Kinetics of binding of [3H]acetylcholine to Torpedo postsynaptic membranes: association and dissociation rate constants by rapid mixing and ultrafiltration

Abstract

An automated rapid-mixing ultrafiltration apparatus was constructed to measure at subsecond times the kinetics of binding of radiolabeled cholinergic ligands to nicotinic postsynaptic membranes isolated from Torpedo electric tissue. The dissociation of [3H]AcCh[3H-Acetylocholine]-receptor complexes at 7 and 23.degree. C was characterized by Kd of 0.05/s and 0.15/s, respectively. The association kinetics at low concentrations of [3H]AcCh were determined at 23.degree. C. They were consistent with a model in which 20% of the AcCh binding sites preexist in a receptor conformation that binds AcCh with high affinity (Kd=3 nM) and with a bimolecular association constant, k+ = 5.7 .times. 107/M s. The association kinetics at higher AcCh concentrations revealed a transient low-affinity binding step that occurred relatively slowly. In the presence of 0.8 .mu.M [3H]AcCh, this component of the association reaction was characterized by rate constant of 2/s. The binding kinetics are discussed with reference to the processes of channel activation and receptor desensitization.