Modulation of glutamate-induced uncompetitive blocker binding to the NMDA receptor by temperature and by glycine

Publisher Website
Google Scholar
Abstract
The effect of temperature on the binding of [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) to the ion channel of the N-methyl-D-aspartate (NMDA) receptors was studied in washed rat brain-cortex membranes. Raising the temperature from 5 to 33.degree. C resulted in a significant increase in the association rates of [3H]TCP binding measured in the presence of 1 .mu.M glutamate and 1 .mu.M glycine, but was less effective in the absence of the added agonists. No such effects of temperature on the dissociation rates of [3H]TCP-receptor complexes were observed. In the absence of agonists, neither the association nor the dissociation binding components varied with temperature, suggesting a diffusion-controlled limitation of access of the ligand to its site within the nonactivated NMDA receptor. No evidence was found for a temperature-dependent change in the density of [3H]TCP binding sites or for heterogeneity of [3H]TCP binding sites associated with the NMDA receptor, even though when approaching equilibrium the binding kinetics in the presence of glutamate and glycine deviated from an ordinary bimolecular reaction scheme. The data were fitted instead to a two-exponent binding function, comprising the sum of a fast and a slow binding component. Their corresponding time constants exhibited an increase with temperature, and the increase of each one was correlated significantly with the corresponding decrease in the equilibrium binding constant; however, there was no temperature-related change in the relative proportions of the two components, with the fast binding component (.alpha.) accounting for 50-70% of the site population. Varying glutamate concentrations (without glycine) resulted in corresponding dose-dependent increase of both the fast and the slow time constants, with no change in .alpha. (28-35%). Glycine increased .alpha. from its glutamte-induced level to 60%, but did not change the fast and slow time constants. The results suggest homogeneity of [3H]TCP-binding domains within the NMDA receptor channel but variability of total channel opening time. The observed effects of glutamate and of glycine on the kinetic components are consistent with this suggestion.