NMDAR developmental switch monitored by TCN 213 BJP Wyllie DJA, Chen PE (2007). Taking the time to study competitive antagonism. Br J Pharmacol 150: 541–551. van Zundert B, Yoshii A, Constantine-Paton M (2004). Receptor compartmentalization and trafﬁcking at glutamate synapses: a developmental proposal. Trends Neurosci 27: 428–437. Supporting information Additional Supporting Information may be found in the online version of this article: Figure S1 Requirement of glycine for NMDA receptor acti- vation and structures of the GluN1 NMDA receptor subunit antagonists, TCN 213 and 5,7 DCKA. (A) TEVC recording from an oocyte expressing recombinant GluN1/GluN2A NMDA receptors and voltage-clamped at -40 mV. Application of glutamate (100 mM) in the absence of glycine (indicated by the grey bar and trace) evokes only a small (<20 nA) inward current. Simultaneous application (indicated by the black bars) of both glutamate and glycine (50 mM) results in a large (2.5 mA) inward current. On average, the mean current evoked by glutamate in the absence of added glycine was 1.2 0.4% (n = 4) of the current recorded when both glutamate and glycine were present. (B) Structure of the novel glycine site antagonist, TCN 213, characterized in this study. (C) Structure of the prototypical glycine site antagonist, 5,7 DCKA. Figure S2 Schild analysis using two-point dose–response curves. (A) Del-Castillo–Katz reaction scheme showing mutu- ally exclusive binding of an agonist, A, and an antagonist, B, to a receptor, R. The active state of the receptor AR* is reached via an intermediate liganded but inactive state AR. The antagonist when bound to R results in the inactive state, BR. Equilibrium constants for agonist and antagonist binding are denoted as KA and KB, respectively, while the equilibrium constant for the isomerization reaction of AR to the AR* state is denoted by E. In the absence of antagonist and using the equilibrium constants as shown, the proportion (pAR*) of E receptors in the AR* state has a maximum value (i.e. ) 1+ E of 0.5 with an EC50 value (i.e. KA ) of 1.5 mM. (B) Series of 1+ E predicted dose-response curves generated in the absence or presence of antagonist for the reaction scheme shown in A. In the presence of the antagonist, the dose–response curves are shifted in a parallel manner to the right and retain the maximum response that is achieved in the absence of the antagonist. In the presence of the antagonist, the EC50 is [B] shifted by a factor of 1 + cB, where cB = . (C) The area KB enclosed by the dashed grey box in panel B is expanded to show the proﬁle of the dose–response curves on a conven- tional semi-log plot (upper graph) and a log–log plot (lower graph). Note the linear transformation of the data when log–log scales are used. Since each of the lines are parallel, dose ratios (r) can easily be obtained by dividing the concen- tration of agonist required to evoke a response in the pres- ence of a given antagonist concentration by the agonist concentration that produces the same response in the absence of antagonist. Thus, only two data points are required to deﬁne each line on the log-log plot when low agonist concentrations are used – this is the basis of our methodology. (D) Schild plot using the dose ratio values obtained from C. The data points are ﬁtted with a straight line with unity slope. The intercept on the abscissa recovers the KB value of 2 mM used in the reaction scheme in panel A. Figure S3 Antagonism of recombinant NMDA receptors at higher glycine concentrations. (A) Upper trace: example of TEVC current trace recorded from an oocyte expressing GluN1/GluN2A NMDA receptors. NMDA receptors were acti- vated by NMDA (50 mM) and glycine (3 mM). Application of TCN 213 (30 mM) results is an approximately 75% reduction in the amplitude of the current response. Lower trace: TEVC current recorded under the same conditions but from an oocyte expressing GluN1/GluN2B NMDA receptors. Note that TCN 213 produces very little block of the NMDA receptor-mediated current and therefore at these concentra- tions of NMDA (50 mM) and glycine (3 mM) discriminates between these two NMDA receptor subtypes. (B) Bar graph illustrating the mean block produced by TCN 213 of GluN1/ GluN2A and GluN1/GluN2B NMDA receptor-mediated cur- rents. On average, GluN1/GluN2A NMDA receptor currents were blocked by 78 4% (n = 5), whereas GluN1/GluN2B NMDA receptor currents were blocked by 2 0.5% (n = 4). Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. British Journal of Pharmacology (2012) 166 924–937 937