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Concept: Opioid receptor


G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (K d  = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.

Concepts: Protein, Signal transduction, Cell membrane, Receptor, Opioid receptor, G protein-coupled receptor, Metabotropic glutamate receptor, Solutions


This issue of Molecular Pharmacology is dedicated to Dr. Avram Goldstein, the journal’s founding Editor and one of the leaders in the development of modern pharmacology. This chapter focuses on his contributions to the discovery of the dynorphins and evidence that members of this family of opioid peptides are endogenous agonists for the kappa opioid receptor. In his original publication describing the purification and sequencing of dynorphin A, Avram described this peptide as ‘extraordinarily potent’ (‘dyn’ from the Greek, dynamis = power and ‘-orphin’ for endogenous morphine peptide). The name originally referred to its high affinity and great potency in the bioassay that was used to follow its activity during purification, but the name has come to have a second meaning: Studies of its physiological function in brain continue to provide powerful insights to the molecular mechanisms controlling the mood disorders and drug addiction. In the 30 years since its discovery, we have learned that the dynorphin peptides are released in brain during stress exposure. Once released, they activate kappa opioid receptors distributed throughout the brain and spinal cord where they trigger cellular responses resulting in different stress responses: analgesia, dysphoria-like behaviors, anxiety-like responses, and increased addiction behaviors in experimental animals. Avram predicted that a detailed molecular analysis of opiate drug actions would someday lead to better treatments for drug addiction, and he would be gratified to know that subsequent studies enabled by his discovery of the dynorphins resulted in insights that hold great promise for new treatments for addiction and depressive disorders.

Concepts: Opioid, Morphine, Opioid receptor, Heroin, Buprenorphine, Endorphin, Codeine, Kappa Opioid receptor


It is well-known that genotypic differences can account for the subject-specific responses to opiate administration. In this regard, the basal activity of the endogenous system (either at the receptor or at the ligand level) can modulate the effects of exogenous agonists as morphine, and vice versa. The μ opioid receptor from zebrafish, dre-oprm1, binds endogenous peptides and morphine with similar affinities. Morphine administration during development altered the expression of the endogenous opioid propeptides proenkephalins and proopiomelanocortin. Treatment with opioid peptides (Met-ENK, MEGY and β-END) modulated dre-oprm1 expression during development. Knocking-down dre-oprm1 gene significantly modified the mRNA expression of the penk and pomc genes, thus indicating that oprm1 is involved in shaping penk and pomc expression. Besides, the absence of a functional oprm1 clearly disrupted the embryonic development, as proliferation was disorganized in the central nervous system of oprm1-morphant embryos: mitotic cells were found widespread through the optic tectum, and not restricted to the proliferative areas of the mid- and hindbrain. TUNEL staining revealed that the number of apoptotic cells in the Central Nervous System (CNS) of morphants was clearly increased at 24 hpf. These findings will help to understand the role of the endogenous opioid system in the CNS development. Our results will also contribute to unravel the complex feedback loops which modulate opioid activity, and which may be involved in establishing a coordinated expression of both receptors and endogenous ligands. Further knowledge of the complex interactions between the opioid system and analgesic drugs will provide insights that may be relevant for analgesic therapy.

Concepts: Central nervous system, Opioid, Morphine, Opioid receptor, Buprenorphine, Hydrocodone, Codeine, Oxycodone


Hypothalamic pro-opiomelanocortin (POMC) neurons promote satiety. Cannabinoid receptor 1 (CB1R) is critical for the central regulation of food intake. Here we test whether CB1R-controlled feeding in sated mice is paralleled by decreased activity of POMC neurons. We show that chemical promotion of CB1R activity increases feeding, and notably, CB1R activation also promotes neuronal activity of POMC cells. This paradoxical increase in POMC activity was crucial for CB1R-induced feeding, because designer-receptors-exclusively-activated-by-designer-drugs (DREADD)-mediated inhibition of POMC neurons diminishes, whereas DREADD-mediated activation of POMC neurons enhances CB1R-driven feeding. The Pomc gene encodes both the anorexigenic peptide α-melanocyte-stimulating hormone, and the opioid peptide β-endorphin. CB1R activation selectively increases β-endorphin but not α-melanocyte-stimulating hormone release in the hypothalamus, and systemic or hypothalamic administration of the opioid receptor antagonist naloxone blocks acute CB1R-induced feeding. These processes involve mitochondrial adaptations that, when blocked, abolish CB1R-induced cellular responses and feeding. Together, these results uncover a previously unsuspected role of POMC neurons in the promotion of feeding by cannabinoids.

Concepts: Receptor, Opioid, Receptor antagonist, Opioid receptor, Cannabinoid receptor, Dopamine, Buprenorphine, Proopiomelanocortin


Exercise is rewarding, and long-distance runners have described a runner’s high as a sudden pleasant feeling of euphoria, anxiolysis, sedation, and analgesia. A popular belief has been that endogenous endorphins mediate these beneficial effects. However, running exercise increases blood levels of both β-endorphin (an opioid) and anandamide (an endocannabinoid). Using a combination of pharmacologic, molecular genetic, and behavioral studies in mice, we demonstrate that cannabinoid receptors mediate acute anxiolysis and analgesia after running. We show that anxiolysis depends on intact cannabinoid receptor 1 (CB1) receptors on forebrain GABAergic neurons and pain reduction on activation of peripheral CB1 and CB2 receptors. We thus demonstrate that the endocannabinoid system is crucial for two main aspects of a runner’s high. Sedation, in contrast, was not influenced by cannabinoid or opioid receptor blockage, and euphoria cannot be studied in mouse models.

