Fig. 2: Kinetic modeling of glutamate binding.

a Population FRET contour plots and its corresponding cumulative population histograms (on the right) for receptors treated by 3 µM CGP and 0, 300 nM, and 1 mM glutamate. Each contour plot contains FRET traces from one movie, N number of molecules. b Representative donor (green) and acceptor (red) intensity traces (top) and the corresponding smFRET trace (blue) with 3-state SKM idealization (magenta, bottom) in 3 µM CGP and 300 nM glutamate. Histograms, assigned to high, middle, and low FRET levels, are plotted on the right in black, red, and blue, respectively. c Glutamate binding increases ATD interdimer distance in two discrete steps whose \(\triangle \)FRET values are not significantly different (P = 0.25, two-tailed paired t test, n = 11, SEM error bars). d Transition-density plots from a representative movie show FRET values before (initial) and after (final) each transition (t/s = average number of transitions per second; N_t = total transitions per movie). Dotted circles indicate void at locations of direct transition between high and low FRET levels, indicating extreme rarity, i.e., transitions between high and low FRET go almost exclusively through the intermediate FRET state. e Dwell-time histograms for high, middle, and low FRET states fitted by kinetic model in (f). Total events indicate the total number of dwell times analyzed in all three histograms. f Kinetic scheme of glutamate binding to NMDAR. Resting (R), single-glutamate-binding site occupied (RA), both glutamate-binding sites occupied (RAA). The reaction mechanism illustrates association and dissociation rate constants (in µM⁻¹ s⁻¹ and s⁻¹) that are consistent with two identical, independent binding events and a Kd similar to the activated-state glutamate EC₅₀ dose –response (Fig. 1d).