Biochemical Studies of the Nicotinic Acetylcholine Receptor from Torpedo Californica Electroplax

The ligand binding properties of the acetylcholine (AcCh) receptor have been investigated using radioactive cholinergic ligands, radiolabelled a-neurotoxins, and fluorescent decamethonium analogues. The radioactive cholinergic ligands appear to bind with high affinity to half as many sites as α-Bungarotoxin (α-BuTx). One possible interpretation of these data is that two distinct classes of sites are present in equal populations. The results of kinetic experiments conducted in this laboratory have shown that the receptor binds a-BuTx with a five fold greater second order rate constant and binds cholinergic ligands with 2-7 fold lower affinity to the "fast" than to the "slow" site. The binding properties of a series of synthetic bis-3-aminopyridinium- 1,n-alkane fluorescent decamethonium analogues to the purified receptor were investigated. The probes bind with substantially greater affinity to the "slow" than to the "fast" site, and therefore served as useful tools to map the ligand binding topography of the uslow11 site of the receptor. This site binds the probes with increasing affinity as the methylene bridge length (n) increases from 4 to 12 carbons and decreases from 18 to 16 carbons. One possible interpretation of these data is that when the "slow" site binds a bifunctional probe such as bis-3-aminopyridiniunr-1,14-tetradecane, both ends of the probe interact strongly with the receptor. Therefore the "slow" site of the receptor may be composed of at least two subsites. Recently published evidence indicates that the venom of the krait Bungarus caeruleus contains unique toxic proteins which affect the function of the AcCh receptor in vivo. Therefore we undertook the fractionation and purification of various proteins from this venom in order to examine their use as probes of the AcCh receptor. Two of the basic components were highly neurotoxic in. mice and had significant levels of phospholipase-A activity. These components appear to be similar to the presynaptic neurotoxin β-BuTx. Two other basic components were toxic in mice and also reduced the rate of α-BuTx binding to the purified acetylcholine receptor. These components appear to be similar to the postsynaptic neurotoxin α-BuTx. Two acidic components display A-type phospholipase activity and perturb the carbamylcholine binding properties of receptor enriched membrane preparations. These data indicate that the fatty acids and/or lysophospholipids released by endogenous or exogenous phospholipase A may affect the receptor conformation such that the receptor binds carbamylcholine with greater affinity.