Acetylcholine receptor inhibition by d-tubocurarine involves both a competitive and a noncompetitive binding site as determined by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles.
The issue of whether d-tubocurarine, the classical acetylcholine receptor inhibitor, inhibits the receptor by a competitive or noncompetitive mechanism has long been controversial. d-Tubocurarine, in this study, has been found to be both a competitive (KC = 120 nM) and a noncompetitive (KNC = 4 microM) inhibitor of receptor-mediated ion flux at zero transmembrane voltage in membrane vesicles prepared from Electrophorus electricus electroplax. A spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, was used to measure both the rate coefficient of ion flux prior to receptor inactivation (desensitization) and the rate coefficient of the rapid inactivation process. Inhibition by d-tubocurarine of the initial rate of ion flux decreased with increasing acetylcholine concentration, consistent with competitive inhibition, but the inhibition by micromolar concentrations of d-tubocurarine could not be overcome with saturating concentrations of acetylcholine, consistent with noncompetitive inhibition. A minimum mechanism is proposed in which d-tubocurarine competes for one of the two acetylcholine activating sites and also binds to a noncompetitive site. The present data do not distinguish between one or two competitive sites, although one successfully accounts for all of the data. By variation of the acetylcholine concentration, the two types of sites could be studied in isolation. Binding of d-tubocurarine to the noncompetitive site does not change the rate of rapid receptor inactivation, whereas binding of d-tubocurarine to the competitive site decreases the rate of rapid inactivation by displacing acetylcholine, in agreement with the observation that d-tubocurarine does not inactivate (desensitize) the E. electricus receptor by itself.(ABSTRACT TRUNCATED AT 250 WORDS)
J W
JW
Karpen
G P
GP
Hess
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003042:Cocaine / Q000494:pharmacology; D004557:Electric Organ / Q000378:metabolism; D004593:Electrophorus; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008564:Membrane Potentials / Q000187:drug effects; D008954:Models, Biological; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*; D014403:Tubocurarine / Q000494:pharmacology*
D007473:Ion Channels; D011950:Receptors, Cholinergic; D003042:Cocaine; D014403:Tubocurarine