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).

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