alpha-Conotoxins selectively inhibit one of the two acetylcholine binding sites of nicotinic receptors.

Muscle subtypes of the nicotinic acetylcholine receptor contain two acetylcholine binding sites that can be distinguished pharmacologically. The affinities of several alpha-conotoxins for the two acetylcholine binding sites on nicotinic receptors from BC3H1 cells and Torpedo electric organ were investigated. alpha-Conotoxins MI, GI, and SIA each inhibited the binding of 125I-alpha-bungarotoxin to nicotinic acetylcholine receptors on BC3H1 cells with two distinct and independent affinities, which differed by > 10,000-fold. The affinities of alpha-conotoxins SI and SII were significantly lower and the differences in the affinities of each of these toxins for the two sites were < 400-fold. alpha-Conotoxins MI, GI, SIA, and SI had higher affinity for the acetylcholine binding site near the alpha/delta subunit interface of nicotinic receptors from BC3H1 cells. However, when assessed using nicotinic receptors from Torpedo electric organ, alpha-conotoxin MI displayed higher affinity for the acetylcholine binding site near the alpha/gamma subunit interface. These observations suggest that species variations in the sequences of the gamma and delta subunits resulted in a dramatic reversal of the relative affinities of the alpha-conotoxins for each acetylcholine binding site. Some of the practical implications of these observations are discussed.

Irreversible inhibition of nicotinic acetylcholine receptors by the bipinnatins. Toxin activation and kinetics of receptor inhibition.

Bipinnatin-A, -B, and -C belong to a family of naturally occurring marine neurotoxins known as the lophotoxins. The lophotoxins are unique in that they irreversibly inhibit nicotinic acetylcholine receptors by forming a covalent bond with a tyrosine residue at position 190 in the alpha-subunit of the receptor. In this study, we show that the inhibitory activity of the bipinnatins against the nicotinic receptor increased with preincubation of the toxins in aqueous buffer prior to incubation with the receptor. The parent species of the bipinnatins displayed little, if any, affinity for the nicotinic receptor. Preincubation of the toxins appeared to produce a single, relatively stable, active toxin species that irreversibly inhibited the two acetylcholine-binding sites on the nicotinic receptor with two distinguishable apparent pseudo first-order rates. The difference in the rates of irreversible inhibition of the two binding sites on the receptor was exploited to selectively inhibit one site for the pharmacological investigation of the other. The bipinnatins preferentially inhibited the binding site near the alpha/delta-subunit interface that displays low affinity for metocurine and high affinity for acetylcholine. The bimolecular reaction constants for the interaction of the bipinnatins with the nicotinic receptor decreased in the order bipinnatin-B > bipinnatin-A > bipinnatin-C for both acetylcholine-binding sites. The ratio of the bimolecular reaction constants for the two binding sites on the receptor was not the same for the three bipinnatins. This indicates that the reaction of the bipinnatins with the nicotinic receptor is sensitive to differences in the structure of the two acetylcholine-binding sites. The bipinnatins may be useful in the design of novel drugs for the nicotinic receptor that exclusively inhibit one of the two binding sites and for the investigation of structural differences between the two acetylcholine-binding sites of the receptor.