Two-electrode voltage clamp (TEVC) on Xenopus oocytes.

Female Xenopus laevis were housed and oocytes harvested according to the European Union guidelines (husbandry authorization #C75–05-31; project authorization #05137.02). Xenopus laevis oocytes were harvested, prepared, injected and perfused according to previously published procedures^[30]. Recombinant NMDA receptors were expressed in Xenopus oocytes by co-injection of 32 or 50 nL of a 1:1 mixture of cDNAs coding for the wt GluN1–1a subunit and the GluN2 subunit of interest (at 10 or 30 ng/μL, nuclear injection). Oocytes were then kept at 18 °C in a Barth solution (in mM: 88 NaCl, 1 KCl, 0.33 Ca(NO₃)₂, 0.41 CaCl₂, 0.82 MgSO₄, 2.4 NaHCO₃, 7.5 HEPES, pH adjusted to 7.6 with NaOH) supplemented with gentamicin (50 μg/mL) and D-(−)-2-Amino-5-phosphonopentanoic acid (APV, 50 μM), and recorded 24–72 h after injection.

Data were collected and analyzed using pClamp 10.5 (Molecular Devices) and fitted using Sigmaplot 11.0 (Systat Software Inc). The standard external recording solution used for recordings at pH 7.3 contained (in mM): 100 NaCl, 0.3 BaCl₂, 5 HEPES and 2.5 KOH. The pH was adjusted to 7.3 with HCl. NMDAR-mediated currents were induced by simultaneous application of saturating concentrations of L-glutamate and glycine (100 μM each). UV uncaging was performed by illuminating from the top the oocyte and the perfusion chamber with a 365 nm LED (pE-2, CoolLED, UK) by the means of a liquid light guide. Unless notified, recordings were performed at a holding potential of −60 mV. All experiments were performed at room temperature.

Compounds 1, 2 and 3 were diluted as stock solutions of 20 (1 and 3) or 10 mM (2) in DMSO. The day of the experiment, they were diluted to the appropriate concentration in the recording solution and kept in the dark during the whole duration of the experiment to avoid photolysis or photoconversion. Due to the poor solubility of 2, DMSO was added to Compound 2 solutions to the final concentration of 1 %. Since DMSO itself induced a small inhibition of NMDAR currents, control and agonist solutions were also supplemented with 1 % DMSO. cis-2 solution was obtained by irradiating from the top 25 mL of trans-2 solution with 365 nm light for 20 min in a graduated cylinder covered with aluminum foil. The UV PSS was checked by UV/vis spectroscopy. The cis-2 solution was then kept in the dark during the whole course of the experiment.

Voltage-sensitivity was measured by performing 10 s voltage ramps from −100 to +40 mV. The oocyte membrane was equilibrated during 2 s at −100 mV before performing the voltage ramp. NMDAR I-V curves were obtained by subtracting offline the current measured in absence of agonists (i.e. leak current) to the current measured in presence of agonists (plus or minus Compound 3). Values are shown as mean ± standard error of the mean.