Investigation of Inactive-State κ Opioid Receptor Homodimerization via Single-Molecule Microscopy Using New Antagonistic Fluorescent Probes

Opioid
receptors (ORs) are among the best-studied G protein-coupled
receptors due to their involvement in neurological disorders and important
role in pain treatment. Contrary to the classical monomeric model,
indirect evidence suggests that ORs might form dimers, which could
be endowed with a distinct pharmacological profile, and, thus, be
targeted to develop innovative pharmacological therapies. However,
direct evidence for the spontaneous formation of OR dimers in living
cells under physiological conditions is missing. Despite a growing
interest in the κ opioid receptor (KOR), KOR-selective fluorescent
probes are particularly scarce in the literature. Herein, we present
the first set of fluorescent KOR-selective probes with antagonistic
properties. Two of these were employed in single-molecule microscopy
(SMM) experiments to investigate KOR homodimerization, localization,
and trafficking. Our findings indicate that most KORs labeled with
the new fluorescent probes are present as apparently freely diffusing
monomers on the surface of a simple cell model.