Bhattacharya S , Khatri A , Swanger SA , DiRaddo JO , Yi F , Hansen KB , Yuan H , Traynelis SF .

R01 NS097536 NINDS NIH HHS , F32 NS078873 NINDS NIH HHS , R37 NS036654 NINDS NIH HHS , R01 NS036654 NINDS NIH HHS , R01 HD082373 NICHD NIH HHS , R01 NS065371 NINDS NIH HHS , P20 GM103546 NIGMS NIH HHS

	Figure 1. PYD-106 Does Not Potentiate NMDA-Evoked Currents in Wild-Type Mouse Granule Cells (A) Left: acute slice of cerebellum maintained in artificial cerebro-spinal fluid (ACSF) (scale bar, 0.5 mm). The inset shows a photomicrograph of granule cells (scale bar, 30 μm). Arrows indicate granule cells. Right: cerebellar architecture with the molecular layer (ML), the Purkinje cell layer (PCL), and the granule cell layer (GCL). The dashed circle indicates a granule cell (GC). (B) Representative current responses to a pulse of NMDA with 30 μM DCS, DCS plus 50 μM PYD-106 (PYD), DCS wash before CIQ application, and DCS plus 20 μM CIQ in the bath. (C) The mean ± SEM current response amplitudes in PYD-106, CIQ, and 200 μM DL-APV (APV) were expressed as the percentage of the mean response in DCS. (D) Left: amplitude for DCS and PYD-106 current responses to NMDA (n = 8 slices, p = 0.01). Center: response amplitude in DCS before CIQ application and CIQ responses (n = 6 slices, p = 0.03). Right: comparison of NMDA responses in PYD-106 and CIQ (the horizontal line is the median, the box is the 25–75 percentile, the whiskers are the 5–95 percentile). (E) Representative current responses to NMDA and glycine in Grin2A−/− and Grin2C−/− slices for control and 50 μM PYD-106. (F) The mean ± SEM current responses in PYD-106 and 200 μM DL-APV were expressed as percent of control. (G) Left: individual responses for control and PYD-106 in Grin2A−/− slices (n = 8, p = 0.0078). Center: response amplitude for control and PYD-106 in Grin2C−/− slices (n = 8, p = 0.058). Right: PYD-106 current responses as box and whisker plots. The broken line in (D) and (G) shows 100%. Wilcoxon matched pair signed-rank test was used for each experiment to compare drug to control.
	Figure 2. PYD-106 Does Not Potentiate NMDA and Glycine-Evoked Currents in Cultured Cortical Neurons (A and D) NMDAR current responses were evoked by pressure-applied NMDA and glycine (1 mM and 0.5 mM) in cultured cortical neurons transfected with GFP or GluN2C and GFP. Current responses were recorded by whole-cell voltage-clamp recording in the presence of 50 μM PYD-106, 10 μM CIQ, 3 μM ifenprodil, or 400 μM DL-APV. (B and C) Comparison of NMDAR responses for control versus PYD-106 followed by CIQ in neurons transfected with GluN2C and GFP (B) or GFP alone (C). (E and F) Comparison of NMDAR responses for control and PYD-106 followed by CIQ in the presence of ifenprodil in neurons transfected with GluN2C and GFP (E) or GFP alone (F). The experiment was terminated in DL-APV and ifenprodil. (B, C, E, and F) Responses from the experiments are shown with the horizontal lines as the median, boxes showing the 25–75 percentile, and whiskers showing the 5–95 percentile.
	Figure 3. Pharmacological Profile of Triheteromeric GluN1/GluN2A/GluN2C Receptors (A–D) Glutamate (A), glycine (B), and DCS (C) concentration-response data for GluN1/GluN2AC1/GluN2CC2 (2AC1/2CC2), GluN1/GluN2AC1/GluN2AC2 (2AC1/2AC2), and GluN1/GluN2CC1/GluN2CC2 (2CC1/2CC2) receptors in Xenopus oocytes. The DCS maximal response at 1,000 μM was compared to that of the glycine response at 30 μM in the presence of 100 μM glutamate for triheteromeric receptors and wild-type GluN1/GluN2A (2A WT) and GluN1/GluN2C (2C WT) receptors (D). The statistical evaluation is given in Table S2. (E and F) Concentration-response curves for Zn2+ at pH 6.8 (E) and Mg2+ (F). (G and H) Current-voltage relationships of NMDAR responses in different concentrations of extracellular Mg2+ (G: 1 mM and H: 0.3 mM; Table S3). Data are expressed as mean ± SEM.
