Cellular signalling of non-synonymous single-nucleotide polymorphisms of the human µ-opioid receptor (OPRM1).

UNLABELLED: There is significant variability in individual responses to opioid drugs, which is likely to have a significant genetic component. A number of non-synonymous single-nucleotide polymorphisms (SNPs) in the coding regions of the µ-opioid receptor gene (OPRM1) have been postulated to contribute to this variability. Although many studies have investigated the clinical influences of these µ-opioid receptor variants, the outcomes are reported in the context of thousands of other genes and environmental factors, and we are no closer to being able to predict individual response to opioids based on genotype. Investigation of how µ-opioid receptor SNPs affect their expression, coupling to second messengers, desensitization and regulation is necessary to understand how subtle changes in receptor structure can impact individual responses to opioids. To date, the few functional studies that have investigated the consequences of SNPs on the signalling profile of the µ-opioid receptor in vitro have shown that the common N40D variant has altered functional responses to some opioids, while other, rarer, variants display altered signalling or agonist-dependent regulation. Here, we review the data available on the effects of µ-opioid receptor polymorphisms on receptor function, expression and regulation in vitro, and discuss the limitations of the studies to date. Whether or not µ-opioid receptor SNPs contribute to individual variability in opioid responses remains an open question, in large part because we have relatively little good data about how the amino acid changes affect µ-opioid receptor function. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Fluorescence-based, high-throughput assays for µ-opioid receptor activation using a membrane potential-sensitive dye.

The development of new and improved opioid analgesics requires high-throughput screening (HTS) methods to identify potential therapeutics from large libraries of lead compounds. Here we describe two simple, real-time fluorescence-based assays of µ-opioid receptor activation that may be scaled up for HTS. In AtT-20 cells expressing the µ-opioid receptor (MOPr), opioids activate endogenous G protein gated inwardly rectifying K channels (GIRK channels), leading to membrane hyperpolarization. In Chinese hamster ovary cells expressing MOPr, adenylyl cyclase activation via forskolin results in membrane hyperpolarization, which is inhibited by opioids. Changes in membrane potential can be measured using a proprietary membrane potential-sensitive dye. In contrast to many HTS methods currently available, these assays reflect naturalistic coupling of the receptor to effector molecules.

A6V polymorphism of the human µ-opioid receptor decreases signalling of morphine and endogenous opioids in vitro.

BACKGROUND AND PURPOSE: Polymorphisms of the µ opioid receptor (MOPr) may contribute to the variation in responses to opioid drugs in clinical and unregulated situations. The A6V variant of MOPr (MOPr-A6V) is present in up to 20% of individuals in some populations, and may be associated with heightened susceptibility to drug abuse. There are no functional studies examining the acute signalling of MOPr-A6V in vitro, so we investigated potential functional differences between MOPr and MOPr-A6V at several signalling pathways using structurally distinct opioid ligands. EXPERIMENTAL APPROACH: CHO and AtT-20 cells stably expressing MOPr and MOPr-A6V were used. AC inhibition and ERK1/2 phosphorylation were assayed in CHO cells; K channel activation was assayed in AtT-20 cells. KEY RESULTS: Buprenorphine did not inhibit AC or stimulate ERK1/2 phosphorylation in CHO cells expressing MOPr-A6V, but buprenorphine activation of K channels in AtT-20 cells was preserved. [D-Ala2, N-MePhe4, Gly-ol]-enkephalin, morphine and ß-endorphin inhibition of AC was significantly reduced via MOPr-A6V, as was signalling of all opioids to ERK1/2. However, there was little effect of the A6V variant on K channel activation. CONCLUSIONS AND IMPLICATIONS: Signalling to AC and ERK via the mutant MOPr-A6V was decreased for many opioids, including the clinically significant drugs morphine, buprenorphine and fentanyl, as well endogenous opioids. The MOPr-A6V variant is common and this compromised signalling may affect individual responses to opioid therapy, while the possible disruption of the endogenous opioid system may contribute to susceptibility to substance abuse.

Buprenorphine signalling is compromised at the N40D polymorphism of the human µ opioid receptor in vitro.

