Interactions of fluorescent cholinergic antagonists with the membrane-bound acetylcholine receptor

The synthesis of pyrenyl-choline derivatives is reported. The interactions of these compounds with the acetylcholine receptor has been studied in developing chick myotubes in culture and in the adult frog neuromuscular junction with electrophysiological techniques. In vivo, the compounds display the pharmacological characteristics of nicotinic antagonists. Similar conclusions can be derived from in vitro experiments using radiolabelled derivatives in equilibrium binding studies with the membrane-bound receptor from Torpedo, steady-state fluorescence titrations, and toxin kinetic inhibition studies. The rapid kinetics of receptor-probe interactions were studied with the temperature-jump method. The fluorescence emission of the probes, excited by energy transfer from the protein chromophores, enabled the observation of two relaxation processes. The concentration dependence of the relaxation times and associated amplitudes can most economically be accounted for in terms of a reaction mechanism consisting of two sequential ligand binding-isomerization steps. The two ligands find themselves in an environment of high electronic polarizability, having a dielectric constant of about 40, after rapidly binding to two sites which differ in their affinities roughly by a factor of 10 (Apparent Kd's 1.5 and 0.2 ?M).