Chapter 4: Opioid receptor antagonists pharmacologically chaperone a
4.4 Discussion
Although efforts continue to reduce pain by actions on non-MOR targets such as the voltage-gated sodium channels and some nicotinic receptors, at present the MOR continues to be the primary target for analgesic drugs.45,46 MOR trafficking is important to the development of opioid use disorder. If we can understand MOR trafficking and manipulate MOR surface densities in predictable ways, we may better treat opioid use disorder. Until now, there have been no studies done on ERES and MORs.
Here we show that, for a mutant MOR that builds to observable levels in the ER, the antagonists Ntx and naloxone increase the fraction of the cytoplasm
occupied by ERES. This is likely an early event in the pathway that results in increased opioid receptor surface densities and supersensitivity (Figure 4.4). By demonstrating that this adverse effect of antagonist treatment is manifested through ERES, we show that ERES can be considered a component in opioid use disorder.
Many agonists cause some endocytosis, but whether these drugs also cause chaperoning is not clear. We find that morphine, fentanyl, buprenorphine, and methadone do not cause a significant change in ERES levels. Our work suggests that the conformation induced by these agonists is less favorable for chaperoning than the conformation induced by the antagonists. In one hypothesis, this may be due to the lack of S375 phosphorylation when an antagonist is bound.36 To test this, we made MOR[N190K][S375A], a MOR mutant that is highly-ER retained and could not be phosphorylated at S375. Despite the inability to phosphorylate S375, ERES levels did not significantly change using this mutant after 12 h incubation in 10 µM morphine or fentanyl (Figure 4.5).
We ruled out that the increases in ERES arise from increases in [cAMP] by measuring [cAMP] in MOR[N190K]-overexpressing SH-SY5Y cell lysates after incubation in Ntx, vehicle, or forskolin using a competitive ELISA assay. We overexpress MOR[N190K] to most closely replicate cells used in the imaging experiments. SH-SY5Y cells endogenously express the MOR, thus there will be MORs exhibiting basal activity at the plasma membrane that could be reduced by antagonist exposure.47 Despite decreasing the basal activity of the MORs, we did not observe a significant change in [cAMP] in cells treated with Ntx (Figure 4.6).
Since [cAMP] rises are no longer a viable reason for the increase in ERES levels, this experiment further supports the chaperoning hypothesis.
Our work does not challenge the prevailing view that, for the wild type MOR, agonists affect MOR via endocytosis at the plasma membrane—a much later stage than studied here.48 The lack of substantial chaperoning by agonists is consistent with prior observations that several of the key steps in ERES formation could well be suppressed by the effects of MOR activation (such as increases in [cAMP]).16,49 The endocytosed MOR plays a role in the cellular response to membrane- permeant opioids since the MOR can signal within recycling endosomes; in addition, MOR may also signal within the Golgi. 50 Fentanyl increases the recycling of MORs after endocytosis.15 This observation, along with our results that antagonists can control trafficking from the ER to the Golgi, emphasizes how the earlier mechanisms that deliver MORs to the plasma membrane from the ER are regulated differently than the later mechanisms that traffic MORs from endosomes.15 Pathways to the plasma membrane vary among MORs, and one may target one route over the other.
These results also give insight into how the mutant MOR matures in the ER and Golgi. The lack of ERES increases with COPI inhibition suggests some recycling between the ER and the Golgi. This observation is consistent with the
~24 hour incubation with naltrexone before observing increased MOR plasma membrane levels.32 We also observe some time dependence with our ERES experiments (Figure 4.4). We hypothesize that after 4 hours, the mutant MOR has not had an opportunity to enter the Golgi-ER pathway, and only after 12 hours do we observe this transition as an increase in ERES levels. Furthermore, these COPI experiments are consistent with prior reports demonstrating the importance of retrograde transport for pharmacological chaperoning.18,43
Our results provide key information regarding MOR trafficking. We find that the antagonists can increase ERES levels, showing that in this case ERES exit events are rate-limiting for downstream MOR plasma membrane densities.
Meanwhile, agonists do not increase ERES levels, consistent with their inability to increase MOR plasma membrane densities. Pharmacological chaperoning starts at the ER in many cases, and our work provides further evidence that Ntx and naloxone are pharmacological chaperones for the MOR.
Our work also continues to make a case for pharmacological chaperoning as an important part of a drug’s efficacy profile.22 This part of “inside-out pharmacology” involves using ligands to increase the trafficking of receptors.51
Our study applies only to the rare MOR[N190K] mutant; in pilot experiments, we found neither upregulation nor effects on ERES levels with wild type MOR. This limits our ability to explain the mechanism of upregulation/supersensitivity of MOR found in studies on intact animals with WT MORs treated with antagonists.11,52,53 Superresolution experiments like those described here cannot yet be applied to native neurons of intact neural pathways.
One would need to invoke additional, unknown aspects of chaperoning to generalize its usefulness as a mechanism for upregulation / supersensitivity. This point resembles the uncertainties about mechanisms that allow nicotine to upregulate nicotinic receptors of only some cells types and only at some subcellular regions.51
To the extent that pharmacological chaperoning does govern upregulation / supersensitivity by naltrexone, we note that Me-Ntx is FDA-approved to suppress opioid-induced constipation (OIC) when opioids are used for chronic noncancer pain. OIC is a major discomfort. We can now suggest that Me-Ntx and other
membrane-impermeant antagonists, unlike membrane-permeant antagonists such as naltrexone and naloxone, are unlikely to cause upregulation / supersensitivity. This difference would arise from the same mechanism that allows Me-Ntx to block peripheral but not CNS MOR: it does not permeate across membranes.54 Perhaps Me-Ntx deserves wider use for its approved indications.
4.5 Materials and Methods