BJP S McKay et al. Figure 1 TCN 213 antagonism of NMDA receptor-mediated responses is both subtype- and glycine-dependent. (A) Upper panel, TEVC currents recorded from an oocyte expressing GluN1/GluN2A NMDA receptors in response to application of glutamate (100 mM) and glycine, which was applied at a concentration indicated as a multiple of its EC50 value (1.5 mM for GluN1/GluN2A NMDA receptors). TCN 213 (10 mM) was applied as indicated and inhibited the glutamate/glycine evoked response, but the extent of the inhibition was dependent on the glycine concentration. Lower panel, a series of similar TEVC current traces but recorded in the presence of a saturating concentration of glycine (50 mM) and variable concentrations of glutamate. The extent of the inhibition produced by TCN 213 is similar in each case. (B) A series of representative TEVC current traces illustrating similar experiments to those shown in panel A but for recordings made from oocytes expressing GluN1/GluN2B NMDA receptors. Note here the modest inhibition produced by TCN 213. (C) Bar graphs summarizing the mean data obtained from a series of experiments (n = 6) that investigated the glycine dependency of TCN 213 antagonism of steady-state responses at both GluN1/GluN2A and GluN1/GluN2B NMDA receptors. (D) As in panel C but for experiments where the glycine concentration was ﬁxed (50 mM) and glutamate was applied at 0.1, 1 and 10 times its EC50 concentration. Calibration bars for TEVC traces illustrated in panels A and B: 750 nA, 50 s. times its EC50 value even when TCN 213 was applied at 30 mM. Indeed, we were unable to determine an IC50 value even for the lowest glycine concentration applied (75 nM = 0.1 ¥ EC50). Thus, TCN 213 appears to preferentially block GluN1/GluN2A over GluN1/GluN2B NMDA receptors and inasmuch as our data can be compared with the high- throughput screening assays reported previously, this antago- nist does discriminate effectively between GluN2A- and GluN2B-containing NMDA receptors (Bettini et al., 2010). In direct comparison, inhibition curves for the prototypical glycine site antagonist, 5,7 DCKA, were similar when it was 928 British Journal of Pharmacology (2012) 166 924–937 used to block responses mediated by GluN1/GluN2A and GluN1/GluN2B NMDA receptors (Figure 2C,D). The IC50 values for 5,7 DCKA obtained at each of the glycine concen- trations are reported in Table 1. Schild analysis of TCN 213 antagonism of GluN1/GluN2A NMDA receptor-mediated currents While IC50 values are commonly reported to describe the potency of an antagonist, they suffer from the fact that they are dependent on both the nature and the concentration of