	Figure 4. Glutamate Deactivation Time Courses of Diheteromeric and Triheteromeric NMDARs (A) Normalized representative current responses to brief (<5 ms) application of 1 mM glutamate in the continuous presence of 100 μM glycine from outside-out patches excised from oocytes expressing of GluN1/GluN2A/GluN2A (blue), GluN1/GluN2AC1/GluN2CC2 (red), or GluN1/GluN2C/GluN2C (green). (B) Deactivation time constants for GluN1/GluN2A/GluN2A and GluN1/GluN2C/GluN2C receptors were obtained by fitting the time course with a single exponential function. Deactivation time constants for GluN1/GluN2AC1/GluN2CC2 receptors were obtained by fitting the response time course with the sum of three exponential functions. (C) The total response from GluN1/GluN2AC1/GluN2CC2 receptors is shown with the GluN1/GluN2A/GluN2A and GluN1/GluN2C/GluN2C receptor components, which were simulated using the deactivation time constant determined from oocytes expressing GluN1/GluN2A/GluN2A and GluN1/GluN2C/GluN2C receptors. The amplitudes of the diheteromeric contribution to the time course were set as the escape current measured from GluN1/GluN2AC1/GluN2AC1 and GluN1/GluN2CC2/GluN2CC2 receptors. The GluN1/GluN2AC1/GluN2CC2 receptor component was isolated by subtracting the GluN1/GluN2A/GluN2A and GluN1/GluN2C/GluN2C receptor components from the total current. (D) Summary of deactivation time constants for the GluN1/GluN2AC1/GluN2CC2 receptor component determined from 4 different batches of oocytes.
	Figure 5. Representative Data for Single-Channel Recordings from Outside-Out Patches (A–C) Unitary currents recorded at −80 mV are shown on a compressed and expanded scale for (A) GluN1/GluN2AC1/GluN2AC2, (B) GluN1/GluN2CC1/GluN2CC2, and (C) GluN1/GluN2AC1/GluN2CC2. Openings of diheteromeric GluN1/GluN2AC1/GluNAC2 and triheteromeric GluN1/GluN2AC1/GluN2CC2 NMDARs channels are clustered into bursts separated by inactive periods. In contrast, openings of diheteromeric GluN1/GluN2CC1/GluN2CC2 NMDARs show no burst structure. (D) Representative open duration histogram from a one-channel outside-out patch for GluN1/GluN2AC1/GluN2AC2 (31,655 events), GluN1/GluN2CC1/GluN2CC2 (3,004 events), and GluN1/GluN2AC1/GluN2CC2 (2,737 events) NMDARs fitted by three exponential components. Also shown are representative shut duration histograms from GluN1/GluN2AC1/GluN2AC2, GluN1/GluN2CC1/GluN2CC2, and GluN1/GluN2AC1/GluN2CC2 NMDARs from single-patch recordings fitted with six exponential components.
