Abstract
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.
Highlights
Bipinnatin-A,B, and -C belong to a family of naturally molecules of acetylcholine must bind to the receptor in orderto occurring marine neurotoxins known as the lophotox- open the channel andallow ion flux across the membrane
The inhibitory activity of the bipinnatins against the Bipinnatin-A, -B, and -C belong to a family of marine neunicotinic receptor increased with preincubation of the rotoxinsthat canbe isolated from various species toxins in aqueous buffer prior to incubation with the of the soft corals Lophogorgia and Pseudopterogorgia (Fenical receptor.The parent species of the bipinnatins dis- et al, 1981;Abramson et al, 1991a)
We investigated changes in the rateof irreversible inhibition of mouse muscle nicotinic acetylcholine receptors by the bipinnatins and report the ratesof the inhibition of nicoand B23) of 1%Triton X-100
Summary
(Received for publication, October 27, 1993,and in revised form, December 28, 1993). From the Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. Threatio of the bimolecular reaction constants for the two binding sites on the receptor was notthe same forthe three bipinnatins This indicates that thereaction of the bipinnatins with the lophotoxins irreversibly inhibit nicotinic receptors by forming a covalent bond with TyrlgOin the a-subunoitf the receptor (Abramson et al.,1989). Nicotinic receptor subunits thatlack a the nicotinic receptor is sensitive to differences in the tyrosine residue a t position 190 in the sequence We investigated changes in the rateof irreversible inhibition of mouse muscle nicotinic acetylcholine receptors by the bipinnatins and report the ratesof the inhibition of nicoand B23) of 1%Triton X-100. Nonspecific binding of lZ5I-a-BTXwasdetermined from cells incubated with 100 ILM a-BTX for 30 min prior t o the 1-h
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