Ligand-gated ion channels bind agonists with higher affinity in the open th
an in the closed state. The kinetic basis of this increased affinity has re
mained unknown, because even though the rate constants of agonist associati
on to and dissociation from closed receptors can be estimated with reasonab
le certainty, the kinetics of the binding steps in open receptors have prov
en to be elusive. To be able to measure the agonist-dissociation rate const
ant from open muscle nicotinic receptors, we increased the probability of l
igand unbinding from the open state by engineering a number of mutations th
at speed up opening and slow down closing but leave the ligand-binding prop
erties unchanged. Single-channel patch-clamp recordings from the wild-type
and mutant constructs were performed at very low concentrations of acetylch
oline (ACh). The durations of individual channel activations were analyzed
assuming that "bursts" of fully liganded (diliganded) receptor openings can
be terminated by ligand dissociation from the closed or open state (follow
ed by fast closure) or by desensitization. This analysis revealed that ACh
dissociates from diliganded open receptors at approximate to 24 s(-1), that
is, approximate to2,500 times more slowly than from diliganded closed rece
ptors. This change alone without a concomitant change in the association ra
te constant to the open state quantitatively accounts for the increased equ
ilibrium affinity of the open channel for ACh. Also, the results predict th
at both desensitization and ACh dissociation from the open state frequently
terminate bursts of openings in naturally occurring gain-of-function mutan
ts (which cause slow-channel congenital myasthenia) and therefore would con
tribute significantly to the time course of the endplate current decay in t
hese disease conditions.