BACKGROUND AND PURPOSE: There is significant variation in individual response to opioid drugs, which may result in inappropriate opioid therapy. Polymorphisms of the µ opioid receptor (MOP receptor) may contribute to individual variation in opioid response by affecting receptor function, and the effect may be ligand-specific. We sought to determine functional differences in MOP receptor signalling at several signalling pathways using a range of structurally distinct opioid ligands in cells expressing wild-type MOP receptors (MOPr-WT) and the commonly occurring MOP receptor variant, N40D. EXPERIMENTAL APPROACH: MOPr-WT and MOPr-N40D were stably expressed in CHO cells and in AtT-20 cells. Assays of AC inhibition and ERK1/2 phosphorylation were performed on CHO cells, and assays of K activation were performed on AtT-20 cells. Signalling profiles for each ligand were compared between variants. KEY RESULTS: Buprenorphine efficacy was reduced by over 50% at MOPr-N40D for AC inhibition and ERK1/2 phosphorylation. Buprenorphine potency was reduced threefold at MOPr-N40D for K channel activation. Pentazocine efficacy was reduced by 50% for G-protein-gated inwardly rectifying K channel activation at MOPr-N40D. No other differences were observed for any other ligands tested. CONCLUSIONS AND IMPLICATIONS: The N40D variant is present in 10-50% of the population. Buprenorphine is a commonly prescribed opioid analgesic, and many individuals do not respond to buprenorphine therapy. This study demonstrates that buprenorphine signalling to several effectors via the N40D variant of MOP receptors is impaired, and this may have important consequences in a clinical setting for individuals carrying the N40D allele.

A real-time, fluorescence-based assay for measuring µ-opioid receptor modulation of adenylyl cyclase activity in Chinese hamster ovary cells.

Inhibition of adenylyl cyclase (AC) activity is frequently used to measure µ-opioid receptor (MOR) activation. We sought to develop a simple, rapid assay of AC activity in whole cells that could be used to study MOR signaling. Chinese hamster ovary cells expressing human MOR (CHO-MOR cells) were grown in 96-well plates and loaded with membrane potential-sensitive fluorescent dye. CHO-MOR cells were treated with the AC activator forskolin (FSK), with or without simultaneous application of MOR agonists, and the resulting change in fluorescence was measured. CHO-MOR cells hyperpolarized in response to application of FSK (pEC50, 7.3) or calcitonin (pEC50, 9.4). A submaximally effective concentration of FSK (300 nM) caused a 52% ± 2% decrease in fluorescence. Simultaneous application of the opioids DAMGO (pEC50, 7.4; E(max), 56%), morphine (pEC50, 7.0; E(max), 61%); and buprenorphine (pEC50, 8.6; E(max), 24%) inhibited the FSK response in a dose-dependent manner while having no effect by themselves. The effects of DAMGO were blocked by pertussis toxin. This assay represents a simple, robust method for real-time observation of AC inhibition by MOR in CHO cells. It represents an appealing alternative to end-point assays that rely on cAMP accumulation and can avoid potential confounds associated with rapid desensitization of MOR signaling.

14q12 microdeletions excluding FOXG1 give rise to a congenital variant Rett syndrome-like phenotype.

Rett syndrome is a clinically defined neurodevelopmental disorder almost exclusively affecting females. Usually sporadic, Rett syndrome is caused by mutations in the X-linked MECP2 gene in ∼90-95% of classic cases and 40-60% of individuals with atypical Rett syndrome. Mutations in the CDKL5 gene have been associated with the early-onset seizure variant of Rett syndrome and mutations in FOXG1 have been associated with the congenital Rett syndrome variant. We report the clinical features and array CGH findings of three atypical Rett syndrome patients who had severe intellectual impairment, early-onset developmental delay, postnatal microcephaly and hypotonia. In addition, the females had a seizure disorder, agenesis of the corpus callosum and subtle dysmorphism. All three were found to have an interstitial deletion of 14q12. The deleted region in common included the PRKD1 gene but not the FOXG1 gene. Gene expression analysis suggested a decrease in FOXG1 levels in two of the patients. Screening of 32 atypical Rett syndrome patients did not identify any pathogenic mutations in the PRKD1 gene, although a previously reported frameshift mutation affecting FOXG1 (c.256dupC, p.Gln86ProfsX35) was identified in a patient with the congenital Rett syndrome variant. There is phenotypic overlap between congenital Rett syndrome variants with FOXG1 mutations and the clinical presentation of our three patients with this 14q12 microdeletion, not encompassing the FOXG1 gene. We propose that the primary defect in these patients is misregulation of the FOXG1 gene rather than a primary abnormality of PRKD1.

A continuous, fluorescence-based assay of µ-opioid receptor activation in AtT-20 cells.

Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.