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

Abstract

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 different expression levels. At high membrane densities, a split GFP assay reveals that {kappa}OR dimerizes, while OR and {delta}OR stay monomeric. In contrast, single-molecule imaging showed no {kappa}OR dimers at low receptor densities. To reconcile our seemingly contradictory results, we used a high-density single-molecule assay to assess membrane protein interactions at densities up to 100x higher than conventional single-molecule imaging. We observe that {kappa}OR is monomeric at low densities and forms dimers at densities that are considered physiological. In contrast, OR and {delta}OR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting {kappa}OR dimers but not higher-order aggregates suggests that ORs dimerize through a single, specific interface.