Selective and Wash-Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single-Molecule Imaging of μ-Opioid...

micro-Opioid receptors (micro-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how micro-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the micro-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single-molecule imaging of micro-ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of micro-ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that micro-ORs interact with each other to form short-lived homodimers on the plasma membrane. This approach provides a new strategy to investigate micro-OR pharmacology at single-molecule level.