NMDAR developmental switch monitored by TCN 213 BJP GluN1 was co-expressed with each of the GluN2 NMDA receptor subunits. This interpretation was consistent with our ﬁndings from Schild analysis, which indicated that the KB values for 5,7 DCKA at GluN1/GluN2A and GluN1/GluN2D NMDA receptors were indistinguishable (Chen et al., 2008). Thus, 5,7 DCKA binds to the GluN1 site in GluN1/GluN2A and GluN1/GluN2D NMDA receptors with equal afﬁnity despite the fact there is a 10-fold difference in the potency of glycine at GluN1/GluN2A and GluN1/GluN2D NMDA recep- tors. Nevertheless, two derivatives of 5,7 DCKA do exhibit some subtype selectivity for GluN2A- and GluN2B- containing NMDA receptors. L-689 560 (Hess et al., 1996) shows a modest (5-fold) selectivity for GluN2A- over GluN2B- containing NMDA receptors, whereas CGP-61594 (Honer et al., 1998) is 10-fold more potent at GluN1/GluN2B NMDA receptors compared with GluN1/GluN2A NMDA receptors. Our data for TCN 213 were quite different and suggested a far greater selectivity of TCN 213 than those reported for those antagonists based on the 5,7 DCKA structure. While the data illustrated in Figure 2B do not allow any estimation of an IC50 value for TCN 213 acting at GluN1/GluN2B NMDA receptors, we would estimate that the IC50 value would need to be in excess of 300 mM (for glycine concentration equal to its EC50). This would indicate a selectivity of around 100-fold, which is considerably greater than that reported for antagonists based on the 5,7 DCKA structure. We need also to consider an alternative explanation for the antagonism produced by TCN 213. The study of Bettini et al. (2010) showed that TCN 213 failed to displace the GluN1 ligand MDL 105 519, which can be taken to indicate that TCN 213 does not act competitively (i.e. mutually exclu- sive binding with glycine), but rather that it acts allosteri- cally to modulate glycine binding in a manner which cannot be distinguished by the types of experiments we have carried out in the present study. For example, if TCN 213 caused an increase in the dissociation rate of glycine from its binding site on the GluN1 subunit, then our experiments and the limited concentration range of TCN 213 we have used could not distinguish between this alternative mode of action and competitive, reversible antagonism (SF Traynelis, K Hansen and K Odgen, pers. comm.). Evidence for either a competi- tive antagonist mechanism or an allosteric modulation of glycine (un)binding will be provided from studies examining crystal structures bound with TCN 213 and detailed investi- gations of the kinetics of TCN 213 blocking and unblocking rates. Conclusion While precise details concerning the mode of action of TCN 213 remain, it is clear that this ligand and the related sulpho- namide derivatives (Bettini et al., 2010; Ogden and Traynelis, 2011) offer us new pharmacological tools to probe NMDA receptor function and speciﬁcally afford us the opportunity to antagonize selectively GluN1/GluN2A NMDA receptors. Our study took advantage of this selective antagonism by TCN 213 and allowed us to use a pharmacological approach to monitor directly changes in subunit composition in func- tional NMDA receptors in developing cortical neurones. Acknowledgements This work was supported by the funds from The Wellcome Trust and the Honours Programmes in Pharmacology and Neuroscience at The University of Edinburgh. We thank Matt Livesey for constructive comments on the manuscript and Steve Traynelis, Kasper Hansen and Kevin Ogden (Emory University) for helpful discussions and for sharing unpub- lished data. Conﬂicts of interest The authors state no conﬂict of interest. References Alexander SPH, Mathie A, Peters JA (2011). Guide to Receptors and Channels (GRAC), 5th Edition. Br J Pharmacol 164 (Suppl. 1): S1–S324. Arunlakshana O, Schild HO (1959). Some quantitative uses of drug antagonists. Br J Pharmacol Chemother 14: 48–57. Auberson YP, Allgeier H, Bischoff S, Lingenhoehl K, Moretti R, Schmutz M (2002). 5-Phosphonomethylquinoxalinediones as competitive NMDA receptor antagonists with a preference for the human 1A/2A, rather than 1A/2B receptor composition. Bioorg Med Chem Lett 12: 1099–1102. Bading H, Greenberg ME (1991). 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