Ligand Binding Dynamics of Ion Channels and GPCRs Using Single-Molecule Fluorescence

Chemical signaling underlies many biological processes, and membrane receptors such as G protein–coupled receptors and ligand-gated ion channels represent two of the most pharmacologically important protein families. Advances in single-molecule fluorescence techniques have transformed our understanding of molecular mechanisms, including protein folding, transcription, and ligand binding. Unlike ensemble measurements, which average over populations and obscure molecular heterogeneity, single-molecule approaches enable direct observation of individual events, revealing rare conformational states and distinguishing between mechanisms that are indistinguishable at the ensemble level. This review highlights how single-molecule FRET (smFRET) and single-molecule fluorescence ligand binding (smFLiB) provide complementary insights into ligand-dependent receptor activation and allosteric coupling. smFRET offers structural information by tracking conformational transitions, but limited observation times can hinder detection of slow or infrequent events. In contrast, smFLiB allows long-duration monitoring of ligand–receptor interactions throughout the activation pathway, though with less direct information about structural rearrangements. Through selected case studies, we illustrate how these techniques have been applied to dissect the complexity of ligand–receptor interactions with unprecedented resolution. These advances hold promise for guiding the rational design of more selective and effective therapeutics targeting membrane proteins.