Two-electrode voltage clamp recordings from Xenopus oocytes

Defolliculated stage V-VI Xenopus oocytes were obtained from EcoCyte Bioscience or oocytes in ovaries were obtained from Xenopus 1, and individual oocytes were defolliculated and injected with GluN1 and GluN2A cRNA at a 1:2 ratio (5–10 ng total in 50 nl water), as previously described (Hansen et al., 2013; Ogden and Traynelis, 2013). Following injection, oocytes were maintained for 2–7 d at 15°C in Barth’s culture medium containing 88 mM NaCl, 2.4 mM NaHCO₃, 1 mM KCl, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, and 5 mM HEPES (pH 7.4) and supplemented with 0.1 mg/ml gentamicin sulfate and 1 µg/ml streptomycin. Oocytes were placed in a recording chamber and continuously perfused with a solution containing 90 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.5–1 mM BaCl₂, and 0.01 mM EDTA (pH 7.4). Responses were measured at room temperature (21–23°C) for 0.10–100 µM glutamate in 100 µM glycine, and 0.03–30 µM glycine in 100 µM glutamate. Microelectrodes were fabricated from borosilicate glass (TW150F-4; World Precision Instruments), filled with 0.3–3 M KCl, and currents were recorded under two-electrode voltage clamp at a holding potential of −40 mV (OC-725C; Warner Instruments). Currents were low pass filtered at 10 Hz and digitized at 20 Hz using custom acquisition software (EasyOocyte). Additionally, channel open probability was calculated from the degree of MTS ethylammonium (MTSEA) potentiation. Currents were evoked by 100 µM glutamate and glycine. In the continued presence of agonists, 200 µM MTSEA was applied and the degree of potentiation was determined. The following equation was used to determine open probability (P_Open) as a function of potentiation:

Potentiation is the current after MTSEA treatment divided by the current before treatment. The control and MTSEA chord conductance γ were estimated from GluN1/GluN2A receptors (Yuan et al., 2005).