	Figure 6. An Expanded View of Single-Channel Recordings Shows Multiple Conductance Levels (A–C) Unitary currents recorded from (A) GluN1/GluN2AC1/GluN2AC2, (B) GluN1/GluN2AC1/GluN2CC2, and (C) GluN1/GluN2CC1/GluN2CC2 NMDARs illustrate multiple conductance levels. Direct sublevel transitions are indicated by asterisks. (D) Fitted amplitude histograms from single-channel analysis of a representative patch with a GluN1/GluN2AC1/GluN2AC2 (A/A, black line, left axis), GluN1/GluN2AC1/GluN2CC2 (A/C, gray, broken line, right axis), or GluN1/GluN2CC1/GluN2CC2 receptor (C/C, black line, right axis) were fitted by the sum of 2–3 Gaussian distributions (smooth lines). (E) All contiguous openings with a direct transition between two sublevels with different amplitudes and durations greater than 2.5 filter rise times were identified from the idealized record, and a scatterplot was made of the first initial amplitude and the second amplitude. The critical amplitude (ACRIT) values (broken lines) were calculated as described in the STAR Methods to determine which conductance level each amplitude was assigned. The relative proportion of all direct transitions (determined by ACRIT) is given for all possible transitions between sublevels in all patches. When multiple direct transitions occurred, only the first two were analyzed.
	Figure S1 (Related to Figure 1). Concentration-response curves for potentiation by PYD-106 in presence of 100 M glutamate and 30 M glycine for GluN1/GluN2BC1/GluN2BC2, GluN1/GluN2CC1/GluN2CC2, GluN1/GluN2BC1/GluN2CC2 and GluN1/GluN2BC1/GluN2CC2(NK). PYD-106 only showed potentiation of GluN1/GluN2CC1/GluN2CC2 receptors (data are mean±SEM; n=5-8 oocytes from 2 frogs).
	Figure S2 (Related to Figure 2). A. 100 M glutamate and 30 M glycine evoked current responses recorded from cultured cortical neurons. 100 M spermine is co-applied with glutamate and glycine to evaluate the presence of alternatively spliced GluN1 exon5. All experiments were terminated with DL-APV. Because spermine shows voltage-dependent block of the channel at high concentrations, outward currents were recorded at VHOLD of +40 mV. B. Current response amplitudes are shown from three cells in the absence and presence of spermine.
	Figure S3 (Related to Figure 3). The graph shows the escape current for GluN1/GluN2AC1/GluN2AC1 from co-expression of GluN1/GluN2AC1/GluN2CC2(RK+TI), and for GluN1/GluN2CC2/GluN2CC2 from co-expression of GluN1/GluN2AC1(RK+TI)/GluN2CC2. Escape currents are also shown for GluN1/GluN2AC1(RK+TI)/GluN2CC2(NK) and GluN1/GluN2AC1/GluN2CC2(NK,RK+TI) in the presences of 1 mM Mg2+. Each response is expressed as percentage of the GluN1/GluN2AC1/GluN2CC2 current on days 1-4 post injection (D1-D4). Data are mean±SEM from 3 separate experiments (n=12-16 oocytes from 3 different frogs).
	Figure S4 (Related to Figure 5 and 6). A, B, C, D. Representative currents for di- and triheteromeric NMDARs after 0.2 mM MTSEA (thick black bar) co-application with glutamate and glycine (100 μM and 30 μM respectively; thin gray bar, Glu/Gly). MTSEA covalently binds to the cysteine residue introduced at GluN1-A652C, which locks the channel open (Wood et al., 1995; Yuan et al., 2005). This increases the amplitude of MTSEA potentiation in a manner inversely proportional to the open probability of the receptor (Yuan et al., 2005). E. The mean±SEM open probability (Popen) is shown for GluN1-A652C (denoted N1A7C, marking the position of the alanine residue) co-expressed with GluN2AC1/GluN2AC2, GluN2AC1/GluN2CC2, GluN2AC1/GluN2CC2(NK), GluN2CC1/GluN2CC2 NMDARs and wild type GluN2A or GluN2C (***=p<0.001, statistical significance compared against GluN1-A652C/GluN2AC1/GluN2AC2; see Table S4).
	Figure S5 (Related to Figure 3). A, B, C. Pharmacological profile of triheteromeric GluN1/GluN2A/GluN2C receptors: (+)-CIQ, PYD-106 and TCN-201 concentration-response data for GluN1/GluN2AC1/GluN2CC2 (2AC1/2CC2), GluN1/GluN2AC1/GluN2AC2 (2AC1/2AC2), and GluN1/GluN2CC1/GluN2CC2 (2CC1/2CC2) receptors in expressed Xenopus oocytes. Data are mean ±SEM (see Table S1).