Concepts: Receptor, Opioid, Morphine, Opioid receptor, Cannabinoid receptor, Tetrahydrocannabinol, Cannabis, Cannabidiol


The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as means to modulate pain perception, stress responses and affective reward states. Therefore, the KOR has become a prominent drug discovery target towards treating pain, depression and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAP kinase activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased towards G protein coupling and away from βarrestin2 recruitment. Here we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent functionally selective, small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

Concepts: Signal transduction, Receptor, Opioid, Morphine, Drug addiction, Opioid receptor, Buprenorphine, Kappa Opioid receptor


Opioid use for pain management has dramatically increased, with little assessment of potential pathophysiological consequences for the primary pain condition. Here, a short course of morphine, starting 10 d after injury in male rats, paradoxically and remarkably doubled the duration of chronic constriction injury (CCI)-allodynia, months after morphine ceased. No such effect of opioids on neuropathic pain has previously been reported. Using pharmacologic and genetic approaches, we discovered that the initiation and maintenance of this multimonth prolongation of neuropathic pain was mediated by a previously unidentified mechanism for spinal cord and pain-namely, morphine-induced spinal NOD-like receptor protein 3 (NLRP3) inflammasomes and associated release of interleukin-1β (IL-1β). As spinal dorsal horn microglia expressed this signaling platform, these cells were selectively inhibited in vivo after transfection with a novel Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Multiday treatment with the DREADD-specific ligand clozapine-N-oxide prevented and enduringly reversed morphine-induced persistent sensitization for weeks to months after cessation of clozapine-N-oxide. These data demonstrate both the critical importance of microglia and that maintenance of chronic pain created by early exposure to opioids can be disrupted, resetting pain to normal. These data also provide strong support for the recent “two-hit hypothesis” of microglial priming, leading to exaggerated reactivity after the second challenge, documented here in the context of nerve injury followed by morphine. This study predicts that prolonged pain is an unrealized and clinically concerning consequence of the abundant use of opioids in chronic pain.

Concepts: Protein, Receptor, Opioid, Pain, Morphine, Opioid receptor, Ketamine, Recreational drug use


Kappa opioid receptors (KORs) are involved in a variety of aversive behavioral states, including anxiety. To date, a circuit-based mechanism for KOR-driven anxiety has not been described. Here, we show that activation of KORs inhibits glutamate release from basolateral amygdala (BLA) inputs to the bed nucleus of the stria terminalis (BNST) and occludes the anxiolytic phenotype seen with optogenetic activation of BLA-BNST projections. In addition, deletion of KORs from amygdala neurons results in an anxiolytic phenotype. Furthermore, we identify a frequency-dependent, optically evoked local dynorphin-induced heterosynaptic plasticity of glutamate inputs in the BNST. We also find that there is cell type specificity to the KOR modulation of the BLA-BNST input with greater KOR-mediated inhibition of BLA dynorphin-expressing neurons. Collectively, these results provide support for a model in which local dynorphin release can inhibit an anxiolytic pathway, providing a discrete therapeutic target for the treatment of anxiety disorders.

Concepts: DNA, Cell, Enzyme inhibitor, Opioid receptor, Inhibitor, Anxiety disorder, Alprazolam, Kappa Opioid receptor


The kappa opioid receptor (KOR) is involved in mediating pruritus; agonists targeting this receptor have been used to treat chronic intractable itch. Conversely, antagonists induce an inch response at the site of injection. As a G protein-coupled receptor (GPCR), the KOR has potential for signaling via G proteins and βarrestins, however, it is not clear which of these pathways are involved in the KOR modulation of itch. In this study asked whether the actions of KOR in pruritus involve βarrestins by using βarrestin2 knockout (βarr2-KO) mice as well as a recently described biased KOR agonist that biases receptor signaling toward G protein pathways over βarrestin2 recruitment. We find that the KOR antagonists nor-binaltorphimine (NorBNI) and 5'-guanidinonaltrindole (5'GNTI) induce acute pruritus in C57BL/6J mice, with reduced effects in KOR-KO mice. βarr2-KO mice display less of a response to KOR antagonist-induced itch compared to wild types, however no genotype differences are observed from chloroquine phosphate (CP)-induced itch, suggesting that the antagonists may utilize a KOR-βarrestin2 dependent mechanism. The KOR agonist U50,488H was equally effective in both WT and βarr2-KO mice in suppressing CP-induced itch. Furthermore, the G protein biased agonist, Isoquinolinone 2.1 was as effective as U50,488H in suppressing the itch response induced by KOR antagonist NorBNI or CP in C57BL/6J mice. Together these data suggest that the antipruritic effects of KOR agonists may not require βarrestins.

Concepts: Signal transduction, Receptor antagonist, Opioid receptor, G protein-coupled receptor, Chloroquine, Inverse agonist, Buprenorphine, G protein


Stress facilitates reinstatement of addictive drug seeking in animals and promotes relapse in humans. Acute stress has marked and long-lasting effects on plasticity at both inhibitory and excitatory synapses on dopamine neurons in the ventral tegmental area (VTA), a key region necessary for drug reinforcement. Stress blocks long-term potentiation at GABAergic synapses on dopamine neurons in the VTA (LTP), potentially removing a normal brake on activity. Here we show that blocking kappa opioid receptors (KORs) prior to forced-swim stress rescues LTP. In contrast, blocking KORs does not prevent stress-induced potentiation of excitatory synapses nor morphine-induced block of LTP. Using a kappa receptor antagonist as a selective tool to test the role of LTP in vivo, we find that blocking KORs within the VTA prior to forced-swim stress prevents reinstatement of cocaine seeking. These results suggest that KORs may represent a useful therapeutic target for treatment of stress-triggered relapse in substance abuse.

Concepts: Receptor, Morphine, Drug addiction, Opioid receptor, Addiction, Long-term potentiation, Dopamine, Butorphanol