Binding and internalization of fluorescent opioid peptide conjugates in living cells

The dynamics of agonist-stimulated opioid receptor internalization and trafficking have been difficult to study in living cells in part because the available probes were inadequate. To overcome this obstacle, six new fluorescent opioid peptides were developed. Dermorphin (DERM), deltorphin (DELT), TIPP and endomorphin were conjugated to BODIPY TR or Alexa Fluor 488, two fluorescent dyes with distinct hydrophobic properties. In membrane binding assays the fluorescent conjugates DERM-A488 or -BTR, DELT-A488 or -BTR and TIPP-A488 displayed good binding affinity and selectivity for μ- and δ-opioid receptor subtypes. Furthermore, the fluorescent conjugates of dermorphin and deltorphin were biologically active as demonstrated by their ability to hyperpolarize locus coeruleus neurons (DERM-A488 or -BTR) and inhibit calcium currents in NG108-15 (DELT-A488). Both of these responses were antagonized by naloxone. In conjunction with confocal fluorescent microscopy the trafficking of these fluorescent ligands was monitored in real-time. The internalization of these ligands by μ- and δ-opioid receptors was found to be naloxone-sensitive and temperature-dependent. Interestingly, once these ligands were internalized the fluorescent puncta that formed became distributed in one of two patterns. In Chinese hamster ovary cells heterologously expressing either μ- or δ-opioid receptors the intracellular puncta were concentrated in the perinuclear region of the cell, whereas they were distributed throughout the cytoplasm in cells derived from either NG108-15 or SH-SY5Y cells. In summary, we have demonstrated that these novel, fluorescent opioid peptide conjugates permit real-time visual tracking of receptor-ligand complexes, including their internalization and trafficking, in living cells.