2.6. HEK‐293T cells & whole‐cell patch‐clamp electrophysiology

Whole‐cell patch‐clamp electrophysiology studies were carried out in the HEK‐293 cells, expressing recombinant NMDARs that lack native functional NMDARs.40, 41, 42 Equal quantity of (1 µg) cDNA for GluN1a, GluN2 (A or B or C or D) subunits were co‐transfected 24–48 h before patch‐clamp electrophysiology assay. Activation of NMDAR by ambient glutamate from the cell culture media was inhibited (to avoid excitotoxicity) by adding 50 µM memantine into the culture media during transfection.43 Cells were carefully washed before performing experiments and were used for the electrophysiology experiments after 24–48 h incubation at 37°C with 5%CO₂. The whole‐cell patch‐clamp electrophysiology assay was performed using semi‐automated patch‐clamp equipment, Port‐a‐Patch (Nanion Technologies GmbH). Following are the constituents of various solutions used for patch‐clamp electrophysiology: internal solution [(mM) NaCl 10, EGTA 20, CsF 110, HEPES 10], Mg‐free external (recording) solution [(mM) NaCl 140, KCl 4, CaCl₂ 2, HEPES 10, D‐GlucoseMonohydrate 5], and Mg‐free seal enhancer solution [(mM) NaCl 80, KCl 3, CaCl₂ 35, HEPES 10]. Nanion NPC chips with 2–3.5 mOhms resistance were used for the HEK‐293 cell recordings. Agonist concentrations used for this set of experiments are provided in the Figure ​Figure55.

CNS4 modulates GluN1/2A currents in glutamate concentration‐dependent manner. Patch‐clamp electrophysiology assays were performed using HEK293T cells expressing GluN1/2A receptors. Traces represent current responses evoked by 0.3 µM (A, C, gray bar) or 100 µM (B, D, back bar) glutamate and 100 µM glycine as an agonist (Ag). (A, B) 100 µM CNS4 (red) was co‐applied with agonist for 4 s. (C, D) 100 µM CNS4 was applied 4 s after exposure to the respective agonist. Each pair of Ag and +CNS4 application events is shown in dot plots. Histograms show statistical significance. I _max, maximum inducible current by Ag. Steady‐state current values were obtained 4s after Ag application and 4s after +CNS4 application. (A) 0.03 µM Glu, n = 8 recordings from four cells. (B) 100 µM Glu, n = 10 recordings from three cells. (C) 0.3 µM Glu, n = 7 recordings from three cells. (D) 100 µM Glu, n = 14 recordings from four cells. Statistics, Wilcoxon matched‐pairs signed rank test. *p < .05; **p < .01. (E) Y‐axis deactivation time constant (τ) in milliseconds. Ag, 100 µM glutamate + 100 µM glycine; +CNS4, 100 µM CNS4 + Ag. CNS4 significantly increased deactivation time constant (Ag, 710.9 ± 98.27 ms, n = 9 (three cells) vs. Ag + CNS4, 1067 ± 104.3 ms, n = 19 (four cells), *p < .05, unpaired t test). NS, not significant