Fig. 1.

6′-GNTI specifically targets KOP/DOP heterodimers. (a) Coexpression of opioid receptor types in HEK-293 cells was visualized by immunofluorescent staining of cells stably coexpressing HA-DOP-R (DOP) and FLAG-KOP-R (KOP) with antibodies directed to the respective epitope tags. (Scale bar = 10 μm.) (b) Ratio of opioid receptor heterodimers versus homomers by serial immunoprecipitation (IP). Cells coexpressing both HA-DOP-R (HADOP) and FLAG-KOP-R (FKOP) or each individually were lysed, and the receptors were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were immunoblotted (IB) with anti-HA antibody to detect KOP/DOP heterodimers (lanes 5 and 7). After the FLAG immunoprecipitation (F), the lysates were immunoprecipitated with anti-HA antibody and immunoblotted for HA to detect the DOP homomers/monomers remaining after immunoprecipitation of the heterodimers. Compare lanes 5 (heterodimers) and 6 (homomers/monomers). (c)6′-GNTI induced Ca²⁺ release in HEK-293 cells expressing one or two opioid receptor types. Agonist-mediated intracellular Ca²⁺ release was measured in cells expressing the chimeric G protein Δ6-G_qi4-myr (200 ng for every 40,000 cells) and MOP-R (▵), DOP-R (□), or KOP-Rs (○) alone or MOP/DOP-Rs (▪), KOP/MOP-Rs (▴), or KOP/DOP-Rs (•). Intracellular Ca²⁺ release was measured in a Flex apparatus (Molecular Devices), where relative light units (RLU) = maximum Ca²⁺ peak/cell number × 1,000. Shown are representative curves carried out in duplicate (n = 4). (Inset) Structure of 6′-GNTI. (d) Effects of receptor type-selective antagonists on 6′-GNTI-induced Ca²⁺ release in cells expressing the KOP/DOP-R heterodimer. Cells expressing the KOP/DOP-R heterodimer were preincubated for 30 min with increasing doses of NTI (○) or NorBNI (•) and stimulated with 100 nM 6′-GNTI. Agonist-induced Ca²⁺ release was assessed as described in c. Data are mean ± SEM measured in duplicates. (e and f) Effect of 6′-GNTI (e) and KOP-R- and DOP-R-selective antagonists (f) on competition for [³H]diprenorphine binding to cells expressing KOP/DOP-R heterodimers. Whole-cell competition binding experiments were performed on cells stably expressing the KOP/DOP-R heterodimers. Cells were incubated with 1.5 nM [³H]diprenorphine and increasing amounts of 6′-GNTI (▪) (e), NorBNI (•)(f), or NTI (○)(f). Shown are representative curves carried out in duplicate (n = 4). Note that error bars in e are too small to be visualized.