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Electrophysiological recordings of diheteromeric and triheteromeric NMDAR-mediated whole-cell currents in HEK-293T cells

Procedure

Electrophysiological recordings were obtained 18 to 24 hours after transfection, perfusing the cells continuously at RT with an extracellular physiological bath solution: 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM glucose, and 10 mM Hepes, adjusted to pH 7.42 with NaOH. Glutamate (1 mM; Sigma-Aldrich), in the presence of glycine (1, 50, or 100 μM depending the experiment type; Tocris) and d-serine (0 to 300 μM) was applied for 0.5 s by piezoelectric translation (P-601.30; Physik Instrumente) of a theta-barrel application tool made from borosilicate glass (1.5 mm outside diameter; Sutter Instruments), and the activated currents were recorded in the whole-cell configuration at a holding potential of −60 mV, acquired at 5 kHz and filtered at 2 kHz by means of Axopatch 200B amplifier, Digidata 1440A interface, and pClamp10 software (Molecular Devices Corporation). Electrodes with open-tip resistances of 2 to 4 megohms were made from borosilicate glass (1.5 mm outside diameter, 0.86 mm outside diameter; Harvard Apparatus), pulled with a PC-10 vertical puller (Narishige), and filled with intracellular pipette solution containing 140 mM CsCl, 5 mM EGTA, 4 mM Na2ATP, 0.1 mM Na3GTP, and 10 mM Hepes, adjusted to pH 7.25 with CsOH. Glutamate and glycine-evoked currents were expressed as current density (in pA/pF; maximum current divided by input capacitance as measured from the amplifier settings) to avoid differences due to surface area in the recorded cells.

The kinetics of deactivation and desensitization of the NMDAR responses were determined by fitting the glutamate/glycine-evoked responses at Vm − 60 mV to a double-exponential function to determine the weighted time constant (τw,des)$τw,des=τf(AfAf+As)+τs(AsAf+As)$where Af and τf are the amplitude and time constant of the fast component of desensitization, respectively, and As and τs are the amplitude and time constant of the slow component of desensitization, respectively.

To infer single-channel conductance values from macroscopic deactivating currents, we used NSFA as previously described (24). The single-channel current (i) was calculated by plotting the ensemble variance against mean current (Ī) and fitting with Sigworth parabolic function (73)$σ2=σB2+(iI‐−(I‐2N))$where σ2B is the background variance and N is the total number of channels contributing to the response. The weighted-mean single-channel conductance was determined from the single-channel current and the holding potential of −60 mV.

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