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/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, 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/µm2 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.

BIOSS Centre for Biological Signalling Studies University of Freiburg Freiburg Germany

CIBSS Centre for Integrative Biological Signalling Studies University of Freiburg Freiburg Germany

Department of Biochemistry Faculty of Science Charles University Prague Czech Republic

Department of Biomathematics Institute of Physiology Czech Academy of Sciences Prague Czech Republic

Faculty of Biology University of Freiburg Freiburg Germany

Institute of Internal Medicine 4 Medical Center of the University of Freiburg Freiburg Germany

Institute of Pharmaceutical Sciences University of Freiburg Freiburg Germany

Prague Czech Republic

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