Desensitization of functional µ-opioid receptors increases agonist off-rate

Abstract

Desensitization of µ-opioid receptors (MORs) develops over 5-15 minutes after the application of some, but not all, opioid agonists and lasts for tens of minutes after agonist removal. The decrease in function is receptor selective (homologous) and could result from 1) a reduction in receptor number or 2) a decrease in receptor coupling. The present investigation used photolysis of two caged opioid ligands to examine the kinetics of MOR-induced potassium conductance before and after MOR desensitization. Photolysis of a caged antagonist, carboxynitroveratryl-naloxone (caged naloxone), blocked the current induced by a series of agonists, and the time constant of decline was significantly decreased after desensitization. The increase in the rate of current decay was not observed after partial blockade of receptors with the irreversible antagonist, β-chlornaltrexamine (β-CNA). The time constant of current decay after desensitization was never more rapid than 1 second, suggesting an increased agonist off-rate rather than an increase in the rate of channel closure downstream of the receptor. The rate of G protein-coupled K(+) channel (GIRK) current activation was examined using photolysis of a caged agonist, carboxynitrobenzyl-tyrosine-[Leu(5)]-enkephalin. After acute desensitization or partial irreversible block of MORs with β-CNA, there was an increase in the time it took to reach a peak current. The decrease in the rate of agonist-induced GIRK conductance was receptor selective and dependent on receptor number. The results indicate that opioid receptor desensitization reduced the number of functional receptor and that the remaining active receptors have a reduced agonist affinity.

Fig. 1.

The rate of current decline induced by photolysis of CNV-NLX (5 µM) increased after desensitization induced by ME (30 µM). (A) Two experiments, one where photolysis of CNV-NLX was applied at the peak of the ME-induced current and the second 10 minutes after the application of ME. Examples of the change in current induced by CNV-NLX at the peak (left) and 10 minutes after the application of ME (right) are shown. (B) Summarized results showing the time constant of decay at the peak (blue), after ME (10 minutes, green), and at the peak of a nonsaturating concentration of ME (1 µM, black).

Fig. 2.

The rate of current decline increased after desensitization with the high-affinity agonist DERM (1 µM). (A) Two experiments showing the inhibition of the DERM current at the peak of the current (left) and 10 minutes after the application of DERM (right). Example traces of the current decline induced by CNV-NLX at the peak (peak), 10 minutes after DERM (DERM, 10 minutes), and after partial blockade of MORs with β-CNA (after β-CNA) are shown. (B) Dot plot of the time constant of CNV-NLX–induced inactivation of the DERM current at the peak (peak; ●), 10 minutes after DERM (10 minutes; ○), and 5–15 minutes after slices were desensitized with ME (30 µM, 1 to 2 hours, after-ME; ○) and after treatment with β-CNA (post–β-CNA; ●). Shaded areas illustrate the standard area of the time constant of decay measured at the peak (top) and after 10 minutes (bottom). Closed squares indicate the time constant of current decay induced by CNV-NLX after the application of DERM (30 nM). Larger squares are from the current experiments, and smaller squares are from published work. Open squares are the time constant of current decay induced by CNV-NLX in slices that were predesensitized by incubation with ME (30 µM, 30–90 minutes and washed before application of CNV-NLX, after ME). *P < 0.05 by unpaired Wilcoxon–Mann–Whitney rank sum test.

Fig. 3.

Application of DERM (1 µM) after desensitization with SST and OFQ does not change the rate of current decline induced by CNV-NLX. (A) Example trace of an experiment in which the current induced by SST (1 µM, 10 minutes) peaked and decayed, followed by the application of DERM and photolysis of CNV-NLX. (B) Dot plot of experiments measuring the time constant of current decay induced by CNV-NLX at the peak of the current induced by DERM (1 µM) in the presence of somatostatin (DERM + SST), OFQ (DERM + OFQ) and after the washout of OFQ (DERM wash OFQ). Shaded areas are the same as in Fig. 2, illustrating the standard error of experiments with DERM alone at the peak (top) and after 10 minutes (bottom). *P < 0.05 by one-way ANOVA compared with DERM alone; Dunnett post hoc. ns, not significant.

