Inhibition of Ionotropic Neurotransmitter Receptors by Antagonists: Strategy to Estimate the Association and the Dissociation Rate Constant of Antagonists with Very Strong Affinity to the Receptors

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Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist. When an antagonist such as gallamine (Gal), (+)-tubocurarine (Tub), or pancuronium (Pan) was applied simultaneously with acetylcholine (ACh), it inhibited the nAChR competitively and apparently accelerated the rate of inactivation of nAChR. On the other hand, it inhibited the receptor in an apparently noncompetitive manner, and very effectively, when it was preapplied to the receptor. These results were analyzed on the basis of our model. Both the association and the dissociation rate constant of the antagonists were estimated from the inhibition experiments on the basis of the model. Moreover, our results suggested the presence of not only competitive binding sites with a high affinity in the nAChR but also a noncompetitive inhibitory site with a low affinity toward the antagonists. Strychnine (Str) also inhibited glycine receptor (GlyR) competitively when applied simultaneously with glycine (Gly), but inhibited GlyR in a noncompetitive manner when it was preapplied.