Studying GPCR Pharmacology in Membrane Microdomains: Fluorescence Correlation Spectroscopy Comes of Age.

G protein-coupled receptors (GPCRs) are organised within the cell membrane into highly ordered macromolecular complexes along with other receptors and signalling proteins. Understanding how heterogeneity in these complexes affects the pharmacology and functional response of these receptors is crucial for developing new and more selective ligands. Fluorescence correlation spectroscopy (FCS) and related techniques such as photon counting histogram (PCH) analysis and image-based FCS can be used to interrogate the properties of GPCRs in these membrane microdomains, as well as their interaction with fluorescent ligands. FCS analyses fluorescence fluctuations within a small-defined excitation volume to yield information about their movement, concentration and molecular brightness (aggregation). These techniques can be used on live cells with single-molecule sensitivity and high spatial resolution. Once the preserve of specialist equipment, FCS techniques can now be applied using standard confocal microscopes. This review describes how FCS and related techniques have revealed novel insights into GPCR biology.