Fig 2: Protein levels of NR1 and NR2A-D in three salivary glands and cerebral cortex in rat. (A) Typical Simple Western image of NR1, NR2A-D and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Values represent mean ± SD in 5 rats. (B) Values were normalized to housekeeping genes GAPDH. Significantly different from cerebral cortex according to Dunn’s post hoc test following Kruskal–Wallis test; * p < 0.05, ** p < 0.01, and *** p < 0.001. PG, parotid gland; SMG, submandibular gland; SLG, sublingual gland; Cx, cerebral cortex. ND; not detected.

Fig 3: mRNA levels of NR1 (a), NR2A (b), NR2B (c), NR2C (d), and NR2D (e) in mixture of three salivary glands and whole brain of rat. Values represent mean ± SD in 5 rats. Significantly different according to Mann–Whitney test between two groups; ** p < 0.01, and *** p < 0.001.

Fig 4: Both GluN2A- and GluN2B-containing NMDARs can functionally couple to BK channels. (A) Representative confocal microscopy images of PLA experiments in HEK293 cells expressing the protein combinations indicated at the top of the images. Each row corresponds to an imaging channel (Top, DAPI, blue; Middle, NMDAR, green; Bottom, PLA, red). (B) Average PLA signals/µm2 for all conditions shown in A. Data points represent individual measurements. Four independent experiments were performed for each condition. Black lines represent mean ± SEM, ***P < 0.001 versus untransfected cells (UNTRANSF), BK alone (BK), and GluN1 alone (GluN1). (C) Normalized G–V relationships obtained from excised inside-out patches expressing BK channels alone (n = 12) in the presence of different intracellular Ca2+ concentrations in symmetrical K+ solutions. Data points represent mean ± SEM. Lines represent the best fit of a Boltzmann equation to the data. Ca2+ concentrations are color-coded as indicated in the legend. (D) Normalized G–V relationships from excised inside-out patches co-expressing BK channels with GluN1/GluN2A (Left; n = 6) or GluN1/GluN2B (Right; n = 5) in the absence of intracellular Ca2+, with 200 µM NMDA/10 µM glycine in the pipette. Data points represent mean ± SEM. Lines represent the best fit of a Boltzmann equation to the data. (E) Vhalf values obtained from experiments depicted in C and D. Data points represent individual measurements, and lines represent mean ± SEM. (F) Representative current traces recorded in physiological Na+ solutions from excised inside-out patches containing BK alone (Left), BK+GluN1/GluN2A (Middle), and BK+GluN1/GluN2B (Right). (Scale bars: 30 ms and 2,000 pA.) Currents recorded at +140 mV are highlighted in red. (G) Normalized G–V relationships from excised inside-out patches containing BK and GluN1/GluN2A (Left, red; n = 6) or GluN1/GluN2B (Right, blue; n = 5) in solutions containing physiological concentrations of Na+ (see Methods). Black traces in both graphs correspond to G–V curves for BK channels expressed alone (n = 11). Data points represent mean ± SEM. Lines represent the best fit of a Boltzmann equation to the data. (H) Summary of Vhalf values from experiments in G. Data points represent individual measurements and lines represent mean ± SEM, ***P < 0.001 versus BK alone.