On the true affinity of glycine for its binding site at the NMDA receptor complex

To determine the exact potency of inhibitors acting at the glycine site of the NMDA receptor complex using [3H]glycine as a radioligand, the true equilibrium constant (KD) of the radioligand has to be known. To achieve this goal, (1) the contamination of water by glycine was studied, (2) the true affinity of glycine for the NMDA receptor was estimated by different methods, and (3) the inhibition constant of a number of antagonists was determined. HPLC analysis of the deionized water used for the preparation of the assay buffers and solutions resulted in glycine concentrations below 6 nmol/L in four out of seven samples analyzed. The observable equilibrium constant of [3H]glycine binding (Kobs) was found to vary, in the presence of 10-mumol glutamate per liter, between 36 and 163 nmol/L (mean +/- SD: 69 +/- 21 nmol/L, 140 determinations). Based on the observation that the polyamine spermine increased the potency of glycine, but not that of glycine antagonists, an algorithm was developed to calculate KD from Kobs- and IC50-values obtained with and without 100-mumol spermine per liter. Using four different antagonists, KD-values between 24 and 57 nmol/L were obtained. Computer analysis of glycine-stimulated [3H]MK-801 binding resulted in EC50-values between 22 and 35 nmol/L. Based on these results, a true affinity constant for [3H]glycine of 40 nmol/L was assumed and used for the determination of Ki-values of 15 different inhibitors of [3H]glycine binding.