2.3. Two-electrode voltage-clamp (TEVC) current recordings from Xenopus laevis oocytes

Unfertilized Xenopus oocytes (defolliculated stage V-VI) were prepared from commercially available ovaries (Xenopus one Inc, Dexter, MI, USA) (W. XiangWei et al., 2019), and were injected with cRNA encoding either WT or mutant NMDAR subunits, and TEVC current recordings were performed (W. Chen et al., 2017). Briefly, cRNA (GluN1:GluN2 ratio 1:1; 5-10 ng in 50 nl of water) was injected into each oocyte, which was maintained at 15-19 °C in Barth’s culture medium (in mM: 2.4 NaHCO₃, 88 NaCl, 1 KCl, 0.41 CaCl₂, 0.33 Ca(NO₃)₂, 0.82 MgSO₄, 5 HEPES; pH was adjusted to 7.4 with NaOH), plus gentamicin sulfate (0.1 mg/mL) and streptomycin (1 μg/mL). Oocytes were transferred to a recording chamber 2-4 days after injection, and perfused with extracellular oocyte recording solution (in mM: 90 NaCl, 1 KCl, 0.5 BaCl₂, 10 HEPES, 0.01 EDTA, pH 7.4; except no EDTA for experiments measuring Mg²⁺ sensitivity). A computer-controlled 8-modular valve positioner controls the solution exchange (Digital MVP Valve, Hamilton, USA). Electrodes were prepared from borosilicate glass (#TW150F-4, World Precision Instruments, Sarasota, FL, USA) by a glass micropipette puller (dual-stage, PC-10, Narishige, Japan). Current responses were recorded under voltage clamp mode (V_HOLD: −40 mV; unless otherwise stated). The recordings were made by current and voltage electrodes (filled with 0.3M and 3M KCl, respectively), using an amplifier (model OC-725C, Warner Instruments, Hamden, USA). A custom software written in LabWindows/CVI (National Instruments, Austin, TX, USA) was used to low-pass filter (10 Hz) and digitize (20 Hz) current responses. Maximal concentrations of agonists (100 μM glutamate and 100 μM glycine) were used in all Xenopus oocyte recordings unless otherwise stated. The agonist (glutamate and glycine) concentration-response curves were fitted by

where EC₅₀ is the glutamate or glycine concentration that evoked a half-maximal current response and nH is the Hill slope. Mg²⁺ potency (IC₅₀ values) was generated by fitting the concentration-response curves by

where IC₅₀ is the Mg²⁺ concentration that evokes a half-maximal response and minimum is the residual blocking at a saturating concentration (e.g. 1 mM) of Mg²⁺. The voltage dependence and affinity were evaluated by fitting the data generated in the presence of Mg²⁺ with the Woodhull equation (McTague, Howell, Cross, Kurian, & Scheffer, 2016)

where I_UNBLOCKED is the current response at a holding potential in the presence of Mg²⁺, I is the current response in the absence of Mg²⁺ at a given holding potential, [Mg²⁺]_o is the extracellular Mg²⁺ concentration (1000 μM), K_{D,0 mV} is the K_D in the absence of an applied electric field, z is the valence (2 for Mg²⁺), δ is the effective fraction of the electric field at the binding site, V is the voltage, Vrev is the reversal potential, and F, R, and T follow their usual meanings. The Woodhull equation was not adjusted for permeation by the blocking ion. The channel open probability (P_OPEN) was estimated (Yuan, Erreger, Dravid, & Traynelis, 2005) from the degree of current potentiation of NMDARs by MTSEA (2-aminoethyl methanethiosulfonate hydrobromide) (Toronto Research Chemicals, Toronto, Ontario, Canada) according to

where γ is the chord conductance for NMDAR channels before and after MTSEA treatment and Potentiation is the amplitude of current response after MTSEA modification divided by the amplitude of current response before the treatment (Yuan et al., 2005).