Akazawa, Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats. 1994, Pubmed

Akazawa, Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats. 1994, Pubmed
Akbarian, Selective alterations in gene expression for NMDA receptor subunits in prefrontal cortex of schizophrenics. 1996, Pubmed
Al-Ghoul, Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in vasopressin and oxytocin neuroendocrine cells. 1997, Pubmed
Al-Hallaq, NMDA di-heteromeric receptor populations and associated proteins in rat hippocampus. 2007, Pubmed
Allgaier, Single-cell RT-PCR analysis of N-methyl-D-aspartate receptor subunit expression in rat locus coeruleus neurones. 2001, Pubmed
Arai, Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering. 2004, Pubmed
Benveniste, Kinetic analysis of antagonist action at N-methyl-D-aspartic acid receptors. Two binding sites each for glutamate and glycine. 1991, Pubmed
Brock, Assembly-dependent surface targeting of the heterodimeric GABAB Receptor is controlled by COPI but not 14-3-3. 2005, Pubmed
Buller, The molecular basis of NMDA receptor subtypes: native receptor diversity is predicted by subunit composition. 1994, Pubmed , Xenbase
Cai, Deficiency of sorting nexin 27 (SNX27) leads to growth retardation and elevated levels of N-methyl-D-aspartate receptor 2C (NR2C). 2011, Pubmed
Cathala, Developmental profile of the changing properties of NMDA receptors at cerebellar mossy fiber-granule cell synapses. 2000, Pubmed
Chazot, Molecular characterization of N-methyl-D-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule. 1994, Pubmed
Cheffings, Single channel analysis of a novel NMDA channel from Xenopus oocytes expressing recombinant NR1a, NR2A and NR2D subunits. 2000, Pubmed , Xenbase
Chen, Growth factor-dependent trafficking of cerebellar NMDA receptors via protein kinase B/Akt phosphorylation of NR2C. 2009, Pubmed
Choi, Identification and mechanism of action of two histidine residues underlying high-affinity Zn2+ inhibition of the NMDA receptor. 1999, Pubmed , Xenbase
Chung, Identification of Novel 14-3-3 Residues That Are Critical for Isoform-specific Interaction with GluN2C to Regulate N-Methyl-D-aspartate (NMDA) Receptor Trafficking. 2015, Pubmed
Clapham, Substance P reduces acetylcholine-induced currents in isolated bovine chromaffin cells. 1984, Pubmed
Clark, Stoichiometry of N-methyl-D-aspartate receptors within the suprachiasmatic nucleus. 2010, Pubmed
Clements, Activation kinetics reveal the number of glutamate and glycine binding sites on the N-methyl-D-aspartate receptor. 1991, Pubmed
Colquhoun, Stochastic properties of ion channel openings and bursts in a membrane patch that contains two channels: evidence concerning the number of channels present when a record containing only single openings is observed. 1990, Pubmed
Cull-Candy, NMDA receptor subunits: diversity, development and disease. 2001, Pubmed
Cull-Candy, Noise and single channels activated by excitatory amino acids in rat cerebellar granule neurones. 1988, Pubmed
Daw, The role of NMDA receptors in information processing. 1993, Pubmed
Dravid, Activation of recombinant NR1/NR2C NMDA receptors. 2008, Pubmed
Dravid, Structural determinants of D-cycloserine efficacy at the NR1/NR2C NMDA receptors. 2010, Pubmed , Xenbase
Erreger, Zinc inhibition of rat NR1/NR2A N-methyl-D-aspartate receptors. 2008, Pubmed
Farrant, NMDA-receptor channel diversity in the developing cerebellum. 1994, Pubmed
Gray, Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo. 2011, Pubmed
Guilarte, Biochemical evidence of an interaction of lead at the zinc allosteric sites of the NMDA receptor complex: effects of neuronal development. 1995, Pubmed
