Use of a Snake Venom Toxin to Characterize the Cholinergic Receptor Protein

Abstract

Abstract. a-Bungarotoxin, a polypeptide of mol wt 8000 purified from the venom of Bungarus multicinctus, blocks irreversibly and specificallythe excitation by cholinergic agonists on the isolated electroplax and on purified membrane fragments in vitro. The toxin also blocks the in vitro binding of decamethonium to a protein recently isolated from electric tissue. This observation strengthens our earlier conclusion that this protein is the cholinergic receptor macromolecule.At a glance, the most evident and simple manner to characterize and identify the physiological receptor of acetylcholine in an excitable membrane is to use compounds that are structurally analogous to acetylcholine and thus present a high affinity for the cholinergic receptor site. However, it is now well established that in most excitable membranes there exist several-distinct classes of sites, all of which are able to bind cholinergic ligands. Among them are, in addition to the physiological receptor site, the catalytic and allosteric sites of the enzyme acetylcholinesterase (AcChE).l The use of cholinergic ligands thus meets with a difficult problem of specificity.2Interestingly enough, in the course of the past few years it has been shown that certain toxins from snake venoms, although completely unrelated structurally to acetylcholine, nevertheless act much like curare. One of them is a-bungarotoxin (a-Bgt), a basic polypeptide of mol wt 8000, which has been extensively studied by Lee and his associates.3-5 a-Bgt is purified from the venom of an elapid snake from Taiwan (Bungarus multicinctus) and the purified toxin gives irreversible neuromuscular blocking effects. In addition, d-tubocurarine protects against the action of a-Bgt. From these findings Lee and Chang4 have concluded that a-Bgt combines irreversibly with the cholinergic receptor at the motor endplate.We report here some experiments carried out with purified a-Bgt and various preparations derived from the electric organ of Electrophorus electricus. It is shown that in vivo, with the isolated electroplax, a-Bgt irreversibly blocks the depolarization caused by bath application of carbamylcholine, a cholinergic agonist. The same effect is observed with purified electric-organ membrane fragments using the in vitro assay of Kasai and Changeux.6 In addition, in agreement with the early observation of Lee and associates, d-tubocurarine protects against a-Bgt blockade. Finally we tested a-Bgt on the binding of 1241

Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin

“…Cervefiansky et al, 1991), form three loops emerging from a dense core containing the disulfide bridges. They belong to the structural family of three-fingered peptidic toxins from Elapidae venoms, which include the nicotinic receptor-blocking a-neurotoxins (Changeux et al, 1970;Endo & Tamiya, 1991), the muscarinic receptor peptide agonists (Adem et al, 1988;Segalas et al, 1995), and the cardiotoxins that interact with cell membranes (Bougis et al, 1981;Dufton & Hider, 1991). Despite a common structural motif, the toxins in this family are directed to diverse targets, yet their modes of action are highly selective.…”

N,O‐di andN,N,O‐tri [3H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

“…The specific and irreversible blocking effect on the cholinergic receptor of the motor endplate of a-bungarotoxin (a-BuTX) isolated from the venom of Bungarus multicinctus (Chang & Lee, 1963) has recently prompted several groups of authors to use this toxin as a specific probe for the characterization of cholinergic recep-the electric organ of Electrophorus and Torpedo (Changeux, Kasai & Lee, 1970;Miledi, Molinoff & Potter, 1971). Preparations of a-BuTX, either iodinated with '3II (Lee & Tseng, 1966;Lee et al, 1967;Miledi & Potter, 1971; or 125I (Berg et al, 1972), or acetylated with [3H] acetic anhydride (Barnard et al, 1971) have been used for this purpose.…”

Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca 2+ -binding sites

“…7-9; see also FruchartGaillard et al, 10). Their extremely high affinity and selectivity permitted the first isolation and purification of the receptor (11).…”