D ap5

HEK-293T and COS-7 cell lines were obtained from the American Type Culture Collection and maintained at 37°C in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin) and D-2-amino-5-phosphonopentanoic acid (D-AP5, Abcam; 0.5–1 mM final concentrations, for HEK-293T and COS-7 cells, respectively) to prevent excitotoxicity. Transient expression of NMDARs in HEK-293T cells was achieved with polyethylenimine (PEI)-based transfection method, and NMDAR-mediated currents were recorded 24 h after transfection. COS-7 cells were transfected with Lipofectamine^TM 2000 (Invitrogen) following the manufacturer’s instructions, and cells were fixed 24 h post-transfection for further immunofluorescence analysis. Cells were transfected with equimolar amounts of GluN1 and GFP-GluN2A/GluN2B subunits (1:1) for immunofluorescence experiments, or with a 1:2 (GluN1:GluN2) ratio for electrophysiological recordings.

Soma voltage-clamp recordings were carried out using a Multiclamp 700B amplifier (Molecular Devices) acquired using a Digidata 1440A (Molecular Devices) and Clampex 10 software (Molecular Devices) recorded at 10 kHz. Patch pipettes (3–5 MΩ) were pulled from borosilicate glass (Sutter Instruments). Neurons were held at −70 mV while series resistance was compensated, and recordings were discarded if the access resistance rose above 15 MΩ by the end of the recording. Tetrodotoxin (TTX, 1 mM, Abcam), D-AP5 (50 mM, Abcam), and picrotoxin (100 mM, Abcam) were included in the bath solution to isolate miniature excitatory events. Recordings of mouse hippocampal neurons at 14–16 DIV were carried out at 34°C in a bath solution containing: 128 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 30 mM glucose, and 25 mM HEPES, pH 7.3 at 305 mOsm. Internal solution contained: 130 mM K-Gluconate, 1 mM EGTA, 10 mM HEPES, 2 mM ATP, 0.3 mM GTP, and 5 mM NA₂phosphocreatine, pH 7.35 at 275 mOsm. Recorded traces were analyzed using Clampfit 10 (Molecular Devices) and down sampled for figure presentation to 2 kHz using Axograph X 1.7.2 (Axograph Scientific).

All drugs used in electrophysiology and imaging experiments were diluted to working concentration in SIF and bath applied. D-AP5, CGP 55845, DNQX, GABAzine (SR 95531), NMDA and gliclazide were purchased from Abcam (Cambridge, MA, USA). Glibenclamide, TFB-TBOA and DL-Dithiothreitol were purchased from Tocris Bioscience (Bristol, UK). Catalase (polyethylene glycol-catalase), aldrithiol-4 and MCS were purchased from Sigma-Aldrich (St. Louis, MO, USA).

mIPSCs were recorded using a Multiclamp 700B amplifier (Molecular Devices) and analyzed as previously described (46 (link)). Briefly, syt1 KO hippocampal neurons expressing WT, Juxta K, F349A, or Juxta K + F349A at day in vitro (DIV) 14 through 19 were transferred to a recording chamber with a bath solution containing the following (in mM): 128 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 30 D-glucose, 25 Hepes, and 1 μM tetrodotoxin, pH 7.3 (305 mOsm). Borosilicate glass pipettes (Sutter Instruments) were pulled by a dual-stage glass micropipette puller (Narishige) and filled with an internal solution containing (in mM) 130 KCl, 1 EGTA, 10 Hepes, 2 ATP, 0.3 GTP, 5 QX-314 (Abcam), and 5 sodium phosphocreatine, pH 7.35 (295 mOsm). mIPSCs were pharmacologically isolated by bath applying D-AP5 (50 µM, Abcam) and cyanquixaline (20 µM, Abcam) and acquired using a Digidata 1440B analog-to-digital converter (Molecular Devices) and Clampex 10 software (Molecular Devices) at 10 kHz. Neurons were held at −70 mV. All cells were equilibrated for ∼1 min after break in before recordings started. Series resistance was compensated, and traces were discarded if the access resistance exceeded 15 ΜΩ for the entire duration. The collected miniature events were detected in Clampfit 11.1 (Molecular Devices) using a template matching search.

Picrotoxin was purchased from Abcam Biochemicals (Cambridge, UK) and prepared in DMSO for use at a final concentration of 100 μM. BRS–015 was prepared in DMSO at a stock concentration of 50 mM. DCG–IV, CGP–52432, and NBQX were purchased from Tocris Bioscience (Bristol, UK). l–glutamic acid, d–AP5 and GYKI 53655 were purchased from Abcam Biochemicals (Cambridge, UK). All other chemicals used for the synthesis of BRS–015 can be sourced in Szulc et al. (2015) .
The following is the supplementary data related to this article.Supplementary Scheme 1

Synthesis of BRS–015. N–Boc–phenylalanine methyl ester was N–methylated and deprotected to generate N–methylphenylalanine methyl ester (2) in 79% yield over two steps. Dehydration using tert–butyl hypochlorite and acylation with diacetoxyacetyl chloride (5) followed by cyclisation in neat boron trifluoride diethyl etherate gave BRS–015 in 72% yield.

Supplementary Scheme 1