Guthmann, Expression of N-methyl-D-aspartate receptor subunits in the rat parabrachial and Kölliker-Fuse nuclei and in selected pontomedullary brainstem nuclei. 1999, Pubmed
Guzman, Robust pacemaking in substantia nigra dopaminergic neurons. 2009, Pubmed
Hansen, Structural determinants of agonist efficacy at the glutamate binding site of N-methyl-D-aspartate receptors. 2013, Pubmed , Xenbase
Hansen, Distinct functional and pharmacological properties of Triheteromeric GluN1/GluN2A/GluN2B NMDA receptors. 2014, Pubmed , Xenbase
Hatton, Modulation of triheteromeric NMDA receptors by N-terminal domain ligands. 2005, Pubmed , Xenbase
Hood, D-cycloserine: a ligand for the N-methyl-D-aspartate coupled glycine receptor has partial agonist characteristics. 1989, Pubmed
Howe, Currents through single glutamate receptor channels in outside-out patches from rat cerebellar granule cells. 1991, Pubmed
Howe, On the kinetics of large-conductance glutamate-receptor ion channels in rat cerebellar granule neurons. 1988, Pubmed
Iijima, Dual regulation of NR2B and NR2C expression by NMDA receptor activation in mouse cerebellar granule cell cultures. 2008, Pubmed
Javitt, Recent advances in the phencyclidine model of schizophrenia. 1991, Pubmed
Jin, Structural basis for partial agonist action at ionotropic glutamate receptors. 2003, Pubmed , Xenbase
Kadotani, Motor discoordination results from combined gene disruption of the NMDA receptor NR2A and NR2C subunits, but not from single disruption of the NR2A or NR2C subunit. 1996, Pubmed
Kaiser, The Bioactive Protein-Ligand Conformation of GluN2C-Selective Positive Allosteric Modulators Bound to the NMDA Receptor. 2018, Pubmed , Xenbase
Kammerer, Heterodimerization of a functional GABAB receptor is mediated by parallel coiled-coil alpha-helices. 1999, Pubmed
Karavanova, Novel regional and developmental NMDA receptor expression patterns uncovered in NR2C subunit-beta-galactosidase knock-in mice. 2007, Pubmed
Kentros, Abolition of long-term stability of new hippocampal place cell maps by NMDA receptor blockade. 1998, Pubmed
Khatri, Structural determinants and mechanism of action of a GluN2C-selective NMDA receptor positive allosteric modulator. 2014, Pubmed , Xenbase
Lagrèze, N-methyl-D-aspartate receptor subunit mRNA expression in human retinal ganglion cells. 2000, Pubmed
Landwehrmeyer, NMDA receptor subunit mRNA expression by projection neurons and interneurons in rat striatum. 1995, Pubmed
Lee, Astrocytic control of synaptic NMDA receptors. 2007, Pubmed
Lichnerova, Distinct regions within the GluN2C subunit regulate the surface delivery of NMDA receptors. 2014, Pubmed
Lisman, Long-term potentiation: outstanding questions and attempted synthesis. 2003, Pubmed
Low, Molecular determinants of coordinated proton and zinc inhibition of N-methyl-D-aspartate NR1/NR2A receptors. 2000, Pubmed , Xenbase
Lu, NMDA receptor subtypes at autaptic synapses of cerebellar granule neurons. 2006, Pubmed
Luo, The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B). 1997, Pubmed
Magleby, Burst kinetics of single calcium-activated potassium channels in cultured rat muscle. 1983, Pubmed
Mizuta, Developmental expression of NMDA receptor subunits and the emergence of glutamate neurotoxicity in primary cultures of murine cerebral cortical neurons. 1998, Pubmed
Monyer, Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. 1994, Pubmed
Monyer, Heteromeric NMDA receptors: molecular and functional distinction of subtypes. 1992, Pubmed
Mullasseril, A subunit-selective potentiator of NR2C- and NR2D-containing NMDA receptors. 2010, Pubmed
O'Hara, GABAA, GABAC, and NMDA receptor subunit expression in the suprachiasmatic nucleus and other brain regions. 1995, Pubmed
Paoletti, High-affinity zinc inhibition of NMDA NR1-NR2A receptors. 1997, Pubmed , Xenbase
