EQUILIBRIUM AND KINETIC-STUDY OF GLYCINE ACTION ON THE N-METHYL-D-ASPARTATE RECEPTOR IN CULTURED MOUSE-BRAIN NEURONS

1. The characteristics of the activation of the N-methyl-D-aspartate (NMDAA) response by glycine were studied using whole-cell and outside-out patch clamp recording techniques. 2. Glycine concentration-response (C-R) curves were measured in the presence of 10-mu-M-NMDA and fitted with the Hill equation modified to account for the response to NMDA observed in the absence of added glycine. The mean value of the apparent dissociation constant (K(D)) was 150 nM, and the mean value of the Hill coefficient (n(H)) was 1.1. When the K(D) was corrected for the concentration of contaminating glycine in nominally glycine-free solutions, estimated assuming that there is no response in the absence of glycine, the value was 130 nM. 3. The question of how many glycine binding sites there are on each NMDA receptor-channel complex was addressed by examining the curvature at the foot of the glycine C-R curve. An equation that allowed estimation of both the concentration of contaminating glycine and of the value of n(H) was fitted to glycine C-R data up to 50 nM. The mean value of n(H) was found to be 1.0 consistent with the idea that there is one glycine binding site. 4. The kinetics of the interaction of glycine with the NMDA receptor were measured by fitting single exponential curves to the current relaxation following a jump in glycine concentration in the presence of 10-mu-M-NMDA. The plot of the inverse of the relaxation time constant as a function of glycine concentration after the concentration jump was linear. The association rate constant was estimated from these data as 1.2 x 10(7) M-1 s-1 and the dissociation rate as 1.0 s-1. 5. Experiments were devised to allowed the evaluation of the K(D) and dissociation rates of glycine in the absence of NMDA. They led to a value for K(D) of 80 nM, slightly but significantly lower than the value of 150 nM estimated in the presence of 10-mu-M-NMDA. The glycine dissociation rate in the absence of NMDA was found to be 0.7 s-1, not significantly different from that measured in the presence of 10-mu-M-NMDA. 6. The results are consistent with a model of the NMDA receptor with a single glycine binding site. The characteristics of glycine binding are similar in the absence and the presence of 10-mu-M-NMDA, although NMDA binding may cause a small increase in the glycine K(D). Under the recording conditions used here, the dominant action of glycine on the NMDA response is not mediated through an effect on agonist binding at the NMDA site.