Opioid Drugs Act as Molecular Chaperones to Reshape Receptor Trafficking

A new study has revealed that some of the most familiar drugs in medicine’s opioid arsenal do far more than simply switch receptors on or off at the cell surface. Naltrexone, naloxone, morphine, and fentanyl can all act as pharmacological chaperones inside the endoplasmic reticulum, physically helping a misfolded mutant of the μ-opioid receptor fold properly and exit the organelle on its way to the plasma membrane. The finding, published in Pharmacology Research & Perspectives, offers an unusually detailed look at the earliest steps of opioid receptor trafficking and suggests that the concept of “inside-out pharmacology” extends to the receptors that mediate both pain relief and addiction.

The μ-opioid receptor, or MOR, is the primary molecular target for most clinically used analgesic opioids, from morphine to fentanyl. Decades of research have focused on what happens at the plasma membrane, where activated receptors are phosphorylated, bind β-arrestin, and are pulled into the cell by endocytosis. Chronic exposure to certain agonists reduces the number of receptors displayed on the cell surface, a process widely believed to contribute to tolerance and dependence. But the new work shifts attention to a much earlier stage of the receptor’s life: its maturation and export from the endoplasmic reticulum, the cellular factory where membrane proteins are synthesized and folded.

Studying this early phase in normal receptors is difficult because MORs traffic efficiently to the plasma membrane, leaving almost nothing in the endoplasmic reticulum to observe. To overcome this obstacle, the research team, led by Matthew J. Mulcahy, Stephen N. Grant, and Henry A. Lester, used a rare mutant called MOR[N190K], which is substantially retained in the endoplasmic reticulum. When cells expressing this mutant were treated with the antagonists naltrexone or naloxone, fluorescently tagged receptors suddenly appeared outlining the cell surface, reaching a distribution resembling that of the wild-type receptor. The drugs, in other words, had rescued the mutant receptor’s journey to the membrane.

The mechanism behind this rescue appears to involve endoplasmic reticulum exit sites, or ERES, specialized regions of the organelle where newly folded cargo is packaged into coat protein vesicles for transport to the Golgi apparatus. To visualize these structures, the researchers used a fluorescently tagged version of Sec24D, a core component of the ERES machinery. First, they needed to confirm that the receptor and Sec24D actually interact. Using sensitized emission Förster resonance energy transfer, a live-cell technique that detects when two fluorescently labeled proteins come within a few nanometers of each other, they showed that MOR[N190K] sits in close proximity to Sec24D inside the endoplasmic reticulum. A control experiment with Rab5, a marker of early endosomes, produced no comparable signal, ruling out the possibility that the interaction was happening after the receptor had already reached the cell surface and been internalized.

With that interaction established, the team turned to three-dimensional confocal microscopy to ask what happens to ERES when cells are flooded with opioid drugs. They measured the fraction of the cytoplasmic volume occupied by ERES, a metric chosen because, as earlier work by Heinzer and colleagues reasoned, expanding the size of exit sites is a more effective way to boost secretory flux than simply adding more of them. After twelve hours of treatment with ten micromolar concentrations of drug, four ligands stood out: naltrexone, naloxone, morphine, and fentanyl all significantly increased the ERES fraction. Buprenorphine, methadone, and two positive allosteric modulators, BMS 986122 and BMS 986124, did not.

The pattern held up under several additional tests. N-methyl-naltrexone, a permanently charged derivative of naltrexone that crosses the plasma membrane far less readily, produced only a modest shift in ERES levels, consistent with the idea that the drug must physically enter the cell and reach the endoplasmic reticulum to chaperone the receptor. A shorter four-hour incubation with naltrexone yielded only a partial increase, suggesting that the process builds over time as receptors cycle through the secretory pathway. And when the researchers blocked COPI-dependent retrograde transport with brefeldin A, naltrexone and naloxone lost their ability to raise ERES levels entirely. That last result implies that the mutant receptor must make multiple round trips between the endoplasmic reticulum and the Golgi before it is fully matured and ready for the plasma membrane, a requirement that mirrors findings from pharmacological chaperoning studies of other receptor families.

