Abstract

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 (ℓ_on) and dissociation (ℓ_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.), ℓ_on = 2.7 ± 0.9 × 10⁸M⁻¹ s⁻¹, ℓ_off = 2.1 ± 0.7 × 10⁸/s. For (+)-tubocurarine: IC₅₀ = 41 ± 2 nM, ℓ_on = 1.2 ± 0.2 × 10⁸ M⁻¹ s⁻¹, ℓ_off = 5.9 ± 1.3/s. The kinetic results are consistent with the equilibrium results in that ℓ_off/ℓ_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.