Single-molecule methods for characterizing receptor dimers reveal metastable opioid receptor homodimers that induce functional modulation

Opioid receptors (ORs) are critical for endogenous and synthetic analgesics. OR homodimerization is considered important for their pharmacological diversity, but whether they form homodimers remains controversial. Here, we establish that the three classical ORs, μ-, κ-, and δ-ORs (MOR, KOR, and DOR, respectively) undergo repeated transient (120-180 ms) homodimerizations every few seconds. This is achieved by using single-molecule imaging and developing theories for analyzing single-molecule colocalization data, which provide key parameters, such as homodimer-monomer dissociation equilibrium constants and rate constants. Their 9-26 amino-acid C-terminal cytoplasmic domains, without sequence similarities, are involved in specific homodimerization, whereas the transmembrane domains provide less specific affinities. Using the membrane-permeable peptides mimicking the C-terminal homodimerization sequences which block homodimerizations, functions of monomers and homodimers were dissected. KOR and DOR homodimers, but not MOR homodimers, activate downstream G-proteins differently from monomers upon agonist addition, without influencing OR internalization. These findings guide strategies to enhance OR-based analgesia. Receptor dimerization is central to many GPCRs signaling, but key rate and equilibrium constants are hard to measure. Here, the authors present single-molecule methods to obtain such constants and reveal transient opioid receptor homodimers modulating function.