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 mar ine neurotoxins known as the lophotoxins. The lophotoxins are unique i n 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 inhibi tory activity of the bipinnatins against the nicotinic receptor increa sed with preincubation of the toxins in aqueous buffer prior to incuba tion with the receptor. The parent species of the bipinnatins displaye d little, if any, affinity for the nicotinic receptor. Preincubation o f the toxins appeared to produce a single, relatively stable, active t oxin species that irreversibly inhibited the two acetylcholine-binding sites on the nicotinic receptor with two distinguishable apparent pse udo first-order rates. The difference in the rates of irreversible inh ibition of the two binding sites on the receptor was exploited to sele ctively 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 metoc urine and high affinity for acetylcholine. The bimolecular reaction co nstants for the interaction of the bipinnatins with the nicotinic rece ptor decreased in the order bipinnatin-B > bipinnatin-A > bipinnatin-C for both acetylcholine-binding sites. The ratio of the bimolecular re action constants for the two binding sites on the receptor was not the same for the three bipinnatins. This indicates that the reaction of t he bipinnatins with the nicotinic receptor is sensitive to differences in the structure of the two acetylcholine-binding sites. The bipinnat ins may be useful in the design of novel drugs for the nicotinic recep tor 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.