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Opiate drugs such as morphine exert their pharmacological effects by binding to opioid receptors (ORs), the endogenous ligands for which are opioid peptides. Whether opiate drugs have additional OR-mediated effects that are not produced by their peptide counterparts is not clear. Stoeber et al. now show that OR activation in neurons occurs at both cell-surface and intracellular sites, and that opioids and opiates activate ORs at different intracellular compartments.

Here, the authors developed ‘conformational biosensors’ that bind specifically to the activated conformation of µ-ORs and δ-ORs (MORs and DORs, respectively). These sensors were fused with a fluorescent protein tag to report when and where ligands activated OR, and enabled precise temporal and spatial monitoring of OR conformational changes at both surface and intracellular locations in real time in living cells.

Binding of opioid peptides to MOR and DOR in the plasma membrane (PM) promotes rapid internalization and targeting to endosomes. On application of a peptide agonist to cultured non-neural cells expressing ORs and the OR sensor, fluorescence accumulated steadily at endosomes over 20 minutes. The signal persisted after washout of the OR agonist, but was reversed by application of a membrane-permeant antagonist. Moreover, DOR-mediated inhibition of cyclic AMP (cAMP) accumulation continued after washout of the peptide agonist, suggesting that internalized ORs are activated and continue to participate in cell signalling.

The authors then expressed fluorescently tagged ORs and the OR sensor in primary cultures of striatal medium spiny neurons, which express ORs at the PM, in endosomes, at somatic Golgi apparatus and at dendritic Golgi outposts. Application of a selective MOR peptide agonist led to a relocation of colocalized MORs and OR sensors from the PM to endosomes, suggesting that active-conformation MORs at endosomes have bound ligands. This was rapidly reversed by membrane-permeant MOR antagonist naloxone. Similar results were obtained for DOR-selective activation and for activation by endogenous opioids β-endorphin and met-enkephalin.

By contrast, application of membrane-permeant, non-peptide agonist morphine resulted in OR-sensor recruitment within seconds at somatic Golgi apparatus and dendritic Golgi outposts — much faster than peptide agonist-induced internalization. Crucially, morphine did not appear to activate endosomal ORs and peptide agonists failed to recruit the OR sensor to Golgi. Again, OR-sensor fluorescence could be reversed by application of a membrane-permeant antagonist.

Overall, these findings show that opioid peptides and opiate drugs produce different spatiotemporal patterns of OR activation in striatal neurons.