Vol. 60, Issue 4, 790-796, October 2001

The Kinetics of Inhibition of Nicotinic Acetylcholine Receptors by (+)-Tubocurarine and Pancuronium

Ingobert Wenningmann and James P. Dilger

Klinik für Anästhesiologie, Universität Bonn, Bonn, Germany (I.W.); and Departments of Anesthesiology and Physiology & Biophysics, State University of New York at Stony Brook, Stony Brook, New York

Equilibrium conditions of neurotransmitter concentration and receptor binding are never achieved during synaptic transmission at the neuromuscular junction. Thus, it is important to determine the binding kinetics of drugs that act this synapse. Previous determinations of the dissociation rate of (+)-tubocurarine have produced inconsistent results ranging from 0.1 to 4000/s. Here, we used a direct approach to measure association (_on) and dissociation (_on) rates for two competitive antagonists (clinically used as nondepolarizing muscle relaxants), pancuronium and (+)-tubocurarine, at nicotinic acetylcholine receptors (nAChR). We made macroscopic current recordings from outside-out patches of BC3H-1 cells expressing embryonic mouse muscle nAChR. We used a three-tube rapid perfusion system to make timed applications of antagonists and acetylcholine to the patch. We made independent measurements of the equilibrium inhibition (IC₅₀) and the kinetics of onset and recovery of antagonist inhibition at 20 to 23°C. Rate constants were calculated from the predictions of a single (high-affinity) site model of competitive inhibition. For pancuronium: IC₅₀ = 5.5 ± 0.5 nM (mean ± S.D.), _on = 2.7 ± 0.9 × 10⁸ M¹ s¹, _off = 2.1 ± 0.7 × 10⁸/s. For (+)-tubocurarine: IC₅₀ = 41 ± 2 nM, _on = 1.2 ± 0.2 × 10⁸ M¹ s¹, _off = 5.9 ± 1.3/s. The kinetic results are consistent with the equilibrium results in that _off/_on is in good agreement with the IC₅₀ values. All differences between the antagonists are significant at the p < 0.001 level. The higher affinity of pancuronium is caused by a faster association rate (2.2-fold) coupled with a slower dissociation rate (2.8-fold). The association rates of both antagonists are comparable with or greater than the association rate for acetylcholine binding to nAChR.