Fig. 2: Dynamic OR homodimer-monomer equilibrium constants (KDs) and homodimer formation and dissociation rate constants (kon and koff, respectively), providing the %protomers existing as dimers as a function of the expression level.
a Experimental PCCFs for KOR, MOR, DOR, CD28, and TMLDLR (mean ± SEM, n = 20 cells; the PCCF for KOR is the same as that shown in Fig. 1c) and simulated PCCFs for given KDs based on the theory described in Supplementary Note 2 (the total numbers of fluorescent spots, \({{sN}}_{{\mbox{TA}}}\) and \({{sN}}_{{\mbox{TB}}}\), labeling efficiency, and σ were determined from experiments; mean ± SEM, n = 20 independent simulations). CD28 and TMLDLR were dimer and monomer references, respectively. The black lines indicate the best-fit functions, and their KD values are shown. The PCCFs with KD values of 3 and 30 obtained by simulation (magenta and cyan open bars, respectively) are also shown for comparison. b Simulated data showing the relationship between the colocalization index and KD. Colocalization index decreases monotonically with an increase of KD, if the labeling efficiency, the precision of single-molecule localization plus image overlaying, and the number density of fluorescent molecules in each movie were the same (0.7, 140 nm, and 0.5, 1, or 1.5 fluorescent spots/µm2, respectively). KD can be roughly estimated from the colocalization index using these curves. (mean ± SEM; n = 20 independent simulations) c Predicted percentages of OR protomers existing as dimers, plotted as a function of expression levels (without agonist; see Eq. 79 in Supplementary Note 2). The curves indicate the mathematical functions calculated from the KD values. Tick marks between the exact digits represent ≈3.16x (100.5x) of the smaller digit. In this study, since labeling efficiencies were 66% and 61% for SNAP-Surface 549 and SNAP-CF660, respectively, and the dimer spots generally represents only 5–10% of the spots (because 10-20% of ORs exist as homodimers), the OR expression levels in the observed cells are expected to be approximately 1.8 molecules/µm2. OR expression levels would greatly vary depending on particular PM domains, nerve circuits, and various pathological and pharmacological conditions26,27,28,29,30,31,32. Source data are provided as a Source Data file.
