrick, J., Neurotoxins distinguish between different neuronal nicotinic acetylcholine receptor subunit combinations, J. Neurochem., 55, 632, 1990. 

Anatoxin-a (3) is a powerful neurotoxin (inhibitor of acetylcholine esterase) found in freshwater blue-green algae. The compound was required as an analytical standard and also for development of an immunoassay. It was synthesized by the 8-step sequence summarized in Scheme 29.1. The key intermediate 4 was resolved by separation of the dibenzoyl tartrates, and the remaining steps then gave both (+)- and (-)-anatoxin-a.2 The efficiency of the resolution was monitored by formation of the BocAla derivatives, which were distinguishable by NMR an ee of >98% was achieved even before recrystallization of the salts. It is also noteworthy that the published absolute configuration of the intermediate 53 was shown to be in error by X-ray crystallography. 

Use of Toxin Binding to Purify a Channel Protein a-Bungarotoxin is a powerful neurotoxin found in the venom of a poisonous snake (Bungarus multicinctus). It binds with high specificity to the nicotinic acetylcholine receptor (AChR) protein and prevents the ion channel from opening. This interaction was used to purify AChR from the electric organ of torpedo fish. 

Acetylcholine receptor from electric organ of Torpedo sp. Receptor protein noncovalently bound on the surface of a planar interdigitated capacitative sensor. Response was concentration dependent and specific for ACh and inhibited by (+ )-tubocurarine, amantidine and a-neurotoxin. [66]... 

Stimulus-evoked, calcium-dependent release of acetylcholine (ACh) from the cholinergic synapse normally occurs through the formation of a fusion complex between ACh-containing vesicles and the intracellular leaflet of the nerve terminal membrane (Amon et al., 2001). This synaptic vesicle fusion complex consists of several proteins of the SNARE family, including a 25 kDa synaptosomal associated protein (SNAP-25), vesicle-associated membrane protein (VAMP, or synaptobrevin), and the synaptic membrane protein syntaxin. Other SNARE proteins have been identified as components of membrane transport systems in yeast and mammals but have not been implicated as targets for BoNTs. Meanwhile, type A and E neurotoxins cleave SNAP-25 while types B, D, F, and G act on VAMP and type C1 toxin cleaves both syntaxin and SNAP-25. Neurotoxin-mediated cleavage of any of these substrates disrupts the processes involved in the exocytotic release of ACh and leads to flaccid paralysis of the affected skeletal muscles. 

Botulism neurotoxins bind with synaptic vesicular proteins and block the release of acetylcholine from the presynaptic membrane (Osborne et al, 2007). Clinical signs of botulism are weakness, tremors, recumbency, laryngeal paresis, and other signs of nervous system dysfunction (Braun et al, 2005). Botulism toxins do not appear to be excreted in milk (Galey et al, 2000). 

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