Fig. 4.

Summary of the results obtained for five agonists showing the increase in the rate of decline after desensitization. (A) Histogram of the time constant of current decay-induced photolysis of CNV-NLX (5 µM) for DERM (1 µM, same data as in Fig. 2), endomorphin-2 (Endo2; 1 µM), DAMGO (1 µM), ME (30 µM, same data as in Fig. 1), and morphine (1 µM). (Open bars) Measured at the peak current; solid bars are 10 minutes after application of the agonist, with the exception of morphine, which was after treatment of the animal for a week. In each case, the increase in the rate of decline after prolonged treatment (black bars) was significantly different from that measured at the peak. Horizontal line and dark and light gray boxes are the mean, S.E.M., and S.D. of the time constant of decline in the current induced by EC₅₀ concentrations of low-affinity agonists (codiene, DAMGO, ME, DSLET, oxycodone, and morphine, taken from Banghart et al., 2013). (B) Histogram of current amplitude before and after desensitization with the indicated agonist (normalized to the current induced by UK14304, 3 µM). *P < 0.05 by unpaired Wilcoxon–Mann–Whitney rank sum test.

Fig. 5.

Desensitization decreased the amplitude and rate of rise of the current induced by photolysis of CYLE. (A) Example trace of an experiment where the current induced by CYLE is shown before and after desensitization with ME (30 µM). Arrows indicate the light flash. (B) Summarized results showing the inhibition and recovery of the amplitude of the current induced by CYLE. (C) Two superimposed traces of the current induced by CYLE before and after recovery from desensitization (10–20 minutes). (D) Summarized results showing the increase in the rise (time constant) before (C) and after recovery from desensitization (des) and before (C) or after application of β-CNA (CNA; 100 nM, 2 minutes). *P < 0.05 by paired Wilcoxon–Mann–Whitney rank sum test.

Fig. 6.

Repeated long flashes decreased amplitude and rate of current rise induced by photolysis of CYLE. (A) Example trace of an experiment showing the current induced by two 5-second flashes of CYLE. (B) Two superimposed traces showing the change in rate of rise of current induced by two flashes of CYLE. (C) Summarized results showing the time constant of current activation induced by CYLE on three separate flashes applied at 3- to 5-minute intervals (closed circles). Open circles summarize experiments where slices were incubated in ME (30 µM, 30–90 minutes) to induce desensitization and washed before photolysis of CYLE (20 µM, 5 seconds, after ME).

Fig. 7.

CYLE-induced current is decreased by a receptor selective mechanism. (A) Example traces of the current induced by CYLE before (black) and after desensitization with OFQ (1 µM, 8–10 minutes, red). (B) Example traces of the current induced by CYLE (black) and in the presence of morphine (1 µM, 8–10 minutes, blue). (C) Summarized results measuring the time constant of the rate of rise. Experiments with OFQ and morphine were paired, and the control tau-On were combined for illustration. Statistics were done with a paired Wilcoxon–Mann–Whitney rank sum test (*P < 0.05). ns, not significant.

Fig. 8.

MOR desensitization does not change the noradrenaline activation of GIRK. (A) Example trace showing the current induced by photolysis of noradrenaline (arrows) during the application of DERM (1 µM) and DERM + naloxone (1 µM). (B) Summarized results showing the NA current decreased in DERM through occlusion, the reversal with naloxone (1 µM), the increase induced by cocaine (10 µM), followed by the inhibition with idazoxan (1 µM). (C) Superimposed traces of the current induced by caged noradrenaline (NA) before, 7–10 minutes in DERM, and after reversal with naloxone (1 µM). At bottom is the summarized rate of rise of the noradrenaline-induced current. Statistics tested with an analysis of variance, Dunnett’s post hoc test.