Perhaps the most intriguing result came from a double mutant. Serine 375, a residue in the receptor’s C-terminal tail, is a well-known phosphorylation site that governs arrestin recruitment and receptor internalization at the cell surface. When the researchers mutated this serine to alanine in the ER-retained background, creating MOR[N190K][S375A], naltrexone still chaperoned the receptor effectively, but morphine and fentanyl lost their chaperoning power altogether. This suggests that the agonists’ ability to promote receptor export depends on S375, possibly because they can induce phosphorylation of the residue even while the receptor is still maturing inside the endoplasmic reticulum. It also reveals an unexpected, “inside-out” role for a residue that has almost exclusively been studied in the context of events at the plasma membrane.

The team also tested and rejected an alternative explanation for the upregulation. One hypothesis held that antagonists raise intracellular cyclic AMP levels, which in other cell types has been shown to coincide with a global increase in protein trafficking. Because MOR activation suppresses cAMP, blocking basal receptor activity with an antagonist could plausibly raise cAMP and thereby accelerate trafficking generally. But a competitive ELISA assay on cell lysates showed no significant cAMP increase after twelve hours of naltrexone treatment, while the positive control, forskolin, produced the expected rise. With the cAMP pathway ruled out, the pharmacological chaperoning hypothesis stands as the leading explanation for how these ligands expand ERES levels and push the mutant receptor toward the cell surface.

The clinical stakes of this basic cell biology are considerable. Naltrexone and naloxone are the two most commonly prescribed opioid antagonists, given to people recovering from opioid use disorder. Chronic antagonist treatment is known to increase the number of opioid receptors on the cell surface, producing a supersensitivity that can persist for weeks after the final dose. If a patient relapses during that window, the expanded receptor population raises the risk of a fatal overdose. Understanding that antagonists may drive this upregulation, at least in part, by chaperoning receptors through the early secretory pathway opens a potential strategy for decoupling the therapeutic benefits of antagonist therapy from its most dangerous side effect.

The authors are careful about the limits of their findings. In pilot experiments, neither naltrexone nor the other ligands increased ERES levels or surface expression of the wild-type receptor, so the chaperoning mechanism demonstrated here applies only to the rare MOR[N190K] mutant, and the microscopy approach cannot yet be applied to native neurons in intact neural circuits. Generalizing the mechanism to explain supersensitivity in animals with normal receptors will require additional, still unknown elements of the chaperoning process, much as the mechanisms behind nicotine’s selective upregulation of nicotinic receptors in certain cell types remain only partly resolved. Interestingly, buprenorphine’s known ability to rescue the mutant receptor’s surface expression despite failing to raise ERES levels suggests that it acts through a different, ERES-independent trafficking step, hinting that multiple routes to the plasma membrane can be targeted by small molecules.

Even with those caveats, the study makes a strong case that pharmacological chaperoning deserves a place in any complete account of a drug’s efficacy profile. Ligands long classified purely by their actions at the cell surface, agonists and antagonists alike, turn out to influence the internal processing of their own receptors, changing how cells respond to drug exposure from the inside out. As the authors note, this principle now appears to span several drugs of abuse, from nicotine to opioids, and the hope is that future experiments will keep following opioid receptors into the endoplasmic reticulum and Golgi, far from the plasma membrane where most of their story has been told until now.

Subject of Research: Pharmacological chaperoning of the μ-opioid receptor by opioid agonists and antagonists via endoplasmic reticulum exit sites

Article Title: Naltrexone, Naloxone, Morphine, and Fentanyl Pharmacologically Chaperone a Mutant μ‐Opioid Receptor via an Endoplasmic Reticulum Exit Site‐Dependent Pathway

Article References: Grant, S. N., Mulcahy, M. J., & Lester, H. A. (2026). Naltrexone, Naloxone, Morphine, and Fentanyl Pharmacologically Chaperone a Mutant μ‐Opioid Receptor via an Endoplasmic Reticulum Exit Site‐Dependent Pathway. Pharmacology Research & Perspectives, 14(5), Article e70315. https://doi.org/10.1002/prp2.70315

Image Credits: AI Generated

DOI: 10.1002/prp2.70315

Keywords: μ-opioid receptor, pharmacological chaperoning, naltrexone, naloxone, fentanyl, morphine, endoplasmic reticulum exit sites, Sec24D, opioid use disorder, receptor trafficking, S375 phosphorylation, inside-out pharmacology