Pharmacology under the microscope: the use of fluorescence correlation spectroscopy to determine the properties of ligand–receptor complexes

Recent years have revealed a high degree of structural organisation in the way in which cell-surface receptors and their associated signalling complexes interact at a molecular level. Fluorescence-based techniques have been at the forefront of methodologies used to investigate this organisation and dissect the pharmacology of drug-receptor interactions at the single-cell level. One such technique, fluorescence correlation spectroscopy (FCS), in conjunction with a fluorescent ligand or receptor, is capable of providing quantitative information about the number of receptors and their mobilities within small areas of the cell membrane that approach the size of some signalling domains. This article describes the use of FCS to perform subcellular quantitative pharmacology, with particular reference to G-protein-coupled receptors (GPCRs). In conjunction with other forms of fluctuation analysis, such as two-colour cross-correlation FCS and molecular brightness analysis, FCS provides the first opportunity to investigate the domain-specific nature of GPCR pharmacology.