Abstract

N-Methyl-D-aspartate (NMDA) receptor ligands regulate the binding of uncompetitive antagonists in membranes prepared from rat brain. To determine the mechanism of this regulation, we examined the kinetics of the binding of the radiolabeled uncompetitive antagonist [3H]N-(1-[thienyl]cyclohexyl) piperidine (TCP). Increasing concentrations of NMDA receptor agonists produced dose-dependent increases in the association and dissociation rate constants of TCP. The NMDA receptor antagonist amino phosphono valeric acid virtually abolished both the association and dissociation of TCP. Linear regression analysis detected a significant (p less than 0.001) correlation between the effect of NMDA receptor ligands on the apparent association and dissociation rate constants. The most parsimonious explanation of the data is that NMDA receptor ligands regulate TCP binding by controlling access of TCP to a transiently accessible or "guarded" binding site located in the receptor-coupled ion channel. An increase in affinity or number of TCP binding sites is neither necessary nor sufficient to explain the potentiation of TCP binding produced by NMDA agonists. This finding validates the use of uncompetitive antagonist binding as a measure of the functional activation of the NMDA receptor-coupled ion channel in isolated membrane preparations.