Nmdar Labeling and Functional Characterization

HEK293T cells were plated into 30 mm dishes and transfected (jetPrime, PolyPlus) with GluN1*F554C and GluN2A* constructs at a mass ratio of 1.5:4.5 μg per 20 ml of media. 300 μM DL-AP5 (abcam) and 30 μM DCKA (abcam) were present in the media and recordings were performed 24 to 48 hours post-transfection. Prior to recording, cells were incubated in 150 nM Alexa 555 maleimide (ThermoFisher) and 600 nM Alexa 647 maleimide (ThermoFisher) for at least 1 hour to mimic smFRET labelling conditions. Outside-out patches were excised and piezo-driven solution exchange was performed as outlined elsewhere^{51 (link)}. The external solution was (in mM) 150 NaCl, 20 HEPES, 10 Tricine, 1 CaCl₂, and 0.1 glycine, pH 7.4 (NaOH). The pipette solution was 135 CsF, 33 CsOH, 11 EGTA, 10 HEPES, 2 MgCl₂ and 1 CaCl₂, pH 7.4. Lifted whole cell recordings were performed as described elsewhere^{52 (link)} using the same solutions as above with the addition of 2.5 mM KCl to the external solution.