Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10–100 × smaller than in classical biochemical assays. At membrane densities around 25/µm 2 , a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm 2 , single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm 2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.