Abstract
Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) were applied to determine diffusion rate and oligomeric size of G‐protein‐coupled receptors at the single molecule level. FCS was used to monitor fluctuations in fluorescence intensity arising from fluorescent‐tagged serotonin 5‐ HT2C receptors freely diffusing within the plasma membrane of HEK293 cells and primary hippocampal neurons. FCS reported diffusion rates of 0.9μm2/s and 1.3μm2/s for GFP‐ and YFP‐tagged receptors, respectively. Since the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins within a cluster it can be used to determine the oligomer state of a protein complex. PCH analysis of fluorescent‐tagged 5‐HT2C receptors provided molecular brightness values that were approximately twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5‐HT. Bimolecular fluorescence complementation (BiFC) of the N‐ and C‐terminal halves of YFP, attached to 5‐HT2C receptors, was observed on the plasma membrane with a brightness equal to the monomeric YFP control. Co‐expression of GFP‐tagged 5‐HT2C receptors with a large excess of non‐tagged 5‐HT2C receptors decreased the brightness by half. These results indicate that the mobile fraction of 5‐HT2C receptors in the plasma membrane is dimeric. Supported by NIH‐ MH86796.
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