Figure 9.

Representation of the ways in which differences in diffusion coefficients (D values) may be generated for A₃AR-GFP and fluorescent ligand-occupied A₃AR complexes. Pink area represents the confocal volume; blue area at the waist is a representation of the cell membrane within the volume. A) If the ligand stays bound to the receptor while it is present in the confocal volume, then the A₃AR-GFP and A₃AR–ligand complexes will have the same diffusion coefficients. B) If the fluorescent ligand associates with, or dissociates from, the receptor during its time in the measurement volume, then the FCS analysis will conclude that the A₃AR–ligand complex was present for a shorter time, and a faster diffusion coefficient will be calculated compared to that obtained for A₃AR-GFP complexes. C, D) Scenarios for a ligand dissociation experiment where the receptor is prelabeled with fluorescent ligand, and during the washout phase, the ligand dissociates during transit through the volume under control conditions (C) or after the addition of an allosteric regulator (D). In the experiments shown in Figs. 5 and 6, there is a decrease in the number of particles showing the characteristics of panel C and an increase in the number of particles showing the characteristics of panel D. Calculation of the diffusion coefficient is necessary to compare the average dwell times of fluorescent species within the confocal volume because of the different sizes of confocal volume illuminated by different laser wavelengths when focused on a diffraction-limited point (such as was used in the FCS experiments). However, for the purposes of this illustration, we assumed that the confocal volumes were the same for the red- and green-labeled species.