a, Whole-cell patch-clamp recordings examining the inhibitory effect of ambient Zn2+ on peak currents of GluN1–GluN2A receptors without or with 10 mM Tricine. b, Bar graph of the I peak1 /I peak2 (Tricine) ratio. I peak2 (Tricine) represents the peak current measured in the presence of 10 mM Tricine; I peak1 represents the peak current without Tricine. Data are presented as mean ± s.e.m. from n = 6 different cells. c,d, Whole-cell patch-clamp recordings from HEK 293 F cells expressing wild-type GluN1–GluN2A (c) or GluN1–GluN2B (d) receptors. e, Box-and-whisker plot showing the I 7 s / I peak ratio of GluN1–GluN2A and GluN1–GluN2B-mediated currents. I 7 s refers to the current measured 7 s after agonist application, and I peak represents the peak current. Data are presented as mean ± s.e.m from n GluN1–GluN2A-(WT) = 5 different cells, n GluN1–GluN2B-(WT) = 5 different cells. P value was determined by two-tailed unpaired Student’s t-test, p < 0.0001. The boxes represent the median, 25th and 75th percentile values and the whiskers represent the minimum and maximum values. f, Box-and-whisker graph showing the effect of GluN2A(K220C) and GluN2B(K221C) mutations on the desensitization rate. Data are presented as mean ± s.e.m from n GluN1–GluN2A-(WT) = 5, n GluN1–GluN2A-(K220C) = 7, n GluN1–GluN2B-(WT) = 5, n GluN1–GluN2B(K221C) = 6, n represents different cells. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test (two-sided). P values are provided in the figure. The boxes represent the median, 25th and 75th percentile values and the whiskers represent the minimum and maximum values. g, Fluorescence gel of the wild-type and cysteine-substituted GluN1–GluN2A and GluN2B receptors. GFP tag was engineered on the GluN2A or GluN2B. Band of dimeric GluN2A-N2A or GluN2B-N2B is indicated. The experiments were repeated for three times with consistent results. For the gel source data, see Source Data Extended Data Fig. 6. h,i, Whole-cell patch-clamp recordings from HEK 293 F cells expressing GluN1–GluN2A(K220C) (h) or GluN1–GluN2B(K221C) (i) mutants. Right panels show the position of N2A-K220 and N2B-K221 at the interface between two R2 lobes.
Source data