Paoletti, NMDA receptor subunits: function and pharmacology. 2007, Pubmed
Paoletti, Molecular organization of a zinc binding n-terminal modulatory domain in a NMDA receptor subunit. 2000, Pubmed , Xenbase
Rauner, Triheteromeric NR1/NR2A/NR2B receptors constitute the major N-methyl-D-aspartate receptor population in adult hippocampal synapses. 2011, Pubmed
Romero-Hernandez, Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal Domain. 2016, Pubmed , Xenbase
Rosenmund, The tetrameric structure of a glutamate receptor channel. 1998, Pubmed
Sheinin, Subunit specificity and mechanism of action of NMDA partial agonist D-cycloserine. 2001, Pubmed , Xenbase
Sheng, Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. 1994, Pubmed
Smart, Zn2+ ions: modulators of excitatory and inhibitory synaptic activity. 2004, Pubmed
Soares, Differential subcellular targeting of glutamate receptor subtypes during homeostatic synaptic plasticity. 2013, Pubmed
Standaert, Expression of NMDA glutamate receptor subunit mRNAs in neurochemically identified projection and interneurons in the striatum of the rat. 1999, Pubmed
Stern, Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors. 1992, Pubmed , Xenbase
Stroebel, Controlling NMDA receptor subunit composition using ectopic retention signals. 2014, Pubmed , Xenbase
Sun, Allosteric Interactions between NMDA Receptor Subunits Shape the Developmental Shift in Channel Properties. 2017, Pubmed , Xenbase
Sun, Expression of NR1, NR2A-D, and NR3 subunits of the NMDA receptor in the cerebral cortex and olfactory bulb of adult rat. 2000, Pubmed
Sundström, Analysis of NMDA receptors in the human spinal cord. 1997, Pubmed
Takahashi, Functional correlation of NMDA receptor epsilon subunits expression with the properties of single-channel and synaptic currents in the developing cerebellum. 1996, Pubmed
Tovar, Triheteromeric NMDA receptors at hippocampal synapses. 2013, Pubmed
Tovar, The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. 1999, Pubmed
Traynelis, Control of voltage-independent zinc inhibition of NMDA receptors by the NR1 subunit. 1998, Pubmed , Xenbase
Traynelis, Glutamate receptor ion channels: structure, regulation, and function. 2010, Pubmed
Tsien, The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. 1996, Pubmed
Tu, DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke. 2010, Pubmed
Ueno, Mossy fiber Zn2+ spillover modulates heterosynaptic N-methyl-D-aspartate receptor activity in hippocampal CA3 circuits. 2002, Pubmed
Vicini, Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors. 1998, Pubmed
Wafford, Preferential co-assembly of recombinant NMDA receptors composed of three different subunits. 1993, Pubmed , Xenbase
Watanabe, Developmental changes in distribution of NMDA receptor channel subunit mRNAs. 1992, Pubmed
Watson, D-cycloserine acts as a partial agonist at the glycine modulatory site of the NMDA receptor expressed in Xenopus oocytes. 1990, Pubmed , Xenbase
Wenzel, NMDA receptor heterogeneity during postnatal development of the rat brain: differential expression of the NR2A, NR2B, and NR2C subunit proteins. 1997, Pubmed
Westbrook, Micromolar concentrations of Zn2+ antagonize NMDA and GABA responses of hippocampal neurons. , Pubmed
Williams, Separating dual effects of zinc at recombinant N-methyl-D-aspartate receptors. 1996, Pubmed , Xenbase
Wood, Structural conservation of ion conduction pathways in K channels and glutamate receptors. 1995, Pubmed , Xenbase
Yuan, Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3. 2005, Pubmed
Zhang, Spermine potentiation of recombinant N-methyl-D-aspartate receptors is affected by subunit composition. 1994, Pubmed , Xenbase