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 (IC(50)) and the kinetics of onset and recovery of antagonist 
inhibition at 20 to 23 degrees C. Rate constants were calculated from the 
predictions of a single (high-affinity) site model of competitive inhibition. 
For pancuronium: IC(50) = 5.5 +/- 0.5 nM (mean +/- S.D.), l(on) = 2.7 +/- 0.9 x 
10(8) M(-1) s(-1), l(off) = 2.1 +/- 0.7/s [corrected] x 10(8)/s. For 
(+)-tubocurarine: IC(50) = 41 +/- 2 nM, l(on) = 1.2 +/- 0.2 x 10(8) 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 IC(50) 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.