μ-Opioid Receptors: Ligand-Dependent Activation of Potassium Conductance, Desensitization, and Internalization

Abstract

micro-Opioid receptor (MOR) desensitization and endocytosis have been implicated in tolerance and dependence to opioids. The efficiency of each process is known to be agonist dependent; however, it is not known what determines the relative efficiency of various agonists at either process. In the present study, homologous MOR desensitization in locus ceruleus (LC) neurons and MOR internalization in HEK293 cells were examined using a series of agonists. The results show that the rank order of this series of agonists was different when comparing the magnitude of hyperpolarization and the ability to cause desensitization in LC neurons. Endocytosis of MOR was also examined in HEK293 cells using the same agonists. The relative ability to cause endocytosis in HEK293 cells correlated with the degree of desensitization in LC cells. This strong correlation suggests that the two processes are closely linked. The results also suggest that agonist efficacy is not necessarily a predictor of the ability to cause MOR desensitization or endocytosis. Identification and characterization of the biophysical properties of agonists that favor desensitization and internalization of receptors will lead to a better understanding of opioid signaling.

Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors

“…Because it is not possible to rapidly equilibrate and remove drugs from brain slices, the determination of efficacy for rapid desensitization in the LC has been somewhat problematic, but the degree of MOP activation and subsequent desensitization are correlated. Methadone is less efficacious than met-enkephalin and more efficacious than morphine (25,58), consistent with the findings of the present study. Morphine induces no measurable macroscopic desensitization of GIRK activation in LC neurons, but it has not been possible to examine morphine-or methadone-induced desensitization with lower concentrations of agonist because of the slow equilibration time of these agonists in brain slices (25,58).…”

“…The potency of all agonists to desensitize FLAG-MOP was at least 10-fold lower than for G protein activation. These results suggest that the capacity of an agonist to produce rapid desensitization might be related to its efficacy for activation of G proteins, although the efficiency of fast desensitization is much lower than receptor activation, as has generally been indicated in previous studies (24,25,58). The desensitization of FLAG-MOP inhibition of I Ca appeared to be homologous, because inhibition of I Ca by moderate concentrations of SRIF was not affected by treatment with concentrations of DAMGO that produced maximum MOP desensitization.…”

“…Some studies have reported that morphine and other agonists such as DAMGO and methadone have similar signaling efficacies but that morphine has a much lower efficacy to induce receptor internalization (11,22). Other studies suggest that the capacity of MOP agonists to induce desensitization or endocytosis is simply a function of their efficacy to induce activation of G proteins (24,25) and that the partial agonist properties of morphine are the reason for its poor efficacy to induce internalization. The uncertainty concerning differential efficacy of MOP agonists for G protein activation, rapid desensitization, and endocytosis have probably arisen for three main reasons.…”

“…First, most studies of these processes have used heterologous expression systems that greatly overexpress receptors, thus disrupting quantitative determination of relative signaling efficacy as well as the stoichiometry of MOP/GRK/␤-arrestin interactions (11,26,27). Second, MOP activation, desensitization, and endocytosis have generally been compared between different cellular expression systems under different conditions (22,24,25). This is mostly because cell lines do not generally express effector proteins that can be used as rapid reporters of MOP activation, whereas receptor internalization is difficult to study quantitatively in native cells because of relatively low levels of native receptor expression.…”

Functional characterization of human variants of the mu-opioid receptor gene

“…This trafficking is thought to contribute to both attenuated opioid signaling during acute agonist stimulation and the recovery of responsiveness on removal of the agonist (6). Although most opiates promote MOR internalization via ␤-arrestin mediated pathways, activation of MORs by morphine results in very little internalization (5,7,8). It has been suggested that this aberrant trafficking of MORs results in prolonged activation of the receptor, leading to compensatory changes that result in tolerance to therapeutic doses of morphine (9,10).…”